U.S. patent application number 10/060019 was filed with the patent office on 2003-01-02 for methods and compositions for modulating ubiquitin dependent proteolysis.
This patent application is currently assigned to Mount Sinai Hospital Corporation. Invention is credited to Tyers, Mike, Willems, Andrew.
Application Number | 20030003564 10/060019 |
Document ID | / |
Family ID | 27370443 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003564 |
Kind Code |
A1 |
Tyers, Mike ; et
al. |
January 2, 2003 |
Methods and compositions for modulating ubiquitin dependent
proteolysis
Abstract
The invention relates to methods and compositions for modulating
ubiquitin dependent proteolysis.
Inventors: |
Tyers, Mike; (Toronto,
CA) ; Willems, Andrew; (Toronto, CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Mount Sinai Hospital
Corporation
Toronto
ON
|
Family ID: |
27370443 |
Appl. No.: |
10/060019 |
Filed: |
January 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10060019 |
Jan 29, 2002 |
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09177165 |
Oct 22, 1998 |
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6426205 |
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60063254 |
Oct 24, 1997 |
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60092443 |
Jul 10, 1998 |
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Current U.S.
Class: |
435/226 ;
424/94.63 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4738 20130101; C07K 14/395 20130101 |
Class at
Publication: |
435/226 ;
424/94.63 |
International
Class: |
A61K 038/48; C12N
009/64 |
Claims
We claim:
1. A method for modulating ubiquitin dependent proteolysis
comprising administering an effective amount of one or more of the
following: (a) a complex comprising an E2 ubiquitin conjugating
enzyme, a protein of the Cullin family, and a F-box binding
protein, and optionally a protein containing an F-box motif; (b) a
complex comprising a protein of the Cullin family and a protein
containing an F-box motif; (c) a peptide derived from the binding
domain of an E2 ubiquitin conjugating enzyme that interacts with a
protein of the Cullin family; (d) a peptide derived from the
binding domain of a protein of the Cullin family that interacts
with an E2 ubiquitin conjugating enzyme; (d) a peptide derived from
the binding domain of a protein of the Cullin family that interacts
with an F-box binding protein; (e) a peptide derived from the
binding domain of an F-box binding protein that interacts with a
protein of the Cullin family; (f) enhancers or inhibitors of the
interaction of an E2 ubiquitin conjugating enzyme or a F-box
binding protein, and a protein of the Cullin family.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods and compositions for
modulating ubiquitin dependent proteolysis.
BACKGROUND OF THE INVENTION
[0002] Ubiquitin-dependent proteolysis is a key regulatory
mechanism that controls diverse cellular processes (reviewed in
Hochstrasser 1996). In this pathway, ubiquitin is transferred via
transthioesterification along a cascade of carrier enzymes,
E1->E2->E3, and ultimately conjugated in an isopeptide
linkage to a lysine residue of a substrate protein. Reiteration of
the ubiquitin transferase reaction results in formation of a
polyubiquitin chain on the substrate, which is then recognized by
the 26S proteasome, and rapidly degraded. Specificity in protein
ubiquitination derives from E3 enzymes, also known as
ubiquitin-ligases (Hershko et al. 1983). In some cases, an E3
facilitates recognition of the target protein by an E2, while in
others an E3 accepts a ubiquitin thioester from an E2 and directly
transfers ubiquitin to the substrate (Scheffner et al. 1995).
Although substrate recognition is a key aspect of ubiquitin
dependent proteolysis, the identification of E3 enzymes has been
problematic because the few known E3 families bear no sequence
relationship to each other.
[0003] Ubiquitin-dependent proteolysis is essential for two major
cell cycle transitions, the G1 to S phase transition and the
metaphase to anaphase transition (reviewed in King et al. 1996).
These transitions mediate alteration between states of high and low
cyclin-dependent kinase (Cdk) activity, which in turn ensures that
DNA replication origins fire only once per cell cycle and that
chromosome segregation follows DNA replication (reviewed in Nasmyth
1996). Key targets of the ubiquitin proteolytic pathway at these
transitions include positive regulators of Cdks, the cyclins, and
negative regulators of Cdks, the Cdk inhibitors. In budding yeast,
a single Cdk, Cdc28 (or Cdk1) is activated in G1 phase by the G1
cyclins Cln1-Cln3, and in S through M phase by the mitotic cyclins,
Clb1-Clb6 (reviewed in Nasmyth, 1996). A motif called the
destruction box targets mitotic cyclins and other proteins to a
cell cycle-regulated E3 ubiquitin-ligase called the Anaphase
Promoting Complex (APC) or cyclosome (reviewed in King et al.
1996). In contrast, phosphorylation targets G1 cyclins and Cdk
inhibitors for degradation via a constitutive ubiquitination
pathway (reviewed in Deshaies, 1997). Genetic analysis in budding
yeast has revealed several components of this pathway: Cdc4, a WD40
repeat protein (Yochem and Byers, 1987), Cdc34, an E2 ubiquitin
conjugating enzyme (Goebl et al. 1988), Cdc53, a protein that forms
a tight complex with phosphorylated Clns (Willems et al. 1996),
Grr1, a leucine rich repeat protein (Flick and Johnston, 1981), and
Skp1, a protein that binds to a motif called the F-box (Bai et al.
1996). The F-box motif occurs in Cdc4, Grr1 and several other yeast
and mammalian proteins (Bai et al. 1996). Cells lacking functional
Cdc4, Cdc34, Cdc53 or Skp1 arrest in G1 because the Cdk inhibitor
Sic1 is not degraded, which prevents the onset of Clb-Cdc28
activity and initiation of DNA replication (Nugroho and Mendenhall
1994; Schwob et al. 1994; Bai et al. 1996). In late G1 phase, Sic1
is phosphorylated by the Cln-Cdc28 kinases and thus targeted for
ubiquitin dependent proteolysis (Schwob et al. 1994; Schneider et
al. 1996; Tyers 1996). Recently, a requirement for Cdc4, Cdc34 and
Cln2-Cdc28 activity in Sic1 ubiquitination has been demonstrated in
an in vitro yeast extract system (Verma et al. 1997). Cdc34, Cdc53
and Skp1 are also required for Cln degradation (reviewed in
Deshaies 1997), as is Grr1 (Barral at al. 1995), although this
protein was originally identified because of its role in glucose
repression (Flick and Johnston 1991).
[0004] Other important regulatory proteins are degraded via the
Cdc34 pathway, including the Cln-Cdc28 inhibitor Far1 (McKinney et
al. 1993; Henchoz et al. 1997), the replication protein Cdc6
(Piatti et al. 1996), and the transcription factor Gcn4 (Komitzer
et al. 1994). Aside from its G1 function, Skp1 also plays a role in
G2 because certain conditional alleles of SKP1 arrest cells in G2,
and because Skp1 is a component of the Cbf3 kinetochore complex
(Bai et al. 1996; Connelly and Hieter 1996; Stemmann and Lechner
1996).
[0005] Genetic and biochemical evidence indicates that Cdc53
interacts with Cdc4 and Cdc34 (Willems et al. 1996; Mathias et al.
1996), and that the F-box of Cdc4 binds Skp1 (Bai et al. 1996).
These interactions, and the fact that Cdc53 physically associates
with phosphorylated forms of Cln2, suggest that Cdc4, Cdc34, Cdc53
and Skp1 may participate in an E2/E3 ubiquitination complex that
recognizes and ubiquitinates phosphorylated substrates (Bai et al.
1996; Willems et al. 1996). Divergence of the Sic1 and Cln
degradation pathways apparently occurs at the level of the two
F-box proteins. Cdc4 is required for degradation of Sic1 (Schwob et
al. 1994), whereas Grr1 is required for Cln1/2 degradation (Barral
et al. 1995). It was therefore hypothesized that distinct F-box
proteins recruit specific substrates to an E3 ubiquitin-ligase
complex that contains Skp1 (Bai et al. 1996). The existence of a
complex in vivo containing F-box proteins and Cdc34, Cdc53 and Skp1
has yet to be demonstrated.
SUMMARY OF THE INVENTION
[0006] Through analysis of Cdc53-interacting proteins the present
inventors determined that Cdc53 forms complexes with Skp1, Cdc34,
and each of the F-box proteins Cdc4, Grr1 and Met30 in vivo. Each
F-box protein confers functional specificity on a core
Cdc34-Cdc53-Skp1 complex for Sic1 degradation, Cln degradation and
methionine biosynthesis gene regulation, respectively. The present
inventors showed that Cdc53 is a scaffold that tethers Skp1/F-box
proteins to Cdc34 within an E2/E3 ubiquitination complex. The
present inventors have also identified a specific region on Cdc53
that binds to Skp1.
[0007] Broadly stated the present invention relates to (a) a
complex comprising an E2 ubiquitin conjugating enzyme, a protein of
the Cullin family, an -F-box binding protein, and optionally a
protein containing an F-box motif; and (b) a complex comprising a
protein of the Cullin family and a protein containing an F-box
motif. The invention is also directed to (a) a peptide derived from
the binding domain of an E2 ubiquitin conjugating enzyme that
interacts with a protein of the Cullin family; (b) a peptide
derived from the binding domain of a protein of the Cullin family
that interacts with an E2 ubiquitin conjugating enzyme; (c) a
peptide derived from the binding domain of a protein of the Cullin
family that interacts with an F-box binding protein; preferably a
peptide of the formula I or Ia or (d) a peptide derived from the
binding domain of an F-box binding protein that interacts with a
protein of the Cullin family. The invention also contemplates
antibodies specific for the complexes and peptides of the
invention.
[0008] The present invention also provides a method of modulating
ubiquitin dependent proteolysis comprising administering an
effective amount of one or more of the following: (a) a complex
comprising an E2 ubiquitin conjugating enzyme, a protein of the
Cullin family, an F-box binding protein, and optionally a protein
containing an F-box motif; (b) a complex comprising a protein of
the Cullin family and a protein containing an F-box motif ; (c) a
peptide derived from the binding domain of an E2 ubiquitin
conjugating enzyme that interacts with a protein of the Cullin
family; (d) a peptide derived from the binding domain of a protein
of the Cullin family that interacts with an E2 ubiquitin
conjugating enzyme; (d) a peptide derived from the binding domain
of a protein of the Cullin family that interacts with an F-box
binding protein; preferably a peptide of the formula I or Ia (e) a
peptide derived from the binding domain of an F-box binding protein
that interacts with a protein of the Cullin family; or (f)
enhancers or inhibitors of the interaction of an E2 ubiquitin
conjugating enzyme or an F-box binding protein, with a protein of
the Cullin family.
[0009] In a preferred embodiment of the invention a method is
provided for modulating ubiquitin dependent proteolysis comprising
administering an effective amount of one or more of the following:
(a) a complex comprising Cdc34-Cdc53-Skp1; (b) a complex comprising
Cdc34-Cdc53-Ckp1-protein containing an F-box motif; (c) a complex
comprising Cdc53-protein containing an F-box motif; (d) a peptide
comprising the binding domain of Cdc34 that interacts with Cdc53 or
the binding domain of Cdc53 that interacts with Cdc34; (e) a
peptide comprising the binding domain of Cdc53 that interacts with
Skp1 or the binding domain of Skp1 that interacts with Cdc53; or,
(f) inhibitors or enhancers of the interaction of Cdc34 or Skp1,
with Cdc53.
[0010] The invention still further provides a method for
identifying a substance that binds to a complex comprising an E2
ubiquitin conjugating enzyme and a protein of the Cullin family, a
complex comprising an E2 ubiquitin conjugating enzyme, a protein of
the Cullin family, an F-box binding protein, and optionally a
protein containing an F-box motif, or a complex comprising a
protein of the Cullin family and an F-box binding protein,
comprising: (a) reacting the complex with at least one substance
which potentially can bind with the interacting molecules in the
complex, under conditions which permit the formation of conjugates
between the substance and complex and (b) assaying for conjugates,
for free substance, or for non-conjugated complexes. The invention
also contemplates methods for identifying substances that bind to
other intracellular proteins that interact with the complexes of
the invention.
[0011] Still further the invention provides a method for evaluating
a compound for its ability to modulate ubiquitin dependent
proteolysis. For example a substance which inhibits or enhances the
interaction of the molecules in a complex of the invention, or a
substance which binds to the molecules in a complex of the
invention may be evaluated. In an embodiment, the method comprises
providing a known concentration of a complex of the invention, with
a substance which binds to the complex, and a test compound under
conditions which permit the formation of conjugates between the
substance and complex, and removing and/or detecting
conjugates.
[0012] The present invention also contemplates a peptide of the
formula I which interferes with the interaction of Cdc53 and
Skp1
A-Tyr-Met-X.sup.1-X.sup.2-Tyr-X.sup.3-X.sup.4-X.sup.5-Tyr-X.sup.6-X.sup.7--
Cys-X.sup.8 I
[0013] wherein A represents one to ten amino acids, X.sup.1
represents Met, Arg, Thr, or Glu, X.sup.2 represents Leu, Phe, or
Val, X.sup.3 represents Asp or Thr, X.sup.4 represents Ala, Ser,
His, or Thr, X.sup.5 represents Ile or Val, X.sup.6 represents Asn
or Asp, X.sup.7 represents Tyr, Ile, or Met, and X.sup.8 represents
Thr, Val, or Ala.
[0014] In an embodiment of the present invention a peptide of the
formula Ia which interferes with the interaction of Cdc53 and Skp1
is provided:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-Tyr-
Met-X.sup.1-X.sup.2-Tyr-X.sup.3-X.sup.4-X.sup.5-Tyr-X.sup.6-X.sup.7-Cys-X-
.sup.8 Ia
[0015] wherein A.sup.1 represents Ile, Asn, His, Ser, or Ala,
A.sup.2 represents Leu, Met or Phe, A.sup.3 represents Ser, Ala,
Thr, or Asp, A.sup.4 represents Pro, Lys, Arg, or Ser, A.sup.5
represents Thr, Lys, Ser, or Glu, A.sup.6 represents Met, Asp, Tyr,
Gln, or Arg, X.sup.7 represents Met, Arg, Thr, or Glu, X.sup.2
represents Leu, Phe, or Val, and X.sup.3 represents Asp or Thr,
X.sup.4 represents Ala, Ser, His, or Thr, X.sup.5 represents Ile or
Val, X.sup.6 represents Asn or Asp, X.sup.7 represents Tyr, Ile, or
Met, and X.sup.8represents Thr, Val or Ala.
[0016] The invention also relates to truncations and analogs of the
peptides of the invention. The invention also relates to the use of
a peptide of the formula I or Ia to interfere with the interaction
of a protein of the Cullin family preferably Cdc53 and an F-box
binding protein preferably Skp1; and, pharmaceutical compositions
for inhibiting the interaction of a protein of the Cullin family
preferably Cdc53 and an F-box binding protein preferably Skp1.
[0017] Further, the invention relates to a method of modulating the
interaction of Cdc53 and Skp1 comprising changing the amino acid
Tyr at position 48 and/or Met at position 49 in Cdc53.
[0018] The peptides and antibodies of the invention, and substances
and compounds identified using the methods of the invention may be
used to modulate ubiquitin dependent proteolysis, and they may be
used to modulate cellular processes of cells (such as
proliferation, growth, and/or differentiation, in particular
glucose and methionine biosynthesis, gene expression, cell
division, and transcription) in which the compounds or substances
are introduced.
[0019] Accordingly, the antibodies, peptides, substances and
compounds may be formulated into compositions for adminstration to
individuals suffering from a proliferative or differentiative
condition. Therefore, the present invention also relates to a
composition comprising one or more of a peptide or antibody of the
invention, or a substance or compound identified using the methods
of the invention, and a pharmaceutically acceptable carrier,
excipient or diluent. A method for modulating proliferation,
growth, and/or differentiation of cells is also provided comprising
introducing into the cells a peptide or antibody of the invention,
a compound or substance identified using the methods of the
invention or a composition containing same. Methods for treating
proliferative and/or differentiative disorders using the
compositions of the invention are also provided.
[0020] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples while indicating preferred
embodiments of the invention are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0021] The invention will be better understood with reference to
the drawings in which:
[0022] FIG. 1. Cdc53 two hybrid interactions. (A) Cdc53 two hybrid
screens were carried out with three Cdc53 fusion proteins:
Gal4.sup.DBD-Cdc53, Gal4.sup.DBD-Cdc53.sup..DELTA.N, and
Gal4.sup.DBD-Cdc53.sup..DELTA.K. (B) Interaction of isolates with
Gal4.sup.DBD-Cdc53 and Gal4.sup.DBD-Cdc53.sup..DELTA.K in a
.beta.-galactosidase filter assay. (C) Schematic of Cdc53
interacting proteins. (D) Two hybrid interactions of
LexA.sup.DBD-Met30 derivatives with VP16.sup.AD-Skp1 Interactions
quantitated by liquid .beta.-galactosidase assay in Miller
units.
[0023] FIG. 2. Genetic interaction between CDC53 and SKP1. (A)
cdc53 Skp1 double mutants are inviable at the semi-permissive
temperature. Spore clones of a representative tetratype tetrad were
grown at 30.degree. C. for two days. (B) Photomicrographs of cells
from a representative cdc53-1 Skp1-12 tetratype grown at 25.degree.
C.
[0024] FIG. 3. Characterization of Cdc53 complexes in yeast
lysates. (A) Effects of temperature sensitive mutations on the
composition of Cdc53 immune complexes. The indicated strains
containing <CDC53 CEN> or <CDC53.sup.M CEN> plasmids
were arrested at 37.degree. C. for 2 h. 9E10 anti-MYC
immunoprecipitates from each strain were immunoblotted and
sequentially probed with anti-Cdc4, anti-Cdc34, anti-Skp1 and
anti-MYC antibodies. The anti-Cdc4 antibody did not reliably detect
Cdc4 in lysates and so the panels were omitted (see part C, below).
(B) Effects of temperature sensitive mutations on the composition
of Skp1 immune complexes. Analysis was as above except that strains
contained either vector or <SKP1.sup.HA CEN> plasmids.
Anti-HA immunoprecipitates were probed with anti-Cdc4, anti-Cdc34,
anti-Cdc53 and anti-HA antibodies. (C) Abundance of Cdc4 and Met30
in Skp1 mutants. Wild type, Skp1-11, Skp1-12 strains containing
either vector, <CDC4.sup.F CEN> or <pADH1-MET30.sup.HA 2
.mu.m> plasmids, were analyzed as above. Anti-FLAG and anti-HA
immunoprecipitates were probed with anti-Cdc4 polyclonal antibody
and anti-HA antibody respectively.
[0025] FIG. 4. Cdc53 interacts with multiple F-box proteins. (A)
The indicated immunoprecipitates from wild type cells containing
either vector, <CDC53.sup.M CEN>, <pADH1-MET30.sup.HA 2
.mu.m>, <PADH1-GRR1.sup.HA 2 .mu.m> and <CDC4.sup.F
CEN> plasmids were probed with anti-Cdc53, anti-Skp1, anti-Cdc4
and anti-HA antibodies. IgG light chain is indicated by an
asterisk. (B) Effects of MET30 or GRR1 overexpression. Strains of
the indicated genotype containing an empty vector plasmid
<pADH1-MET30.sup.HA> (left panel), or
<pADH1-GRR1.sup.HA> (right panel) were grown at 30.degree. C.
for 3 days and photographed.
[0026] FIG. 5. Mutational analysis of CDC53. (A) Deletion analysis
of Cdc53 protein-protein interaction domains. Cells were
transformed with untagged (lane 1) or MYC-tagged (lane 2) CDC53 or
MYC-tagged versions of the indicated CDC53 mutants (lanes 3-8) all
expressed from the wild type promoter on CEN plasmids. Lysates from
each strain were immunoprecipitated with 9E10 anti-MYC antibodies,
immunoblotted and probed with 9E10 (top panel) and polyclonal
antibodies specific to each of the indicated proteins (lower
panels). (B) Schematic representation of Cdc53 mutant proteins and
their ability to rescue a cdc53 deletion strain. Regions of amino
acid sequence conservation in the Cdc53 family are indicated in
black (see Methods). The positions of the cdc53-1 (R488C) and
cdc53-2 (G340D) point mutations, and the regions required for
binding to Skp1/F-box proteins and Cdc34 are also indicated. (C)
Cdc53 does not contain essential cysteine residues. A cdc53
deletion strain containing a <CDC53.sup.HA URA3 CEN> plasmid
was transformed with <CDC53.sup.6C TRP CEN>, <CDC53.sup.M
TRP CEN>, or an empty vector plasmid, plated on 5-FOA medium to
select for Ura cells, and photographed after 2 days.
[0027] FIG. 6. Specificity of F-box protein function. (A)
Methionine repression is mediated by Met30, Cdc34, Cdc53 and Skp1
but not Cdc4. The indicated strains were grown in methionine- free
medium and repressed with 1.0 mM methionine for the indicated
times. MET25 expression was determined by Northern analysis and
normalized to ACT1 expression. (B) Grr1 specifically mediates Cln2
degradation. The indicated strains were incubated at 37.degree. C.
for two hours, at which time <pGAL1-CLN2.sup.HA> was
expressed by the addition of galactose for 1.5 hours, and then
repressed with the addition of glucose for the indicated times.
Cln2.sup.HA was detected by immunoblotting with 12CA5 monoclonal
antibody. Exposures were adjusted to give equal Cln2.sup.HA signals
at time zero. Cln2.sup.HA was quantitated and normalized to Cdc28
signals from the same blot probed with anti-Cdc28 antibody.
[0028] FIG. 7 shows the amino acid sequence of a Cdc34 protein.
[0029] FIG. 8 shows the amino acid sequences of a Cdc53 protein and
a Cul-2 protein.
[0030] FIG. 9 shows the amino acid sequence of a Skp1 protein.
[0031] FIG. 10 shows the amino acid sequences of a Cdc4 protein, a
Met30 protein, and a Grr1 protein.
[0032] FIG. 11 shows that amino acids Y48 and M49 in Cdc53 are
required for binding to Skp1. Wild type and Y48W, M49E mutants of
Cdc.sub.53.sup.MYC6 were immunoprecipitated and (A) western blotted
for Cdc53.sup.MYC6, Cdc53, and Skp1, and (B) silver stained. On the
silver-stained gel, bands that exist in the wild type but not the
mutant Cdc53 IP are marked with an open triangle.
[0033] FIG. 12A Amino acids Y48 and M49 in Cdc53 are required for
binding to Skp1. Wild type and Y48W, M49E mutants of
Cdc.sub.53.sup.MYC6 were immunoprecipitated and western blotted for
Cdc53.sup.MYC6, Cdc34, and Skp1.
[0034] FIG. 12B Amino acids Y48 and M49 in Cdc53 are required for
binding to Skp1. Wild type and Y48W, M49E mutants of Cdc53.sup.MYC6
were immunoprecipitated and western blotted for Cdc53.sup.MYC6,
Cdc34, and Skp1, and (B) silver stained. On the silver-stained gel,
bands that exist in the wild type but not the mutant Cdc53 IP are
marked with an open triangle.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Definitions
[0036] Unless otherwise indicated, all terms used herein have the
same meaning as they would to one skilled in the art of the present
invention. Practitioners are particularly directed to Current
Protocols in Molecular Biology (Ansubel) for definitions and terms
of the art.
[0037] Abbreviations for amino acid residues are the standard
3-letter and/or 1-letter codes used in the art to refer to one of
the 20 common L-amino acids. Likewise abbreviations for nucleic
acids are the standard codes used in the art.
[0038] "E2 ubiquitin conjugating enzyme" refers to one of the
components involved in ubiquitin transfer reactions to form
ubiquitin-protein conjugates which are recognized by the 26S
proteasome. An example of an E2 ubiquitin conjugating enzyme is
Cdc34, and homologs or portions thereof. (See FIG. 7 for a Cdc34
amino acid sequence.)
[0039] "Protein of the Cullin family" refers to the family of
proteins involved in the regulation of cell division. The
archetypal member of the family is Cdc53. The family also includes,
homologs and portions of Cdc53, including the proteins regulating
cell division in C. elegans and mammalian cells such as Cul-1,
Cul-2, and the metazoan Cdc53 homologs described in Kipreos et al.,
1996. (See FIG. 8 for sequences for Cdc53 and Cul-2).
[0040] "F-box binding protein" refers to proteins that bind to
proteins containing an F-box motif. Examples of F-box binding
proteins are Skp1 and Scon C and homologs, and portions, thereof.
(See FIG. 9 for a Skp1 sequence.)
[0041] "Proteins containing an F-box motif" refers to proteins have
a characteristic structural motif called the F-box as described in
Bai et al, 1996. Examples of the proteins include Cdc4, Grr1, pop1,
Met30 , Scon2/Scon3, and several other yeast and mammalian proteins
(Bai et al, 1996), and homologs or portions thereof. (See FIG. 10
for a Cdc4 sequence, a Met30 sequence, and a Grr1 sequence.)
[0042] A "binding domain" is that portion of the molecule in a
complex of the invention (i.e. E2 ubiquitin conjugating enzyme,
protein of the Cullin family, F-box binding protein, or protein
containing an F-box motif) which interacts directly or indirectly
with another molecule in a complex of the invention. The binding
domain may be a sequential portion of the molecule i.e. a
contiguous sequence of amino acids, or it may be conformational
i.e. a combination of non-contiguous sequences of amino acids which
when the molecule is in its native state forms a structure that
interacts with another molecule in a complex of the invention.
[0043] By being "derived from" a binding domain is meant any
molecular entity which is identical or substantially equivalent to
the native binding domain of a molecule in a complex of the
invention (i.e. E2 ubiquitin conjugating enzyme, protein of the
Cullin family, F-box binding protein; or protein containing an
F-box motif). A peptide derived from a specific binding domain may
encompass the amino acid sequence of a naturally occurring binding
site, any portion of that binding site, or other molecular entity
that functions to bind to an associated molecule. A peptide derived
from such a binding domain will interact directly or indirectly
with an associated molecule in such a way as to mimic the native
binding domain. Such peptides may include competitive inhibitors,
peptide mimetics, and the like.
[0044] The term "interacting" refers to a stable association
between two molecules due to, for example, electrostatic,
hydrophobic, ionic and/or hydrogen-bond interactions under
physiological conditions. Certain interacting molecules interact
only after one or more of them has been stimulated. For example, a
protein containing an F-box motif may only bind to a substrate if
the substrate is phosphorylated (eg. phosphorylated Sic1).
[0045] An enhancer or inhibitor of the interaction of an E2
ubiquitin conjugating enzyme or a F-box binding protein, and a
protein of the Cullin family is intended to include a peptide or
peptide fragment derived from the binding domain of an E2 ubiquitin
conjugating enzyme, an F-box binding protein, or a protein of the
Cullin family. The enhancer or inhibitor will not include the full
length sequence of the wild-type molecule. Peptide mimetics,
synthetic molecules with physical structures designed to mimic
structural features of particular peptides, may serve as inhibitors
or enhancers. Inhibitors or enhancers affect ubiquitin-dependent
proteolysis. The enhancement or inhibition may be direct, or
indirect, or by a competitive or non-competitive mechanism.
[0046] "Peptide mimetics" are structures which serve as substitutes
for peptides in interactions between molecules (See Morgan et al
(1989), Ann. Reports Med. Chem. 24:243-252 for a review ). Peptide
mimetics include synthetic structures which may or may not contain
amino acids and/or peptide bonds but retain the structural and
functional features of a peptide, or enhancer or inhibitor of the
invention. Peptide mimetics also include peptoids, oligopeptoids
(Simon et al (1972) Proc. Nati. Acad, Sci USA 89:9367); and peptide
libraries containing peptides of a designed length representing all
possible sequences of amino acids corresponding to a peptide, or
enhancer or inhibitor of the invention.
[0047] Sequences are "homologous" or considered "homologs" when at
least about 70% (preferably at least about 80 to 90%, and most
preferably at least 95%) of the nucleotides or amino acids match
over a defined length of the molecule. Substantially homologous
also includes sequences showing identity to the specified sequence.
Preferably, the amino acid or nucleic acid sequences have an
alignment score of greater than 5 (in standard deviation units)
using the program ALIGN with the mutation gap matrix and a gap
penalty of 6 or greater (Dayhoff).
[0048] Peptides of the Invention
[0049] The invention provides peptide molecules which bind to and
inhibit the interactions of the molecules in the complexes of the
invention. The molecules are derived from the binding domain of an
E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an
F-box binding protein; or a protein containing an F-box motif. For
example, peptides of the invention include the following amino
acids of Cdc53 (see FIG. 8): amino acids 448 to 748 (comprising the
binding domain for Cdc34) and amino acids 9 to 280 (comprising the
binding domain for Skp1), or portions thereof that bind to Cdc34
and Skp1. Other proteins containing these binding domain sequences
may be identified with a protein homology search, for example by
searching available databases such as GenBank or SwissProt and
various search algorithms and/or programs may be used including
FASTA, BLAST (available as a part of the GCG sequence analysis
package, University of Wisconsin, Madison, Wis.), or ENTREZ
(National Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, Bethesda, Md.).
[0050] In accordance with an embodiment of the invention; specific
peptides are contemplated that mediate the binding of a protein of
the Cullin family preferably Cdc53, and an F-box binding protein
preferably Skp1.
[0051] Therefore, the invention relates to a peptide of the formula
I which interferes with the interaction of Cdc53 and Skp1
A-Tyr-Met-X.sup.1-X.sup.2-Tyr-X.sup.3-X.sup.4-X.sup.5-Tyr-X.sup.6-X.sup.7--
Cys-X.sup.8 I
[0052] wherein A represents one to ten amino acids, X.sup.1
represents Met, Arg, Thr, or Glu, X.sup.2 represents Leu, Phe, or
Val, X.sup.3 represents Asp or Thr, X.sup.4 represents Ala, Ser,
His, or Thr, X.sup.5 represents Ile or Val, X.sup.6 represents Asn
or Asp, X.sup.7 represents Tyr, Ile, or Met, and X.sup.8 represents
Thr, Val, or Ala.
[0053] In an embodiment of the present invention a peptide of the
formula Ia which interferes with the interaction of Cdc53 and Skp1
is provided:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-Tyr-
Met-X.sup.1-X.sup.2-Tyr-X.sup.3-X.sup.4-X.sup.5-Tyr-X.sup.6-X.sup.7-Cys-X-
.sup.8 Ia
[0054] wherein A.sup.1 represents Ile, Asn, His, Ser, or Ala,
A.sup.2 represents Leu, Met or Phe, A.sup.3 represents Ser, Ala,
Thr, or Asp, A.sup.4 represents Pro, Lys, Arg, or Ser, A.sup.5
represents Thr, Lys, Ser, or Glu, A.sup.6 represents Met, Asp, Tyr,
Gln, or Arg, X.sup.1 represents Met, Arg, Thr, or Glu, X.sup.2
represents Leu, Phe, or Val, and X.sup.3 represents Asp or Thr,
X.sup.4 represents Ala, Ser, His, or Thr, X.sup.5 represents Ile or
Val, X.sup.6 represents Asn or Asp, X.sup.7 represents Tyr, Ile, or
Met, and X.sup.8represents Thr, Val or Ala.
[0055] All of the peptides of the invention, as well as molecules
substantially homologous, complementary or otherwise functionally
or structurally equivalent to these peptides may be used for
purposes of the present invention. In addition to full-length
peptides of the invention, truncations of the peptides are
contemplated in the present invention. Truncated peptides may
comprise peptides of about 7 to 10 amino acid residues.
[0056] The truncated peptides may have an amino group (--NH2), a
hydrophobic group (for example, carbobenzoxyl, dansyl, or
T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl
(PMOC) group, or a macromolecule including but not limited to
lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates
at the amino terminal end. The truncated peptides may have a
carboxyl group, an amido group, a T-butyloxycarbonyl group, or a
macromolecule including but not limited to lipid-fatty acid
conjugates, polyethylene glycol, or carbohydrates at the carboxy
terminal end.
[0057] The peptides of the invention may also include analogs of a
peptide of the invention, and/or truncations of the peptide, which
may include, but are not limited to the peptide of the invention
containing one or more amino acid insertions, additions, or
deletions, or both. Analogs of the peptide of the invention exhibit
the activity characteristic of the peptide e.g. interference with
the interaction of Cdc53 with Skp1, and may further possess
additional advantageous features such as increased bioavailability,
stability, or reduced host immune recognition.
[0058] One or more amino acid insertions may be introduced into a
peptide of the invention. Amino acid insertions may consist of a
single amino acid residue or sequential amino acids.
[0059] One or more amino acids, preferably one to five amino acids,
may be added to the right or left termini of a peptide of the
invention. Deletions may consist of the removal of one or more
amino acids, or discrete portions from the peptide sequence. The
deleted amino acids may or may not be contiguous. The lower limit
length of the resulting analog with a deletion mutation is about 7
amino acids.
[0060] It is anticipated that if amino acids are inserted or
deleted in sequences outside the
Tyr-Met-X.sup.1-X.sup.2-Tyr-X.sup.3-X.sup.4-X.sup.5- -Tyr sequence
that the resulting analog of the peptide will exhibit the activity
of a peptide of the invention.
[0061] Preferred peptides of the invention include the following:
MEVTAIYNYCV, YMEVTAIYNYCVNKS, ILSPTMYMEVYTAIYNYCVNKS,
YMTLYTSVYDYCT, YMTLYTSVYDYCTSIT, MAPKDYMTLYTSVYDYCTSIT,
YMMLYDAVYNICT, YMMLYDAVYNICTTTT, HMSKKYYMMLYDAVYNICTTTT,
YMRFYTHVYDYCT, YMRFYTHVYDYCTSVS, SLTRSQYMRFYTHVYDYCTSVS,
YMELYTHVYNYCT, YMELYTHVYNYCTSVH, SMAKSRYMELYTHVYNYCTSVH,
YMMLYTTIYNMCT, YMMLYTTIYNMCTQKP, AFDSEQYMMLYTTIYNMCTQKP,
YMELYTAIHNTCA, YMELYTAIHNTCADAS, and GMITFYMELYTAIHNTCADAS.
[0062] The invention also includes a peptide conjugated with a
selected protein, or a selectable marker (see below) to produce
fusion proteins.
[0063] The peptides of the invention may be prepared using
recombinant DNA methods. Accordingly, nucleic acid molecules which
encode a peptide of the invention may be incorporated in a known
manner into an-appropriate expression vector which ensures good
expression of the peptide. Possible expression vectors include but
are not limited to cosmids, plasmids, or modified viruses so long
as the vector is compatible with the host cell used. The expression
vectors contain a nucleic acid molecule encoding a peptide of the
invention and the necessary regulatory sequences for the
transcription and translation of the inserted protein-sequence.
Suitable regulatory sequences may be obtained from a variety of
sources, including bacterial, fungal, viral, mammalian, or insect
genes (For example, see the regulatory sequences described in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Selection of appropriate
regulatory sequences is dependent on the host cell chosen, and may
be readily accomplished by one of ordinary skill in the art. Other
sequences, such as an origin of replication, additional DNA
restriction sites, enhancers, and sequences conferring inducibility
of transcription may also be incorporated into the expression
vector.
[0064] The recombinant expression vectors may also contain a
selectable marker gene which facilitates the selection of
transformed or transfected host cells. Suitable selectable marker
genes are genes encoding proteins such as G418 and hygromycin which
confer resistance to certain drugs, .beta.-galactosidase,
chloramphenicol acetyltransferase, firefly luciferase, or an
immunoglobulin or portion thereof such as the Fc portion of an
immunoglobulin preferably IgG. The selectable markers may be
introduced on a separate vector from the nucleic acid of
interest.
[0065] The recombinant expression vectors may also contain genes
which encode a fusion portion which provides increased expression
of the recombinant peptide; increased solubility of the recombinant
peptide; and/or aid in the purification of the recombinant peptide
by acting as a ligand in affinity purification. For example, a
proteolytic cleavage site may be inserted in the recombinant
peptide to allow separation of the recombinant peptide from the
fusion portion after purification of the fusion protein. Examples
of fusion expression vectors include pGEX (Amrad Corp., Melboume,
Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharrnacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GSY), maltose E binding protein, or protein A, respectively, to
the recombinant protein.
[0066] Recombinant expression vectors may be introduced into host
cells to produce a transformant host cell. Transformant host cells
include prokaryotic and eukaryotic cells which have been
transformed or transfected with a recombinant expression vector of
the invention. The terms "transformed with", "transfected with",
"transformation" and "transfection" are intended to include the
introduction of nucleic acid (e.g. a vector) into a cell by one of
many techniques known in the art. For example, prokaryotic cells
can be transformed with nucleic acid by electroporation or
calcium-chloride mediated transformation. Nucleic acid can be
introduced into mammalian cells using conventional techniques such
as calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofectin, electroporation or
microinjection. Suitable methods for transforming and transfecting
host cells may be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press
(1989)), and other laboratory textbooks.
[0067] Suitable host cells include a wide variety of prokaryotic
and eukaryotic host cells. For example, the peptides of the
invention may be expressed in bacterial cells such as E. coli,
insect cells (using baculovirus), yeast cells or mammalian cells.
Other suitable host cells can be found in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1991).
[0068] The peptides of the invention may be tyrosine phosphorylated
using the method described in Reedijk et al. (The EMBO Journal
11(4):1365, 1992). For example, tyrosine phosphorylation may be
induced by infecting bacteria harbouring a plasmid containing a
nucleotide sequence encoding a peptide of the invention, with a
.lambda.gt11 bacteriophage encoding the cytoplasmic domain of the
Elk tyrosine kinase as a LacZ-Elk fusion. Bacteria containing the
plasmid and bacteriophage as a lysogen are isolated. Following
induction of the lysogen, the expressed peptide becomes
phosphorylated by the Elk tyrosine kinase.
[0069] The peptides of the invention may be synthesized by
conventional techniques. For example, the peptides may be
synthesized by chemical synthesis using solid phase peptide
synthesis. These methods employ either solid or solution phase
synthesis methods (see for example, J. M. Stewart, and J. D. Young,
Solid Phase Peptide Synthesis, 2.sup.nd Ed., Pierce Chemical Co.,
Rockford Ill. (1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis Synthesis, Biology editors E. Gross and J.
Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for
solid phase synthesis techniques; and M Bodansky, Principles fo
Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and
J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biologu,
suprs, Vol 1, for classical solution synthesis.) By way of example,
the peptides may be synthesized using 9-fluorenyl methoxycarbonyl
(Fmoc) solid phase chemistry with direct incorporation of
phosphotyrosine as the N-fluorenylmethoxy-carbonyl-O-dimethyl
phosphono-L-tyrosine derivative.
[0070] N-terminal or C-terminal fusion proteins comprising a
peptide of the invention conjugated with other molecules may be
prepared by fusing, through recombinant techniques, the N-terminal
or C-terminal of the peptide, and the sequence of a selected
protein or selectable marker with a desired biological function.
The resultant fusion proteins contain the peptide fused to the
selected protein or marker protein as described herein. Examples of
proteins which may be used to prepare fusion proteins include
immunoglobulins, glutathione-S-transferase (GST), hemagglutinin
(HA), and truncated myc.
[0071] Cyclic derivatives of the peptides of the invention are also
part of the present invention. Cyclization may allow the peptide to
assume a more favorable conformation for association with molecules
in complexes of the invention. Cyclization may be achieved using
techniques known in the art. For example, disulfide bonds may be
formed between two appropriately spaced components having free
sulfhydryl groups, or an amide bond may be formed between an amino
group of one component and a carboxyl group of another component.
Cyclization may also be achieved using an azobenzene-containing
amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc.
1995, 117, 8466-8467. The side chains of Tyr and Asn may be linked
to form cyclic peptides. The components that form the bonds may be
side chains of amino acids, non-amino acid components or a
combination of the two. In an embodiment of the invention, cyclic
peptides are contemplated that have a beta-turn in the right
position. Beta-turns may be introduced into the peptides of the
invention by adding the amino acids Pro-Gly at the right
position.
[0072] It may be desirable to produce a cyclic peptide which is
more flexible than the cyclic peptides containing peptide bond
linkages as described above. A more flexible peptide may be
prepared by introducing cysteines at the right and left position of
the peptide and forming a disulphide bridge between the two
cysteines. The two cysteines are arranged so as not to deform the
beta-sheet and turn. The peptide is more flexible as a result of
the length of the disulfide linkage and the smaller number of
hydrogen bonds in the beta-sheet portion. The relative flexibility
of a cyclic peptide can be determined by molecular dynamics
simulations.
[0073] Peptide mimetics may be designed based on information
obtained by systematic replacement of L-amino acids by D-amino
acids, replacement of side chains with groups having different
electronic properties, and by systematic replacement of peptide
bonds with amide bond replacements. Local conformational
constraints can also be introduced to determine conformational
requirements for activity of a candidate peptide mimetic. The
mimetics may include isosteric amide bonds, or D-amino acids to
stabilize or promote reverse turn conformations and to help
stabilize the molecule. Cyclic amino acid analogues may be used to
constrain amino acid residues to particular conformational states.
The mimetics can also include mimics of inhibitor peptide secondary
structures. These structures can model the 3-dimensional
orientation of amino acid residues into the known secondary
conformations of proteins. Peptoids may also be used which are
oligomers of N-substituted amino acids and can be used as motifs
for the generation of chemically diverse libraries of novel
molecules.
[0074] Peptides that interact with an E2 ubiquitin conjugating
enzyme, a protein of the Cullin family, an F-box binding protein;
or a protein containing an F-box motif may be developed using a
biological expression system. The use of these systems allows the
production of large libraries of random peptide sequences and the
screening of these libraries for peptide sequences that bind to
particular proteins. Libraries may be produced by cloning synthetic
DNA that encodes random peptide sequences into appropriate
expression vectors. (see Christian et al 1992, J. Mol. Biol.
227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990,
Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be
constructed by concurrent synthesis of overlapping peptides (see
U.S. Pat. No. 4,708,871).
[0075] Peptides of the invention may be used to identify lead
compounds for drug development. The structure of the peptides
described herein can be readily determined by a number of methods
such as NMR and X-ray crystallography. A comparison of the
structures of peptides similar in sequence, but differing in the
biological activities they elicit in target molecules can provide
information about the structure-activity relationship of the
target. Information obtained from the examination of
structure-activity relationships can be used to design either
modified peptides, or other small molecules or lead compounds which
can be tested for predicted properties as related to the target
molecule. The activity of the lead compounds can be evaluated using
assays similar to those described herein.
[0076] Information about structure-activity relationships may also
be obtained from co-crystallization studies. In these studies, a
peptide with a desired activity is crystallized in association with
a target molecule, and the X-ray structure of the complex is
determined. The structure can then be compared to the structure of
the target molecule in its native state, and information from such
a comparison may be used to design compounds expected to possess
desired activities.
[0077] The peptides of the invention may be converted into
pharmaceutical salts by reacting with inorganic acids such as
hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric
acid, etc., or organic acids such as formic acid, acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic
acid, succinic acid, malic acid, tartaric acid, citric acid,
benzoic acid, salicylic acid, benezenesulfonic acid, and
toluenesulfonic acids.
[0078] The peptides of the invention may be used to prepare
monoclonal or polyclonal antibodies. Conventional methods can be
used to prepare the antibodies. As to the details relating to the
preparation of monoclonal antibodies reference can be made to
Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd
Ed., Academic Press, London, 1986. As discussed below the
antibodies may be used to identify proteins binding sites for
Skp1.
[0079] The peptides and antibodies specific for the peptides of the
invention may be labelled using conventional methods with various
enzymes, fluorescent materials, luminescent materials and
radioactive materials. Suitable enzymes, fluorescent materials,
luminescent materials, and radioactive material are well known to
the skilled artisan. Labeled antibodies specific for the peptides
of the invention may be used to screen for proteins with Skp1
binding sites, and labeled peptides of the invention may be used to
screen for Skp1 binding site containing proteins such as Cdc53.
[0080] Computer modelling techniques known in the art may also be
used to observe the interaction of a peptide of the invention, and
truncations and analogs thereof with a molecule in a complex of the
invention e.g. Skp1 (for example, Homology Insight II and Discovery
available from BioSym/molecular Simulations, San Diego, Calif.,
U.S.A.). If computer modelling indicates a strong interaction, the
peptide can be synthesized and tested for its ability to interfere
with the binding of Cdc53 and Skp1 as discussed above.
[0081] Complexes of the Invention
[0082] The complexes of the invention include the following: (a) a
complex comprising an E2 ubiquitin conjugating enzyme, a protein of
the Cullin family, and a F-box binding protein, and optionally a
protein containing an F-box motif; and (b) a complex comprising a
protein of the Cullin family and a protein containing an F-box
motif. Complexes also containing molecules that bind to a protein
containing an F-box motif (eg. Sic1, Cln, Met 4 or activated forms
thereof) are also contemplated. It will be appreciated that the
complexes may comprise only the binding domains of the interacting
molecules and such other flanking sequences as are necessary to
maintain the activity of the complexes.
[0083] The invention also contemplates antibodies specific for
complexes of the invention. The antibodies may be intact monoclonal
or polyclonal antibodies, and immunologically active fragments
(e.g. a Fab or (Fab).sub.2 fragment), an antibody heavy chain, and
antibody light chain, a genetically engineered single chain Fv
molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric
antibody, for example, an antibody which contains the binding
specificity of a murine antibody, but in which the remaining
portions are of human origin. Antibodies including monoclonal and
polyclonal antibodies, fragments and chimeras, may be prepared
using methods known to those skilled in the art.
[0084] Antibodies specific for the complexes of the invention may
be used to detect the complexes in tissues and to determine their
tissue distribution. In vitro and in situ detection methods using
the antibodies of the invention may be used to assist in the
prognostic and/or diagnostic evaluation of proliferative and/or
differentiative disorders. Antibodies specific for the complexes of
the invention may also be used therapeutically to decrease the
degradation of proteins that interact with F-box containing
proteins such as Sic1, Cln, and Met4.
[0085] The complexes of the invention play a central role in
ubiquitin-dependent proteolysis and some genetic diseases may
include mutations at the binding domain regions of the interacting
molecules in the complexes of the invention. Therefore, if a
complex of the invention is implicated in a genetic disorder, it
may be possible to use PCR to amplify DNA from the binding domains
to quickly check if a mutation is contained within one of the
domains. Primers can be made corresponding to the flanking regions
of the domains and standard sequencing methods can be employed to
determine whether a mutation is present. This method does not
require prior chromosome mapping of the affected gene and can save
time by obviating sequencing the entire gene encoding a defective
protein.
[0086] Methods for Identifying or Evaluating
Substances/Compounds
[0087] The methods described herein are designed to identify
substances that modulate the activity of a complex of the invention
thus affecting ubquitin dependent proteolysis. Novel substances are
therefore contemplated that bind to molecules in the complexes, or
bind to other proteins that interact with the molecules, to
compounds that interfere with, or enhance the interaction of the
molecules in a complex, or other proteins that interact with the
molecules.
[0088] The substances and compounds identified using the methods of
the invention include but are not limited to peptides such as
soluble peptides including Ig-tailed fusion peptides, members of
random peptide libraries and combinatorial chemistry-derived
molecular libraries made of D- and/or L-configuration amino acids,
phosphopeptides (including members of random or partially
degenerate, directed phosphopeptide libraries), antibodies [e.g.
polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single
chain antibodies, fragments, (e.g. Fab, F(ab).sub.2, and Fab
expression library fragments, and epitope-binding fragments
thereof)], and small organic or inorganic molecules. The substance
or compound may be an endogenous physiological compound or it may
be a natural or synthetic compound.
[0089] Substances which modulate the activity of a complex of the
invention can be identified based on their ability to bind to a
molecule in the complex. Therefore, the invention also provides
methods for identifying novel substances which bind molecules in
the complex. Substances identified using the methods of the
invention may be isolated, cloned and sequenced using conventional
techniques.
[0090] Novel substances which can bind with a molecule in a complex
of the invention may be identified by reacting one of the molecules
with a test substance which potentially binds to the molecule,
under conditions which permit the formation of substance-molecule
conjugates and removing and/or detecting the conjugates. The
conjugates can be detected by assaying for substance-molecule
conjugates, for free substance, or for non-complexed molecules.
Conditions which permit the formation of substance-molecule
conjugates may be selected having regard to factors such as the
nature and amounts of the substance and the molecule.
[0091] The substance-molecule conjugate, free substance or
non-complexed molecules may be isolated by conventional isolation
techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof. To facilitate the assay of the components, antibody
against the molecule or the substance, or labelled molecule, or a
labelled substance may be utilized. The antibodies, proteins, or
substances may be labelled with a detectable substance as described
above.
[0092] A molecule, or complex of the invention, or the substance
used in the method of the invention may be insolubilized. For
example, a molecule, or substance may be bound to a suitable
carrier such as agarose, cellulose, dextran, Sephadex, Sepharose,
carboxymethyl cellulose polystyrene, filter paper, ion-exchange
resin, plastic film, plastic tube, glass beads, polyamine-methyl
vinyl-ether-maleic acid copolymer, amino acid copolymer,
ethylene-maleic acid copolymer, nylon, silk, etc: The carrier may
be in the shape of, for example, a tube, test plate, beads, disc,
sphere etc. The insolubilized protein or substance may be prepared
by reacting the material with a suitable insoluble carrier using
known chemical or physical methods, for example, cyanogen bromide
coupling.
[0093] The invention also contemplates a method for evaluating a
compound for its ability to modulate the biological activity of a
complex of the invention, by assaying for an agonist or antagonist
(i.e.enhancer or inhibitor) of the binding of molecules in the
complex. The basic method for evaluating if a compound is an
agonist or antagonist of the binding of molecules in a complex of
the invention, is to prepare a reaction mixture containing
molecules and the substance under conditions which permit the
formation of complexes, in the presence of a test compound. The
test compound may be initially added to the mixture, or may be
added subsequent to the addition of molecules. Control reaction
mixtures without the test compound or with a placebo are also
prepared. The formation of complexes is detected and the formation
of complexes in the control reaction but not in the reaction
mixture indicates that the test compound interferes with the
interaction of the molecules. The reactions may be carried out in
the liquid phase or the molecules, or test compound may be
immobilized as described herein.
[0094] It will be understood that the agonists and antagonists i.e.
inhibitors and enhancers that can be assayed using the methods of
the invention may act on one or more of the binding sites on the
interacting molecules in the complex including agonist binding
sites, competitive antagonist binding sites, non-competitive
antagonist binding sites or allosteric sites.
[0095] The invention also makes it possible to screen for
antagonists that inhibit the effects of an agonist of the
interaction of molecules in a complex of the invention. Thus, the
invention may be used to assay for a compound that competes for the
same binding site of a molecule in a complex of the invention.
[0096] The invention also contemplates methods for identifying
novel compounds that bind to proteins that interact with a molecule
of a complex of the invention. Protein-protein interactions may be
identified using conventional methods such as
co-immunoprecipitation, crosslinking and co-purification through
gradients or chromatographic columns. Methods may also be employed
that result in the simultaneous identification of genes which
encode proteins interacting with a molecule. These methods include
probing expression libraries with labeled molecules. Additionally,
x-ray crystallographic studies may be used as a means of evaluating
interactions with substances and molecules. For example, purified
recombinant molecules in a complex of the invention when
crystallized in a suitable form are amenable to detection of
intra-molecular interactions by x-ray crystallography. Spectroscopy
may also be used to detect interactions and in particular, Q-TOF
instrumentation may be used.
[0097] Two-hybrid systems may also be used to detect protein
interactions in vivo. Generally, plasmids are constructed that
encode two hybrid proteins. A first hybrid protein consists of the
DNA-binding domain of a transcription activator protein fused to a
molecule in a complex of the invention, and the second hybrid
protein consists of the transcription activator protein's activator
domain fused to an unkown protein encoded by a cDNA which has been
recombined into the plasmid as part of a cDNA library. The plasmids
are transformed into a strain of yeast (e.g. S. cerevisiae) that
contains a reporter gene (e.g. lacZ, luciferase, alkaline
phosphatase, horseradish peroxidase) whose regulatory region
contains the transcription activator's binding site. The hybrid
proteins alone cannot activate the transcription of the reporter
gene. However, interaction of the two hybrid proteins reconstitutes
the functional activator protein and results in expression of the
reporter gene, which is detected by an assay for the reporter gene
product.
[0098] It will be appreciated that fusion proteins and recombinant
fusion proteins may be used in the above-described methods. It will
also be appreciated that the complexes of the invention may be
reconstituted in vitro using recombinant molecules and the effect
of a test substance may be evaluated in the reconstituted
system.
[0099] The reagents suitable for applying the methods of the
invention to evaluate substances and compounds that modulate
ubiquitin dependent proteolysis may be packaged into convenient
kits providing the necessary materials packaged into suitable
containers. The kits may also include suitable supports useful in
performing the methods of the invention.
[0100] Compositions and Treatments
[0101] The peptides of the invention, and substances and compounds
identified using the methods of the invention may be used to
modulate ubiquitin dependent proteolysis, and they may be used to
modulate cellular processes such as proliferation, growth, and/or
differentiation of cells in which the compounds or substances are
introduced. Thus, the substances may be used for the treatment of
proliferative disorders including various forms of cancer such as
leukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas,
melanomas, adenomas, carcinomas of solid tissue, hypoxic tumors,
squamous cell carcinomas of the mouth, throat, larynx, and lung,
genitourinary cancers such as cervical and bladder cancer, breast,
ovarian, colon, hematopoietic cancers, head and neck cancers, and
nervous system cancers, benign lesions such as papillomas,
arthrosclerosis, angiogenesis, and viral infections, in particular
HIV infections, psoriasis, bone diseases, fibroproliferative
disorders such as involving connective tissue, atherosclerosis and
other smooth muscle proliferative disorders, chronic inflammation,
and arthropathies such as arthritis. In addition to proliferative
disorders, the treatment of differentiative disorders which result
from, for example, de-differentiation of tissue which may be
accompanied by abnormal reentry into mitosis. Such degenerative
disorders that may be treated using the peptides and compositions
of the invention include neurodegenerative disorders such as
chronic neurodegenerative diseases of the nervous system, including
Alzheimer's disease, Parkinson's disease, Huntington's chorea,
amylotrophic lateral sclerosis and the like, as well as
spinocerebellar degeneration.
[0102] Accordingly, the peptides, substances, antibodies, and
compounds may be formulated into pharmaceutical compositions for
adminstration to subjects in a biologically compatible form
suitable for administration in vivo. By "biologically compatible
form suitable for administration in vivo" is meant a form of the
substance to be administered in which any toxic effects are
outweighed by the therapeutic effects. The substances may be
administered to living organisms including humans, and animals.
Administration of a therapeutically active amount of the
pharmaceutical compositions of the present invention is defined as
an amount effective, at dosages and for periods of time necessary
to achieve the desired result. For example, a therapeutically
active amount of a substance may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of antibody to elicit a desired response in the individual.
Dosage regima may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered
daily or the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation.
[0103] The acute substance may be administered in a convenient
manner such as by injection (subcutaneous, intravenous, etc.), oral
administration, inhalation, transdermal application, or rectal
administration. Depending on the route of administration, the
active substance may be coated in a material to protect the
compound from the action of enzymes, acids and other natural
conditions that may inactivate the compound.
[0104] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). On this basis, the compositions include,
albeit not exclusively, solutions of the substances or compounds in
association with one or more pharmaceutically acceptable vehicles
or diluents, and contained in buffered solutions with a suitable pH
and iso-osmotic with the physiological fluids.
[0105] The activity of the substances, compounds, antibodies, and
compositions of the invention may be confirmed in animal
experimental model systems.
[0106] The invention also provides methods for studying the
function of a complex of the invention. Cells, tissues, and
non-human animals lacking in the complexes or partially lacking in
molecules in the complexes may be developed using recombinant
expression vectors of the invention having specific deletion or
insertion mutations in the molecules. A recombinant expression
vector may be used to inactivate or alter the endogenous gene by
homologous recombination, and thereby create complex deficient
cells, tissues or animals. Null alleles may be generated in cells
and may then be used to generate transgenic non-human animals.
[0107] The following non-limiting examples are illustrative of the
present invention:
EXAMPLE 1
[0108] The following materials and methods were used in the
investigations described in the example.
[0109] Methods
[0110] Plasmids
[0111] Plasmids were constructed using standard molecular cloning
techniques (Table 1). For two hybrid screens, the CDC53 open
reading frame was cloned into the BamH1 site of pAS2 (provided by
S. Elledge) to create a Gal4.sup.DBD-Cdc53 fusion. Versions that
lacked either the N-terminal 280 residues
(Gal4.sup.DBD-Cdc53.sup..DELTA.N) or internal residues 581-664
(Gal4.sup.DBD-Cdc53.sup..DELTA.K) were created by digestion with
NcoI or KpnI respectively and religating.
Gal4.sup.AD-Cdc4.sup..DELTA.3WD is derived from a truncated CDC4
PCR product (Skowyra et al., 1997) cloned into the BamHI site of
pGAD424. To test the Skp1-Met30 interaction in the two hybrid
system, a SKP1 fragment was cloned into the BamHI site of pVAD1, to
create a VP16-Skp1 fusion. LexA-Met30 derivatives were based on
pLEXM30-4 (Thomas et al. 1995). Met30 was tagged at the N-terminus
with an HA epitope by insertion of a MET30 fragment encoding amino
acids 7-640 from pLEXM30-4 into a pADH1-HA expression plasmid (Li
and Johnston 1997). Cdc4 was tagged at the N-terminus with a FLAG
epitope by site directed mutagenesis. A CDC53 deletion construct
was made by replacing an internal BgIII fragment of pGEM53-8
(Mathias et al. 1996) with an ADE2 fragment. To allow for negative
selection of wild type CDC53 in the cdc53.sup..DELTA. shuffle
strain a 3.6 kbp EcoRI fragment of CDC53 was cloned into a <URA3
CEN> plasmid. Charged to alanine mutagenesis of Cdc53 was
carried out in pMT843, as described previously (Willems et al.
1996). Although none of the mutations caused any obvious phenotype,
restriction sites incorporated during mutagenesis were used to
construct the deletions shown in FIG. 5. The version of Cdc53 in
which all six cysteine residues are replaced by alanines (C59A,
C157A, C412A, C606A, C754A, C774A) was created in a single
site-directed mutagenesis reaction.
[0112] Yeast Strains and Culture
[0113] Yeast strains are listed in Table 2. All strains were
isogenic with the W303 background. Standard methods were used for
yeast culture and transformation (Kaiser et al. 1994). A
cdc53.sup..DELTA. shuffle strain was constructed by deleting one
copy of CDC53 with pMT1514 in K699 a/.alpha. transforming with
pMT951, sporulating and isolating a Ura+Ade+ segregant.
Complementation of the shuffle strain by various <CDC53.sup.M
CEN> plasmids was tested by plating on 0.1% FOA medium. cdc53
Skp1 double mutants were generated in crosses of MTY871 with Y553
and Y555 (Bai et al. 1996). The cln2::pGAL1-CLN2M -LEU2 strain was
created by integrating pMT1111 into K699a. The MET30-1 strain
(CC786-1A) was created by crossing W303-1B with CM100-1A (Thomas et
al. 1995). MET25 mRNA expression was assayed in cultures grown in B
media supplemented with 0.1 mM sulfate as the sulfur source (Thomas
et al. 1995). At a density of 0.5.times.10.sup.7 cells/ml cultures
were shifted to 37.degree. C. for 2 hours, repressed with 1.0 mM
methionine, and time points taken for RNA extraction Cln2 halflife
was determined in pGAL1-CLN2.sup.HA strains as described previously
(Willems et al. 1996).
[0114] Two Hybrid Analysis
[0115] Strain Y187 expressing a Gal4.sup.DBD fusion was transformed
with a yeast Gal4.sup.AD-cDNA library (provided by S. Elledge) or a
Gal4.sup.AD genomic DNA library (James et al. 1996) and screened as
described (Durfee et al. 1993). With the cDNA library,
Gal4.sup.DBD-Cdc53 recovered 1 positive clone (A10) from 140,000
transformants, and Gal4.sup.DBD-Cdc53.sup..DELTA.K recovered 2
positive clones (C23 and C24) from 225,000 transformants.
Gal4.sup.DBD-Cdc53.sup..DELTA.N recovered no positive clones from
427,000 transformants. With the genomic DNA library,
Gal4.sup.DBD-Cdc53 recovered 5 positive clones (F15, F19, F20, F23,
F24) from 1 million transformants and
Gal4.sup.DBD-Cdc53.sup..DELTA.K recovered 7 positive clones (H1,
H6, H8. H9, H11, H13, H17) from 500,000 transformants. Some clones
were isolated several independent times but all unique clones are
represented in FIG. 1.
[0116] Protein and RNA Analysis
[0117] Preparation of yeast lysates and analysis of total RNA were
carried out as described previously (Willems et al. 1996). Northern
blots were probed with a 1.3 kbp MET25 fragment and a 0.6 kbp ACTI
fragment. mRNA abundance was quantitated on a Molecular Dynamics
Storm Phosphorlmager. Immunoblots were processed for ECL detection
as described (Willems et al. 1996) and where indicated signals were
quantitated by densitometry. Affinity purified anti-Cdc4,
anti-Cdc34 and anti-Cdc53 antibodies (provided by M. Goebl), and
anti-Cdc28 antibodies (Tyers et al. 1992) were used at dilutions
between 1:100 to 1:1,000, depending on the particular antibody.
Anti-Skp1 antibodies were used at 1:1,000 (Bai et al. 1996).
Anti-Grr1 antibodies were adsorbed against polyacrylamide to
eliminate background binding and used at 1:100 (Flick and Johnston
1991). Anti-Met30 antibodies were raised against recombinant
Gst-Met30 (residues 297-640 encompassing the WD40 repeats),
affinity purified and used at a dilution of 1:100. The 9E10
anti-MYC and 12CA5 anti-HA monoclonal antibodies were produced as
ascites fluid and used at 1:10,000. Anti-FLAG M2 antibody
conjugated to Sepharose beads was from Kodak. HRP-conjugated
secondary antibodies (Amersham) were used at a dilution of
1:10,000.
[0118] Sequence Analysis
[0119] Regions of sequence conservation between Cdc53 homologs
identified in database searches were determined by amino acid
alignment with ClustalW (Thompson 1994). Conserved residues with a
weight of 10 or higher were identified by analysis of 15 full
length homologs with the Wisconsin Package program Pretty. Black
lines in FIG. 5B indicate the central residue of an 11 residue
window containing four or more such conserved residues.
[0120] Results
[0121] Interactions of Cdc53, Skp1, Cdc4 and Met30 in the Two
Hybrid System
[0122] To identify proteins that interact with Cdc53, two hybrid
screens were carried out with full length Cdc53 and two Cdc53
deletion mutants (FIG. 1A). Two Cdc53 fusion proteins,
Gal4.sup.DBD-Cdc53 and Gal4.sup.DBD-Cdc53.sup..DELTA.K, recovered
multiple independent isolates of Skp1, Cdc4 and Met30 from
Gal4.sup.AD genomic and cDNA libraries (FIG. 1B, 1C). None of the
positive clones recovered interacted with
Gal4.sup.DBD-Cdc53.sup..DELTA.N, suggesting that the N-terminal
region of Cdc53 was important for these interactions (see below).
Met30 was originally isolated as a methionine-dependent repressor
of methionine biosynthesis gene expression, and has a similar
overall structure as Cdc4, with an N-terminal F-box and C-terminal
WD40 repeats (Thomas et al. 1995; Bai et al. 1996). All of the
Met30 and Cdc4 isolates that interacted with Cdc53 contained the
F-box motif, suggesting the F-box may mediate interactions with
Cdc53. In fact, two of three independent Met30 isolates contained
just the F-box and a small amount of flanking region (FIG. 1C).
Similarly, three independent Cdc4 isolates encompassed the F-box
but lacked more N-terminal sequences. Cdc4 and Met30 isolates
missing some or all of the WD40 repeats did however interact more
weakly with Cdc53 than the full length proteins (FIG. 1B, C), which
may reflect an auxiliary role for the WD40 repeats. Since Cdc4
binds Skp1 via the F-box motif (Bai et al. 1996), a Met30-Skp1
interaction was directly tested for in the two hybrid system. The
F-box of Met30 was both necessary and sufficient for interaction of
Met30 with Skp1 (FIG. 1D). As for the Cdc53-Met30 interaction, the
WD40 repeats of Met30 were required for maximal interaction with
Skp1. In summary, two hybrid analysis revealed a Cdc53-Skp1
interaction and suggested the possibility that Cdc53-F-box protein
interactions may be bridged by Skp1.
[0123] Cdc53 and Skp1 Interact Genetically
[0124] To assess the in vivo relevance of the Cdc53-SkpI two hybrid
interaction,-genetic interactions were tested between CDC53 and
SKP1. The cdc53-1 mutation was combined with the Skp1-11 and
Skp1-12 mutations. At a semi-permissive temperature of 30.degree.
C. both tht cdc53-1 Skp1-11 and cdc53-1 Skp1-12 double mutants were
inviable, whereas either single mutant grew as well as the wild
type strain (FIG. 2A). Even at a permissive temperature of
25.degree. C., cdc53-1 Skp1-12 double mutants had a severe growth
defect, and accumulated multiple hyperpolarized buds (FIG. 2B),
akin to the arrest phenotype of single mutants in the Cdc34 pathway
(Mathias et al. 1996). In addition, overproduction of CDC53 was
found to rescue Skp1 temperature sensitive strains (E. Patton,
unpublished data), as reported elsewhere (Skowrya et al. 1997).
This genetic evidence suggests that the Cdc53-Skp1 two hybrid
interaction reflects a common function of Cdc53 and Skp1 in
vivo.
[0125] Cdc53 Associates with Skp1 and Cdc4 in Yeast Lysates
[0126] Next it was determined whether endogenous levels of Cdc53
and Skp1 form a complex in yeast lysates. To minimize possible
disruption of complexes by antibodies, epitope-tagged versions of
Cdc53 and Skp1 were used. Immunoprecipitation of MYC-tagged Cdc53,
followed by immunoblotting with polyclonal antibodies directed
against Skp1, revealed a specific association between Cdc53 and
Skp1 (FIG. 3A, lane 2). Cdc4 and Cdc34 were also present in the
Cdc53 complexes, consistent with the observation that Cdc4 and
Cdc53 cofractionate with polyhistidine tagged Cdc34 (Mathias et al.
1996). In the reciprocal coimmunoprecipitation experiment, Cdc53
specifically associated with HA-tagged Skp1, as did Cdc4 and Cdc34
(FIG. 3B, lane 2). Taken together, these results indicate that
Cdc53 likely forms a multiprotein complex in vivo with Skp1, Cdc4
and Cdc34.
[0127] To determine if any of these protein-protein interactions
correlated with function in vivo, the composition of the Cdc53
complex was examined in various temperature sensitive strains. In
one set of experiments, Cdc53 immune complexes were immunoblotted
with anti-Cdc4, anti-Cdc34 and anti-Skp1 antibodies (FIG. 3A). In
cdc4-1 and Skp1-11 mutants, Cdc4 was not detected in Cdc53 immune
complexes. Although this observation was consistent with a bridging
role for Skp1, the absence of Cdc4 from the complexes was due at
least in part to decreased Cdc4 abundance in the mutants (see FIG.
3C). The Skp1-12 mutation severely decreased the abundance of Cdc4,
Cdc53 and Skp1 itself, and so the absence of associated proteins in
Cdc53 complexes from Skp1-12 cells was not informative.
[0128] In another set of experiments, Skp1 immune complexes from
temperature sensitive strains were immunoblotted with anti-Cdc4,
anti-Cdc34 and anti-Cdc53 antibodies (FIG. 3B). In this
configuration, the amount of Cdc4 in the complex was also reduced
by the cdc4-1 mutation. In contrast, the amount of Cdc4 in the
complex was increased by both the cdc34-2 and cdc53-1 mutations.
Relative to the abundance of Cdc34 in lysates, the amount of Cdc34
in Skp1 complexes was severely compromised by the cdc53-1 mutation.
Cdc53 may therefore bridge the Cdc34-Skp1 interaction (see
below).
[0129] As the anti-Cdc4 antibodies used could not reliably detect
Cdc4 in yeast lysates, it was not possible to determine directly if
the Skp1 mutations reduced the abundance of Cdc4. However,
immunoprecipitation of a FLAG-tagged version of Cdc4 followed by
immunoblotting with anti-Cdc4 polyclonal antibody revealed that
Cdc4 abundance is greatly diminished in Skp1-11 and Skp1-12 strains
(FIG. 3C). The abundance of another F-box protein, Met30, was
similarly reduced by the Skp1-11 and Skp1-12 mutations (FIG. 3C).
As noted above, the abundance of Cdc53 is also decreased by the
Skp1-12 mutation. Thus, Skp1 may function at least in part to
stabilize both Cdc53 and F-box proteins. Overall, each of
temperature sensitive mutations perturbs the mutual interactions,
by altering the abundance of a given component in lysates and/or
the immune complexes.
[0130] Cdc53 Interacts with Two Other F Box Proteins, Met30 and
Grr1
[0131] To corroborate the Cdc53-Met30 and Skp1-Met30 two hybrid
interactions, studies were carried out to determine if Met30 formed
complexes with Cdc53 and Skp1 in yeast lysates. For this purpose an
HA-tagged version of Met30 expressed from the constitutive ADH1
promoter was used. Immunoprecipitation of Met30 followed by
immunoblotting against Cdc53 and Skp1 revealed the presence of both
Cdc53 and Skp1 in Met30 immune complexes (FIG. 4A).
[0132] Because several lines of evidence suggest that Grr1 may
function with Skp1 and Cdc53 to mediate Cln1/2 degradation (Barral
et al. 1995; Bai et al. 1996; Willems et al. 1996), studies were
carried out to test if Grr1 interacts with Cdc53. Indeed, both
Cdc53 and Skp1 were specifically immunoprecipitated with an
HA-tagged version of Grr1 (FIG. 4A). In a control experiment,
FLAG-tagged Cdc4 immune complexes also contained Cdc53 and Skp1,
thereby completing the set of pairwise coimmunoprecipitations
between Cdc4, Cdc34, Cdc53 and Skp1 (FIG. 3A, B; Mathias et al.
1996). It was not possible to reproducibly detect Cdc34 in the
F-box protein immune complexes, perhaps because each of these
complexes necessarily contains only a fraction of the total Cdc34,
Cdc53 and Skp1. Within the limits of the antibodies it was not
possible to detect Cdc4 in Met30 and Grr1 immune complexes,
suggesting that F-box proteins form mutually exclusive complexes
(data not shown). Thus, Skp1 and Cdc53 form independent complexes
with at least three different F-box proteins in vivo.
[0133] The ability of Cdc53 to interact with multiple F-box
proteins raised the possibility that different F-box proteins may
compete for binding to a Cdc34-Cdc53-Skp1 core complex. This
possibility was tested by overexpressing MET30 or GRR1 in cdc4-1,
cdc34-2 and cdc53-1 temperature sensitive strains. Overexpression
of MET30 dramatically impaired growth of a cdc4-1 strain at
30.degree. C., and caused a mild growth defect in cdc53 and cdc34
strains (FIG. 4B) but had no effect on either Skp1-11 or Skp1-12
strains (data not shown). Although overexpression of GRR1 did not
affect growth of a cdc4-1 strain, the growth of cdc34-2 and cdc53-1
strains was retarded at 30.degree. C. (FIG. 4B). It has been noted
previously that high level expression of GRR1 is lethal in Skp1-12
strains at 30.degree. C. (Li and Johnston 1997), and high level
expression of Cdc4 causes inviability of cdc34 and cdc53 strains at
23.degree. C. (Mathias et al. 1996). Taken together, the above
results suggest various F-box proteins may compete for binding to a
core Cdc34-Cdc53-Skp1 complex in vivo, and that the relative
stoichiometry of the various complexes is critical for
viability.
[0134] Cdc53 is a Scaffold Protein for Cdc34 and Skp1-F-Box Protein
Complexes
[0135] To identify potential protein-protein interaction domains of
Cdc53, a series of Cdc53 deletion mutants were constructed using
natural and engineered restriction sites (see Methods). Each of the
mutant proteins was expressed to similar levels as wild type Cdc53
(FIG. 5A). The ability of each Cdc53 mutant protein to interact
with Cdc34. Skp1 and the three F-box proteins Cdc4, Grr1, Met30 was
assessed by immunoblot analysis of MYC-tagged Cdc53 immune
complexes with specific polyclonal antibodies (FIG. 5A). In this
experiment, each of the interactions detected involved
approximately wild type levels of Cdc53 (which was expressed from a
low copy plasmid) and endogenous levels of each of the associated
proteins. Deletion of an N-terminal region of Cdc53 (residues
9-280) completely disrupted Skp1 binding. In parallel, the binding
of all three F-box proteins was specifically disrupted.
Importantly, Cdc34 still interacted with Cdc53.sup..DELTA.9-280,
eliminating the possibility that the truncated protein was simply
misfolded and entirely non-functional. Conversely, deletion of an
internal region of Cdc53 (residues 448-748) abrogated Cdc34 binding
but did not affect binding of Skp1 or any of the F-box proteins.
The strict correlation between the Cdc53-Skp1 interaction and
Cdc53-F-box protein interactions is most easily explained by a
bridging function for Skp1. Furthermore, the independent
non-overlapping binding regions in Cdc53 indicate that the
protein-protein interactions within Cdc53 complexes occur in a
modular fashion.
[0136] Importantly, Cdc53 mutants that were unable to bind either
Skp1/F-box proteins or Cdc34 could not complement a cdc53 deletion
strain, while mutants unaffected in protein-protein interactions
could complement (FIG. 5B). In order to determine if the Skp1/F-box
protein and Cdc34 binding domains of Cdc53 corresponded to
conserved regions of Cdc53, 15 different members-of the Cdc53
family were aligned (FIG. 5B, see Methods for details of the
sequence alignment). Sequence similarity within the Cdc53 family is
restricted to a broad internal region and a narrow region at the
extreme C-terminus. Surprisingly, the latter region is not required
for binding to Skp1/F-box proteins or Cdc34, nor for viability
(FIG. 5A, B). However, the internal conserved region overlaps with
the Cdc34 binding site. There is relatively poor conservation in
the N-terminus of Cdc53, despite the fact that this region contains
the Skp1 binding site. The interaction with Skp1 may possibly be
limited to a subset of the Cdc53 family.
[0137] Based on the sequence alignment many conserved charged
residues in Cdc53 were mutated to alanines but none of the mutants
had any overt phenotype. For instance, mutation of the most
conserved stretch in the entire protein, IVRIMK (residues 755-760),
to polyalanine did not cause an obvious defect in Cdc53 function or
in binding to Skp1/F-box proteins or Cdc34 (FIG. 5A, B). To further
explore the structure/function relationship of Cdc53, the sequence
of two temperature sensitive alleles of CDC53 were determined. The
cdc53-1 mutation causes an R488C substitution while the cdc53-2
mutation causes a G340D substitution. Both mutations alter highly
conserved residues, even though G340 does not lie within a window
of conserved residues. Interestingly, the cdc53-1 mutation occurs
within the Cdc34 binding region. In conjunction with the defective
Skp1-Cdc34 interaction in cdc53-1 strains (FIG. 3B), this result
strongly suggests that the cdc53-1 mutation specifically perturbs
the Cdc34 binding site.
[0138] In addition to target protein recognition, some E3 ligases
form ubiquitin thioester intermediates on catalytic cysteine
residues (Scheffner et al. 1995). As Cdc53 is a component of an E3
ligase complex, studies were carried out to determine whether any
of the cysteine residues in the Cdc53 sequence were required for
function in vivo. Simultaneous mutation of all six cysteine
residues in Cdc53 to alanine did not impair complementation of a
cdc53 deletion strain (FIG. 5C). Although this mutational analysis
does not rule out thioester formation on Cdc53, such reactions
cannot be essential for viability. The primary function of Cdc53 is
therefore to act as a scaffold protein for Skp1/F-box proteins and
Cdc34.
[0139] Cdc34, Cdc53 and Skp1 are Mediators of Methionine
Repression
[0140] To assess the biological significance of the Cdc53-Met30 and
Skp1-Met30 interactions, experiments were carried out to determined
if Cdc34, Cdc53, or Skp1 were required for proper regulation of
methionine biosynthesis genes. The regulation of MET25, which
encodes homocysteine synthase and is representative of methionine
regulated genes was examined. MET25 is activated by the
Cbf1-Met4-Met28 transcriptional complex and repressed by Met30
(Thomas et al. 1995; Kuras et al. 1996). As expected, methionine
repressed MET25 expression in wild type cells (FIG. 6A). As MET30
is an essential gene, an antimorphic allele called MET30-1 was used
as a positive control for methionine derepression (Thomas et al.
1995). As shown previously, MET25 is incompletely repressed by
methionine in MET30-1 cells. Strikingly, repression of MET25 by
methionine was severely compromised in cdc53-1 cells and completely
defective in cdc34-2, Skp1-11 and Skp1-12 cells (FIG. 6A). In
contrast, MET25 was effectively repressed with wild type kinetics
in cdc4-1 cells, thereby demonstrating the specificity of F-box
protein function in methionine biosynthesis gene regulation. The
derepression of MET25 observed in cdc34, cdc53 and Skp1 mutants did
not depend on G1 phase cell cycle arrest because derepression did
not occur in cdc4-1 cells which arrest at the identical point in
G1, and yet did occur in Skp1-12 mutants which arrest in G2
phase.
[0141] Specificity of F-Box Protein Function in Cln2
Degradation
[0142] It has been shown previously that Cln2 is stabilized in
Grr1.DELTA., cdc34-2, cdc53-1 and Skp1-12 strains (Barral et al.
1995; Deshaies et al. 1995; Bai et al. 1996; Willems et al. 1996).
To directly assess the specificity of F-box protein function in
Cln2 degradation, the halflife of Cln2 in cdc4-1, Grr1.DELTA. and
MET30-1 strains was compared. We used glucose repression of a
pGAL1-CLN2.sup.HA construct to measure Cln2 decay rates, as
described previously (Willems et al. 1996). Cln2 was strongly
stabilized in Grr1.DELTA.cells, slightly stabilized in cdc4-1 cells
and not stabilized at all in MET30-1 cells (FIG. 6B). Thus Grr1 is
the primary mediator of Cln2 degradation, at least under the
conditions employed in these experiments. In contrast to Cln2
degradation, and consistent with previous results (Schwob et al.
1994; Bai et al. 1996), Sic1 degradation was found to require Cdc4,
but not Grr1 or Met30 (E. Patton, unpublished data).
[0143] Discussion
[0144] Modular Protein-Protein Interactions Allow Combinatorial
Control of Skp1-Cdc53-F-Box Protein (SCF) Complexes
[0145] Cdc53 was shown to form a multiprotein complex in vivo with
Cdc4, Cdc34 and Skp1. Furthermore, two other F-box proteins, Grr1
and Met30 , form analogous complexes with Skp 1 and Cdc53.
Consistent with these in vivo observations, recombinant Cdc4 and
Grr1 assemble into a complex with Cdc34, Cdc53 and Skp1 (Skowrya et
al. 1997). To simplify description of the various F-box containing
complexes, the generic term SCF, for Skp1-Cdc53-F-box protein
complex has been adopted(Skowrya et al. 1997; Feldman et al. 1997).
The specific F-box complexes described above are thus designated
SCF.sup.Cdc4, SCF.sup.Grr1 and SCF.sup.Met30. Formally, SCF
complexes are E3 ubiquitin-ligases, as they interact with both
substrates and an E2 enzyme, Cdc34 (Willems et al., 1996; Feldman
et al. 1997; Skowrya et al. 1997). In another sense, the
Cdc34-Cdc53-Skp1 triad forms a core complex that adapts to various
F-box proteins via Skp1; this complex is referred to as the E2/E3
core complex.
[0146] Substantial evidence indicates that Skp1 bridges F-box
proteins to Cdc53. First, the F-box of Met30 is sufficient for
interaction with Skp1 and Cdc53 in the two hybrid system. Second,
analysis of Cdc53 complexes from Skp1 and cdc4 strains shows that
Cdc4 is dispensable for the Cdc53-Skp1 interaction. Third, deletion
analysis of Cdc53 reveals that the interaction domain for Skp1
matches that of three different F-box proteins, Cdc4, Grr1 and
Met30. Furthermore, like the Cdc4-Skp1 interaction, the Cdc53-Skp1
interaction occurs in the absence of other proteins in vitro
(Skowrya et al. 1997). However, the interaction of Skp1 with Grr1
in the two hybrid system requires both the F-box and the leucine
rich repeats of Grr1 (Li and Johnston 1997). Similarly, the WD40
repeats of Cdc4 and Met30 are required for maximal interaction with
Skp1. Overall, it is certain that one function of Skp1 is to help
recruit F-box proteins to Cdc53 complexes, perhaps in conjunction
with other domains. As noted above, Skp1 may also be required for
stabilization of F-box proteins and Cdc53 in vivo.
[0147] In addition to the Skp1-F-box interaction, protein-protein
interactions within the E2/E3 core complex are of a modular nature.
Skp1 binds to the N-terminal region of Cdc53, whereas Cdc34 binds a
conserved internal region of Cdc53. The modular nature of these
protein-protein interactions and the absence of cysteine-dependent
functions in vivo indicates that Cdc53 is a scaffold protein that
may anchor Cdc34, Skp1, F-box protein and substrate in the
appropriate orientation for ubiquitin transfer.
[0148] F-Box Proteins Confer Specificity on SCF Function
[0149] Despite identification of Cdc34, Cdc53 and Skp1 through
defects in Sic1 degradation, it is now clear that SCF complexes
also control Cln degradation, glucose repression and methionine
repression. SCF.sup.Cdc4 regulates the G1 to S phase transition
through proteolysis of several key cell cycle regulators. The
dramatic cell cycle arrest phenotype caused by loss of SCF.sup.Cdc4
obscures the pleiotropic functions of the E2/E3 core complex,
despite the fact that Met30 and Grr1 play crucial roles in cellular
metabolism (Flick and Johnston 1991; Thomas et al. 1995).
SCF.sup.Grr1 has a role in both nutrient sensing and cell division,
through regulation of glucose repression and Cln degradation,
respectively (Flick and Johnston 1991; Barral et al. 1995; Li and
Johnston 1997). The present inventors have discovered the existence
of a third SCF complex, SCF.sup.Met30, and demonstrated that in
addition to Met30, each component of the E2/E3 core complex is
required for regulation of methionine biosynthesis genes.
[0150] The specificity of each SCF complex for different cellular
processes is demonstrated by a remarkable absence of cross-talk
between some of the pathways. For instance, the cdc4-1 mutation
does not affect MET25 repression and conversely, the MET30-1
mutation does not affect Cln2 degradation. Although Cdc4 appears
not to mediate Cln2 degradation under the experimental conditions
employed here, Cdc4 does interact weakly with Cln2 (Skowrya et al.
1997), so a role for Cdc4 in Cln degradation should not yet be
excluded. The growth defects caused by high level expression of
CDC4, GRR1 or MET30 in various SCF mutants suggests that different
F-box proteins may be in equilibrium with a limiting amount of the
E2/E3 core complex. If this is so, then F-box proteins may
themselves be subject to stringent regulation. The decreased
abundance of Cdc4 and Met30 in Skp1 temperature sensitive strains
is consistent with this possibility, as is the regulation of Grr1
abundance by glucose (Li and Johnston 1997).
[0151] It is likely that other SCF complexes regulate yet other
processes in yeast. A possible G2 function is suggested by the G2
arrest phenotype of Skp1-12 cells (Bai et al. 1996), and by
interactions of Skp1 with the Cbf3 kinetochore complex (Connelly
and Hieter 1996; Stemmann and Lechner 1996). Finally, because yeast
contains two Cdc53 homologs and one Skp1 homolog, orthologous SCF
pathways may also exist.
[0152] Substrates of SCF Complexes
[0153] To date, only Sic1 has been unequivocally identified as a
direct target for ubiquitination by a SCF complex. Reconstitution
of phosphorylation dependent Sic1 ubiquitination has been achieved
in vitro, in both a yeast extract system and in a purified system
with recombinant proteins (Verma et al. 1997, Skowrya et al. 1997;
Feldman et al. 1997). Strong circumstantial evidence suggests that,
in addition to Sic1, SCF.sup.Cdc4 also targets Fab, Cdc6 and Gcn4
for degradation (Henchoz et al. 1997; McKinney and Cross 1994;
Piatti et al. 1996; D. Kornitzer, personal communication). Although
ubiquitination of Cln1/2 has not yet been reconstituted,
SCF.sup.Grr1 specifically binds to phosphorylated Cln1/2,
consistent with Grr1-dependent degradation of Cln1/2 in vivo
(Skowyra et al., 1997). Genetic analysis suggests that a negative
regulator of glucose repressed genes called Rgt1 could be a
possible target of the SCF.sup.Grr1 complex (Erickson and Johnston
1994; Vallier et al. 1994). However, it is not known if Rgt1
physically interacts with Grr1, nor if Rgt1 is regulated by
ubiquitin dependent proteolysis. The requirement for SCF.sup.Met30
function in methionine repression implicates ubiquitin-dependent
proteolysis. Because Met30 forms a complex with the transactivator
Met4, it is possible that Met30 targets Met4 for degradation,
although other components of the Met4 transcriptional complex, Cbf1
and Met28, are also candidate targets (Kuras et al. 1996). The
mechanisms whereby SCF complex activity is regulated in response to
glucose and methionine are unknown, but could involve
phosphorylation, subcellular localization, F-box protein abundance
and complex assembly (Li and Johnston 1997; Pause et al. 1997).
[0154] SCF Complexes in Other Species
[0155] SCF complexes have recently emerged as key regulators in
other organisms. In S. pombe, a Cdc4 homolog, pop1, controls the
initiation of S phase by targeting the Cdk inhibitor rum1 and the
Cdc6 homolog cdc18 for ubiquitin-dependent proteolysis (Kominami
and Toda 1997). In C. elegans, null mutants of a Cdc53 homolog
called Cul-1 cause hyperplasia in all tissues, suggesting that it
too may target activators of division for degradation (Kipreos et
al. 1996). In human cells, Skp1 binds to cyclin A-Cdk2 through its
associated F-box protein, Skp2, (Zhang et al. 1995) and also forms
a specific complex with human Cul-1 (Y. Xiong, personal
communication). Another human cullin, Cul-2, physically associates
with the VHL tumour suppressor protein, and may thus also regulate
cell proliferation (Pause et al. 1997). As in yeast, degradation of
mammalian G1 cyclins and Cdk inhibitors is phosphorylation and
ubiquitin dependent (Clurman et al. 1996; Won and Reed 1996; Diehl
et al. 1997; Sheaff et al. 1997) and so it will be of prime
importance to determine the role of SCF complexes in these
pathways. The control of gene expression by proteolysis is now well
documented in several systems (Pahl and Baeuerle 1996), and by
analogy to glucose and methionine regulation in yeast, SCF
complexes may prove to be general transcriptional regulators.
Indeed, the Met30 homologs Scon2/SconB and the Skp1 homolog SconC
regulate sulfur metabolism in other fungi (Natorff et al. 1993;
Kumar and Paietta 1995), suggesting that control of methionine
biosynthesis by SCF complexes may be conserved. As metazoans
contain at least six Cdc53 homologs (Kipreos et al. 1996), and as
SCF complexes control multiple processes in yeast, it is likely
that analogous SCF complexes will have both cell cycle and non-cell
cycle functions in higher species. The combinatorial control of SCF
ubiquitin-ligase complexes provides an adaptable regulatory system
that controls cell function through ubiquitin-dependent protein
degradation.
EXAMPLE 2
Identification of a Skp1 Binding Region in the Cdc53 Protein of the
Budding Yeast Saccharomyces cerevisiae
[0156] Amino acids 9-280 of the Cdc53 protein in Saccharomyces
cerevisiae have been shown to be necessary for association with
Skp1 (Patton et al, 1998 Genes Dev. 12:692). Similar experiments
have narrowed the region necessary for the Cdc53/Skp1 interaction
to amino acids 9 to 99 of Cdc53. The amino acid sequence of this
region of Cdc53 was aligned with other Cdc53 cullin homologues
(FIG. 11). The amino acids corresponding to tyrosine (Y) 48 and
methionine (M) 49 in Cdc53 were absolutely conserved in a subset of
homologues more closely related to Cdc53 but not present in any of
the other more distantly related homologues (with the exception of
Y50 in C3A11.08 in Schizosaccharomyces pombe). These two amino
acids in Cdc53 were mutated by Kunkel mutagenesis to tryptophan (W)
and glutamic acid (E) respectively, the homologous residues in
Caenorhabditus elegans Cul-3. Five independent TRP1 plasmid
isolates (pMT1939-1943) of this mutagenesis reaction, as well as a
plasmid carrying a wild type CDC53 gene (pMT843), were transformed
into a CDC53 "shuffle strain" (strain Mty1243, genotype ura3 trp1
leu2 his3 ade2 cdc53::ADE2<CDC53.sup.HA3 URA3 CEN6 ARSH4>).
These mutant and wild type CDC53 genes were tagged with a MYC.sub.6
epitope. These strains were grown on complete minimal medium agar
plates containing 1 g/L of 5-fluoroorotic acid, which kills cells
that produce Ura3, thus killing any cells that do not lose the
<CDC53.sup.HA3 URA3> plasmid. None of the five
CDC53-Y48W,M49E.sup.MYC6 mutants conferred viability on the shuffle
strain, while the wild type CDC53.sup.MYC6 did. Cells containing
either the wild type CDC53.sup.MYC.sub.6 or one of three isolates
of the mutant CDC53.sup.MYC6 were grown to late-log phase
(2.times.10.sup.7 cells/ml), harvested, washed, resuspended in
lysis buffer (50 mM Tris-Cl pH 7.5, 250 mM NaCl, 50M NaF, 5 mM
EDTA, 0.1% NP-40, 1 mM DTT) plus protease inhibitors, snap frozen
in liquid nitrogen, and ground into a powder under liquid nitrogen.
This powder was thawed, spun to remove cellullar debris, and
cleared by spinning in an ultracentrifuge. Protein concentration in
the lysate was adjusted to 24 mg/ml in a volume of 1.25 ml lysis
buffer +10 mM N-ethyl maleimide, for a final mass of 30 mg protein
for each strain. 25 .mu.l of a 50% slurry in lysis buffer of
protein-A beads (Pierce) cross-linked with dimethyl suberimidate to
the anti-MYC monoclonal antibody 9E10 was added to each lysate,
incubated with gentle rocking at 4.degree. C. for several hours,
washed several times with lysis buffer, aspirated, resuspended in
protein sample buffer, and run on two 10% polyacrylamide gels. One
gel was transferred to polyvinylidene fluoride membrane and western
blotted with anti-MYC, anti-Cdc34, and anti-Skp1 antibodies (FIG.
12A). The mutant Cdc53 still binds to Cdc34 but not to Skp1. The
second gel was silver stained (see W. Wray et al. 1981, Anal.
Biochem. 118:197) (FIG. 12B). A number of bands that are present in
the Cdc53.sup.MYC6 immunoprecipitation disappear in the Cdc53-Y48W,
M.sub.49E.sup.MYC6 immunoprecipitation.
[0157] Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated to
those skilled in the art that the invention can be modified in
arrangement and detail without departure from such principles. All
modifications coming within the scope of the following claims are
claimed.
[0158] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety.
1TABLE 1 Plasmids used in this study Plasmid Relevant
characteristics Source pMT634 pGAL1-CLN2-HA LEU2 URA3 CEN Willems
et al. (1997) pMT817 CDC53-C-NorI TRP1 CEN Willems et al. (1996)
pMT843 CDC53.sup.M TRP1 CEN Willems et al. (1996) pMT915 GAL4
.sup.AD-CDC4.sup..DELTA.3WD LEU2 2.mu.m This study pMT918 CDC53 in
pAS1-CYH2 TRP1 2.mu.m This study pMT951 CDC53.sup.HA URA3 CEN This
study pMT954 pGAL4.sup.DBD-CDC53 .DELTA.N TRP1 2.mu.m This study
pMT955 pGAL4.sup.DBD-CDC53 .DELTA.K TRP1 2.mu.m This study pMT1111
pUC119 cln2::GAL-CLN2.sup.M-LEU2 B. Schneider pMT1511 SKP1.sup.HA
LEU2 CEN P. Heiter pMT1514 cdc53::ADE2 in pGEM3 This study pBF339
pADH1.sup.HA TRP1 2.mu.m Li and Johnston (1997) pBF494
pADH1.sup.HA-GRR1.sup..DELTA.N TRP1 2.mu.m Li and Johnston (1997)
pMT1707 pADH1.sup.HA-MET3O TRP1 2.mu.m This study pMT1850
CDC53.sup.M.DELTA.9-280 TRP1 CEN This study pMT1854
CDC53.sup.M.DELTA.448-763,H767A TRP1 CEN This study pMT1856
CDC53.sup.M.DELTA.448-748 TRP1 CEN This study pMT1857
CDC53.sup.M.DELTA.757-815 TRP1 CEN This study pMT1858
CDC53.sup.M.DELTA.794-815 TRP1 CEN This study pMT1859
CDC53.sup.MIVRIMK TRP1 CEN This study pMT1861 CDC53.sup.M6CTRP1 CEN
This study pLexM30-4 pLEXA.sup.DBD-MET30 TRP1 2.mu.m This study
pLexM30-4(297-540) pLEXA.sup.DBD-MET30 .sup..DELTA.297-540 TRP1
2.mu.m This study pLexM30-4(158-297) pLEXA.sup.DBD-MET30
.sup..DELTA.158-297 TRP1 2.mu.m This study pLexM30-4(158-540)
pLEXA.sup.DBD-MET30 .sup..DELTA.158-540 TRP1 2.mu.m This study
pVAD1-SKP1 pVAD-SKP1 LEU2 2.mu.m This study pSE1111
GAL4.sup.AD-SNF1 LEU2 2.mu.m S. Elledge pSE1112 GAL4.sup.DBD-SNF4
TRP1 2.mu.m S. Elledge pRS314 TRP1 CEN Sikorski and Hieter
(1989)
[0159]
2TABLE 2 Yeast strains used in this study Strain Relevant genotype
Source K699 MATa ade2-1 can1-100 his3-1,15S leu2-3,112 trp1-1 ura3
K. Nasmyth K699 a/.alpha. MATa/MAT.alpha. ade2-1/ade2-1
can1-100/can1-100 his3-1,15/his3-1,15 K. Nasmyth
leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3/ura3 MTY668 MATa cdc4-1
This study MTY670 MATacdc34-2 Willems at el. (1996) MTY871 MATa
cdc53-1 Willems at el. (1996) MTY1243 cdc53::ADE2, pMT951 plasmid
This study MTY1293 cdc53-1 skp1-11 This study MTY1294 cdc53-1
skp1-12 This study MTY1295 cln2::pGAL1-CLN2.sup.M LEU2 This study
Y187 MATa ade2-101 his3-.DELTA.200 leu2-3,112 trp1-901 ura3-52
gal4.DELTA.gal80.DELTA. S. Elledge URA3::GAL-lacZ LYS2::GAL-HIS3
Y190 as for Y187 but MAT.alpha. S. Elledge Y553 MAT.alpha. skp1-11
Bai et al. (1996) Y555 Mat.alpha. skp1-12 Bai et al. (1996)
WX131-2c MAT.alpha. cdc53-2 trp1-7 ura3-52 ade2 M. Goebl CC786-1A
ade2 his3 leu2 ura3 trpI MET30-1 This study C170 ade2 his3 leu2
trp1 met4::TRP1 ura3::lexAop-lacZ::URA3 Kuras and Thomas (1995)
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Sequence CWU 1
1
50 1 11 PRT Saccharomyces cerevisiae 1 Met Glu Val Thr Ala Ile Tyr
Asn Tyr Cys Val 1 5 10 2 15 PRT Saccharomyces cerevisiae 2 Tyr Met
Glu Val Thr Ala Ile Tyr Asn Tyr Cys Val Asn Lys Ser 1 5 10 15 3 22
PRT Saccharomyces cerevisiae 3 Ile Leu Ser Pro Thr Met Tyr Met Glu
Val Tyr Thr Ala Ile Tyr Asn 1 5 10 15 Tyr Cys Val Asn Lys Ser 20 4
13 PRT Saccharomyces cerevisiae 4 Tyr Met Thr Leu Tyr Thr Ser Val
Tyr Asp Tyr Cys Thr 1 5 10 5 16 PRT Saccharomyces cerevisiae 5 Tyr
Met Thr Leu Tyr Thr Ser Val Tyr Asp Tyr Cys Thr Ser Ile Thr 1 5 10
15 6 21 PRT Saccharomyces cerevisiae 6 Met Ala Pro Lys Asp Tyr Met
Thr Leu Tyr Thr Ser Val Tyr Asp Tyr 1 5 10 15 Cys Thr Ser Ile Thr
20 7 13 PRT Saccharomyces cerevisiae 7 Tyr Met Met Leu Tyr Asp Ala
Val Tyr Asn Ile Cys Thr 1 5 10 8 16 PRT Saccharomyces cerevisiae 8
Tyr Met Met Leu Tyr Asp Ala Val Tyr Asn Ile Cys Thr Thr Thr Thr 1 5
10 15 9 22 PRT Saccharomyces cerevisiae 9 His Met Ser Lys Lys Tyr
Tyr Met Met Leu Tyr Asp Ala Val Tyr Asn 1 5 10 15 Ile Cys Thr Thr
Thr Thr 20 10 13 PRT Saccharomyces cerevisiae 10 Tyr Met Arg Phe
Tyr Thr His Val Tyr Asp Tyr Cys Thr 1 5 10 11 16 PRT Saccharomyces
cerevisiae 11 Tyr Met Arg Phe Tyr Thr His Val Tyr Asp Tyr Cys Thr
Ser Val Ser 1 5 10 15 12 22 PRT Saccharomyces cerevisiae 12 Ser Leu
Thr Arg Ser Gln Tyr Met Arg Phe Tyr Thr His Val Tyr Asp 1 5 10 15
Tyr Cys Thr Ser Val Ser 20 13 13 PRT Saccharomyces cerevisiae 13
Tyr Met Glu Leu Tyr Thr His Val Tyr Asn Tyr Cys Thr 1 5 10 14 16
PRT Saccharomyces cerevisiae 14 Tyr Met Glu Leu Tyr Thr His Val Tyr
Asn Tyr Cys Thr Ser Val His 1 5 10 15 15 22 PRT Saccharomyces
cerevisiae 15 Ser Met Ala Lys Ser Arg Tyr Met Glu Leu Tyr Thr His
Val Tyr Asn 1 5 10 15 Tyr Cys Thr Ser Val His 20 16 13 PRT
Saccharomyces cerevisiae 16 Tyr Met Met Leu Tyr Thr Thr Ile Tyr Asn
Met Cys Thr 1 5 10 17 16 PRT Saccharomyces cerevisiae 17 Tyr Met
Met Leu Tyr Thr Thr Ile Tyr Asn Met Cys Thr Gln Lys Pro 1 5 10 15
18 22 PRT Saccharomyces cerevisiae 18 Ala Phe Asp Ser Glu Gln Tyr
Met Met Leu Tyr Thr Thr Ile Tyr Asn 1 5 10 15 Met Cys Thr Gln Lys
Pro 20 19 13 PRT Saccharomyces cerevisiae 19 Tyr Met Glu Leu Tyr
Thr Ala Ile His Asn Thr Cys Ala 1 5 10 20 16 PRT Saccharomyces
cerevisiae 20 Tyr Met Glu Leu Tyr Thr Ala Ile His Asn Thr Cys Ala
Asp Ala Ser 1 5 10 15 21 21 PRT Saccharomyces cerevisiae 21 Gly Met
Ile Thr Phe Tyr Met Glu Leu Tyr Thr Ala Ile His Asn Thr 1 5 10 15
Cys Ala Asp Ala Ser 20 22 295 PRT Saccharomyces cerevisiae 22 Met
Ser Ser Arg Lys Ser Thr Ala Ser Ser Leu Leu Leu Arg Gln Tyr 1 5 10
15 Arg Glu Leu Thr Asp Pro Lys Lys Ala Ile Pro Ser Phe His Ile Glu
20 25 30 Leu Glu Asp Asp Ser Asn Ile Phe Thr Trp Asn Ile Gly Val
Met Val 35 40 45 Leu Asn Glu Asp Ser Ile Tyr His Gly Gly Phe Phe
Lys Ala Gln Met 50 55 60 Arg Phe Pro Glu Asp Phe Pro Phe Ser Pro
Pro Gln Phe Arg Phe Thr 65 70 75 80 Pro Ala Ile Tyr His Pro Asn Val
Tyr Arg Asp Gly Arg Leu Cys Ile 85 90 95 Ser Ile Leu His Gln Ser
Gly Asp Pro Met Thr Asp Glu Pro Asp Ala 100 105 110 Glu Thr Trp Ser
Pro Val Gln Thr Val Glu Ser Val Leu Ile Ser Ile 115 120 125 Val Ser
Leu Leu Glu Asp Pro Asn Ile Asn Ser Pro Ala Asn Val Asp 130 135 140
Ala Ala Val Asp Tyr Arg Lys Asn Pro Glu Gln Tyr Lys Gln Arg Val 145
150 155 160 Lys Met Glu Val Glu Arg Ser Lys Gln Asp Ile Pro Lys Gly
Phe Ile 165 170 175 Met Pro Thr Ser Glu Ser Ala Tyr Ile Ser Gln Ser
Lys Leu Asp Glu 180 185 190 Pro Glu Ser Asn Lys Asp Met Ala Asp Asn
Phe Trp Tyr Asp Ser Asp 195 200 205 Leu Asp Asp Asp Glu Asn Gly Ser
Val Ile Leu Gln Asp Asp Asp Tyr 210 215 220 Asp Asp Gly Asn Asn His
Ile Pro Phe Glu Asp Asp Asp Val Tyr Asn 225 230 235 240 Tyr Asn Asp
Asn Asp Asp Asp Asp Glu Arg Ile Glu Phe Glu Asp Asp 245 250 255 Asp
Asp Asp Asp Asp Asp Ser Ile Asp Asn Asp Ser Val Met Asp Arg 260 265
270 Lys Gln Pro His Lys Ala Glu Asp Glu Ser Glu Asp Val Glu Asp Val
275 280 285 Glu Arg Val Ser Lys Lys Ile 290 295 23 298 PRT
Saccharomyces cerevisiae 23 Ile Ala Ala Ala Pro Glu Leu Leu Glu Arg
Ser Gly Ser Pro Gly Gly 1 5 10 15 Gly Gly Gly Ala Glu Glu Glu Ala
Gly Gly Gly Pro Gly Gly Ser Pro 20 25 30 Pro Asp Gly Ala Arg Pro
Gly Pro Ser Arg Glu Leu Ala Val Val Ala 35 40 45 Arg Pro Arg Ala
Ala Pro Thr Pro Gly Pro Ser Ala Ala Ala Met Ala 50 55 60 Arg Pro
Leu Val Pro Ser Ser Gln Lys Ala Leu Leu Leu Glu Leu Lys 65 70 75 80
Gly Leu Gln Glu Glu Pro Val Glu Gly Phe Arg Val Thr Leu Val Asp 85
90 95 Glu Gly Asp Leu Tyr Asn Trp Glu Val Ala Ile Phe Gly Pro Pro
Asn 100 105 110 Thr Tyr Tyr Glu Gly Gly Tyr Phe Lys Ala Arg Leu Lys
Phe Pro Ile 115 120 125 Asp Tyr Pro Tyr Ser Pro Pro Ala Phe Arg Phe
Leu Thr Lys Met Trp 130 135 140 His Pro Asn Ile Tyr Glu Thr Gly Asp
Val Cys Ile Ser Ile Leu His 145 150 155 160 Pro Pro Val Asp Asp Pro
Gln Ser Gly Glu Leu Pro Ser Glu Arg Trp 165 170 175 Asn Pro Thr Gln
Asn Val Arg Thr Ile Leu Leu Ser Val Ile Ser Leu 180 185 190 Leu Asn
Glu Pro Asn Thr Phe Ser Pro Ala Asn Val Asp Ala Ser Val 195 200 205
Met Tyr Arg Lys Trp Lys Glu Ser Lys Gly Lys Asp Arg Glu Tyr Thr 210
215 220 Asp Ile Ile Arg Lys Gln Val Leu Gly Thr Lys Val Asp Ala Glu
Arg 225 230 235 240 Asp Gly Val Lys Val Pro Thr Thr Leu Ala Glu Tyr
Cys Val Lys Thr 245 250 255 Lys Ala Pro Ala Pro Asp Glu Gly Ser Asp
Leu Phe Tyr Asp Asp Tyr 260 265 270 Tyr Glu Asp Gly Glu Val Glu Glu
Glu Ala Asp Ser Cys Phe Gly Asp 275 280 285 Asp Glu Asp Asp Ser Gly
Thr Glu Glu Ser 290 295 24 815 PRT Saccharomyces cerevisiae 24 Met
Ser Glu Thr Leu Pro Arg Ser Asp Asp Leu Glu Ala Thr Trp Asn 1 5 10
15 Phe Ile Glu Pro Gly Ile Asn Gln Ile Leu Gly Asn Glu Lys Asn Gln
20 25 30 Ala Ser Thr Ser Lys Arg Val Tyr Lys Ile Leu Ser Pro Thr
Met Tyr 35 40 45 Met Glu Val Tyr Thr Ala Ile Tyr Asn Tyr Cys Val
Asn Lys Ser Arg 50 55 60 Ser Ser Gly His Phe Ser Thr Asp Ser Arg
Thr Gly Gln Ser Thr Ile 65 70 75 80 Leu Val Gly Ser Glu Ile Tyr Glu
Lys Leu Lys Asn Tyr Leu Lys Asn 85 90 95 Tyr Ile Leu Asn Phe Lys
Gln Ser Asn Ser Glu Thr Phe Leu Gln Phe 100 105 110 Tyr Val Lys Arg
Trp Lys Arg Phe Thr Ile Gly Ala Ile Phe Leu Asn 115 120 125 His Ala
Phe Asp Tyr Met Asn Arg Tyr Trp Val Gln Lys Glu Arg Ser 130 135 140
Asp Gly Lys Arg His Ile Phe Asp Val Asn Thr Leu Cys Leu Met Thr 145
150 155 160 Trp Lys Glu Val Met Phe Asp Pro Ser Lys Asp Val Leu Ile
Asn Glu 165 170 175 Leu Leu Asp Gln Val Thr Leu Gly Arg Glu Gly Gln
Ile Ile Gln Arg 180 185 190 Ser Asn Ile Ser Thr Ala Ile Lys Ser Leu
Val Ala Leu Gly Ile Asp 195 200 205 Pro Gln Asp Leu Lys Lys Leu Asn
Leu Asn Val Tyr Ile Gln Val Phe 210 215 220 Glu Lys Pro Phe Leu Lys
Lys Thr Gln Glu Tyr Tyr Thr Gln Tyr Thr 225 230 235 240 Asn Asp Tyr
Leu Glu Lys His Ser Val Thr Glu Tyr Ile Phe Glu Ala 245 250 255 His
Glu Ile Ile Lys Arg Glu Glu Lys Ala Met Thr Ile Tyr Trp Asp 260 265
270 Asp His Thr Lys Lys Pro Leu Ser Met Ala Leu Asn Lys Val Leu Ile
275 280 285 Thr Asp His Ile Glu Lys Leu Glu Asn Glu Phe Val Val Leu
Leu Asp 290 295 300 Ala Arg Asp Ile Glu Lys Ile Thr Ser Leu Tyr Ala
Leu Ile Arg Arg 305 310 315 320 Asp Phe Thr Leu Ile Pro Arg Met Ala
Ser Val Phe Glu Asn Tyr Val 325 330 335 Lys Lys Thr Gly Glu Asn Glu
Ile Ser Ser Leu Leu Ala Met His Lys 340 345 350 His Asn Ile Met Lys
Asn Glu Asn Ala Asn Pro Lys Lys Leu Ala Leu 355 360 365 Met Thr Ala
His Ser Leu Ser Pro Lys Asp Tyr Ile Lys Lys Leu Leu 370 375 380 Glu
Val His Asp Ile Phe Ser Lys Ile Phe Asn Glu Ser Phe Pro Asp 385 390
395 400 Asp Ile Pro Leu Ala Lys Ala Leu Asp Asn Ala Cys Gly Ala Phe
Ile 405 410 415 Asn Ile Asn Glu Phe Ala Leu Pro Ala Gly Ser Pro Lys
Ser Ala Thr 420 425 430 Ser Lys Thr Ser Glu Met Leu Ala Lys Tyr Ser
Asp Ile Leu Leu Lys 435 440 445 Lys Ala Thr Lys Pro Glu Val Ala Ser
Asp Met Ser Asp Glu Asp Ile 450 455 460 Ile Thr Ile Phe Lys Tyr Leu
Thr Asp Lys Asp Ala Phe Glu Thr His 465 470 475 480 Tyr Arg Arg Leu
Phe Ala Lys Arg Leu Ile His Gly Thr Ser Thr Ser 485 490 495 Ala Glu
Asp Glu Glu Asn Ile Ile Gln Arg Leu Gln Ala Ala Asn Ser 500 505 510
Met Glu Tyr Thr Gly Lys Ile Thr Lys Met Phe Gln Asp Ile Arg Leu 515
520 525 Ser Lys Ile Leu Glu Asp Asp Phe Ala Val Ala Leu Lys Asn Glu
Pro 530 535 540 Asp Tyr Ser Lys Ala Lys Tyr Pro Asp Leu Gln Pro Phe
Val Leu Ala 545 550 555 560 Glu Asn Met Trp Pro Phe Ser Tyr Gln Glu
Val Glu Phe Lys Leu Pro 565 570 575 Lys Glu Leu Val Pro Ser His Glu
Lys Leu Lys Glu Ser Tyr Ser Gln 580 585 590 Lys His Asn Gly Arg Ile
Leu Lys Trp Leu Trp Pro Leu Cys Arg Gly 595 600 605 Glu Leu Lys Ala
Asp Ile Gly Lys Pro Gly Arg Met Pro Phe Asn Phe 610 615 620 Thr Val
Thr Leu Phe Gln Met Ala Ile Leu Leu Leu Tyr Asn Asp Ala 625 630 635
640 Asp Val Leu Thr Leu Glu Asn Ile Gln Glu Gly Thr Ser Leu Thr Ile
645 650 655 Gln His Ile Ala Ala Ala Met Val Pro Phe Ile Lys Phe Lys
Leu Ile 660 665 670 Gln Gln Val Pro Pro Gly Leu Asp Ala Leu Val Lys
Pro Glu Thr Gln 675 680 685 Phe Lys Leu Ser Arg Pro Tyr Lys Ala Leu
Lys Thr Asn Ile Asn Phe 690 695 700 Ala Ser Gly Val Lys Asn Asp Ile
Leu Gln Ser Leu Ser Gly Gly Gly 705 710 715 720 His Asp Asn His Gly
Asn Lys Leu Gly Asn Lys Arg Leu Thr Glu Asp 725 730 735 Glu Arg Ile
Glu Lys Glu Leu Asn Thr Glu Arg Gln Ile Phe Leu Glu 740 745 750 Ala
Cys Ile Val Arg Ile Met Lys Ala Lys Arg Asn Leu Pro His Thr 755 760
765 Thr Leu Val Asn Glu Cys Ile Ala Gln Ser His Gln Arg Phe Asn Ala
770 775 780 Lys Val Ser Met Val Lys Arg Ala Ile Asp Ser Leu Ile Gln
Lys Gly 785 790 795 800 Tyr Leu Gln Arg Gly Asp Asp Gly Glu Ser Tyr
Ala Tyr Leu Ala 805 810 815 25 745 PRT Saccharomyces cerevisiae 25
Met Ser Leu Lys Pro Arg Val Val Asp Phe Asp Glu Thr Trp Asn Lys 1 5
10 15 Leu Leu Thr Thr Ile Lys Ala Val Val Met Leu Glu Tyr Val Glu
Arg 20 25 30 Ala Thr Trp Asn Asp Arg Phe Ser Asp Ile Tyr Ala Leu
Cys Val Ala 35 40 45 Tyr Pro Glu Pro Leu Gly Glu Arg Leu Tyr Thr
Glu Thr Lys Ile Phe 50 55 60 Leu Glu Asn His Val Arg His Leu His
Lys Arg Val Leu Glu Ser Glu 65 70 75 80 Glu Gln Val Leu Val Met Tyr
His Arg Tyr Trp Glu Glu Tyr Ser Lys 85 90 95 Gly Ala Asp Tyr Met
Asp Cys Leu Tyr Arg Tyr Leu Ser Thr Gln Phe 100 105 110 Ile Lys Lys
Asn Lys Leu Thr Glu Ala Asp Leu Gln Tyr Gly Tyr Gly 115 120 125 Gly
Val Asp Met Asn Glu Pro Leu Met Glu Ile Gly Glu Leu Ala Leu 130 135
140 Asp Met Trp Arg Lys Leu Met Val Glu Pro Leu Gln Ala Ile Leu Ile
145 150 155 160 Arg Met Leu Leu Arg Glu Ile Lys Asn Asp Arg Gly Gly
Glu Asp Pro 165 170 175 Asn Gln Lys Val Ile His Gly Val Ile Asn Ser
Phe Val His Val Glu 180 185 190 Gln Tyr Lys Lys Lys Phe Pro Leu Lys
Phe Tyr Gln Glu Ile Phe Glu 195 200 205 Ser Pro Phe Leu Thr Glu Thr
Gly Glu Tyr Tyr Lys Gln Glu Ala Ser 210 215 220 Asn Leu Leu Gln Glu
Ser Asn Cys Ser Gln Tyr Met Glu Lys Val Leu 225 230 235 240 Gly Arg
Leu Lys Asp Glu Glu Ile Arg Cys Arg Lys Tyr Leu His Pro 245 250 255
Ser Ser Tyr Thr Lys Val Ile His Glu Cys Gln Gln Arg Met Val Ala 260
265 270 Asp His Leu Gln Phe Leu His Ala Glu Cys His Asn Ile Ile Arg
Gln 275 280 285 Glu Lys Lys Asn Asp Met Ala Asn Met Tyr Val Leu Leu
Arg Ala Val 290 295 300 Ser Thr Gly Leu Pro His Met Ile Gln Glu Leu
Gln Asn His Ile His 305 310 315 320 Asp Glu Gly Leu Arg Ala Thr Ser
Asn Leu Thr Gln Glu Asn Met Pro 325 330 335 Thr Leu Phe Val Glu Ser
Val Leu Glu Val His Gly Lys Phe Val Gln 340 345 350 Leu Ile Asn Thr
Val Leu Asn Gly Asp Gln His Phe Met Ser Ala Leu 355 360 365 Asp Lys
Ala Leu Thr Ser Val Val Asn Tyr Arg Glu Pro Lys Ser Val 370 375 380
Cys Lys Ala Pro Glu Leu Leu Ala Lys Tyr Cys Asp Asn Leu Leu Lys 385
390 395 400 Lys Ser Ala Lys Gly Met Thr Glu Asn Glu Val Glu Asp Arg
Leu Thr 405 410 415 Ser Phe Ile Thr Val Phe Lys Tyr Ile Asp Asp Lys
Asp Val Phe Gln 420 425 430 Lys Phe Tyr Ala Arg Met Leu Ala Lys Arg
Leu Ile His Gly Leu Ser 435 440 445 Met Ser Met Asp Ser Glu Glu Ala
Met Ile Asn Lys Leu Lys Gln Ala 450 455 460 Cys Gly Tyr Glu Phe Thr
Ser Lys Leu His Arg Met Tyr Thr Asp Met 465 470 475 480 Ser Val Ser
Ala Asp Leu Asn Asn Lys Phe Asn Asn Phe Ile Lys Asn 485 490 495 Gln
Asp Thr Val Ile Asp Leu Gly Ile Ser Phe Gln Ile Tyr Val Leu 500 505
510 Gln Ala Gly Ala Trp Pro Leu Thr Gln Ala Pro Ser Ser Thr Phe Ala
515 520 525 Ile Pro Gln Glu Leu Glu Lys Ser Val Gln Met Phe Glu Leu
Phe Tyr 530 535 540 Ser Gln His Phe Ser Gly Arg Lys Leu Thr Trp
Leu
His Tyr Leu Cys 545 550 555 560 Thr Gly Glu Val Lys Met Asn Tyr Leu
Gly Lys Pro Tyr Val Ala Met 565 570 575 Val Thr Thr Tyr Gln Met Ala
Val Leu Leu Ala Phe Asn Asn Ser Glu 580 585 590 Thr Val Ser Tyr Lys
Glu Leu Gln Asp Ser Thr Gln Met Asn Glu Lys 595 600 605 Glu Leu Thr
Lys Thr Ile Lys Ser Leu Leu Asp Val Lys Met Ile Asn 610 615 620 His
Asp Ser Glu Lys Glu Asp Ile Asp Ala Glu Ser Ser Phe Ser Leu 625 630
635 640 Asn Met Asn Phe Ser Ser Lys Arg Thr Lys Phe Lys Ile Thr Thr
Ser 645 650 655 Met Gln Lys Asp Thr Pro Gln Glu Met Glu Gln Thr Arg
Ser Ala Val 660 665 670 Asp Glu Asp Arg Lys Met Tyr Leu Gln Ala Ala
Ile Val Arg Ile Met 675 680 685 Lys Ala Arg Lys Val Leu Arg His Asn
Ala Leu Ile Gln Glu Val Ile 690 695 700 Ser Gln Ser Arg Ala Arg Phe
Asn Pro Ser Ile Ser Met Ile Lys Lys 705 710 715 720 Cys Ile Glu Val
Leu Ile Asp Lys Gln Tyr Ile Glu Arg Ser Gln Ala 725 730 735 Ser Ala
Asp Glu Tyr Ser Tyr Val Ala 740 745 26 301 PRT Saccharomyces
cerevisiae 26 Lys Lys Ala Thr Lys Pro Glu Val Ala Ser Asp Met Ser
Asp Glu Asp 1 5 10 15 Ile Ile Thr Ile Phe Lys Tyr Leu Thr Asp Lys
Asp Ala Phe Glu Thr 20 25 30 His Tyr Arg Arg Leu Phe Ala Lys Arg
Leu Ile His Gly Thr Ser Thr 35 40 45 Ser Ala Glu Asp Glu Glu Asn
Ile Ile Gln Arg Leu Gln Ala Ala Asn 50 55 60 Ser Met Glu Tyr Thr
Gly Lys Ile Thr Lys Met Phe Gln Asp Ile Arg 65 70 75 80 Leu Ser Lys
Ile Leu Glu Asp Asp Phe Ala Val Ala Leu Lys Asn Glu 85 90 95 Pro
Asp Tyr Ser Lys Ala Lys Tyr Pro Asp Leu Gln Pro Phe Val Leu 100 105
110 Ala Glu Asn Met Trp Pro Phe Ser Tyr Gln Glu Val Glu Phe Lys Leu
115 120 125 Pro Lys Glu Leu Val Pro Ser His Glu Lys Leu Lys Glu Ser
Tyr Ser 130 135 140 Gln Lys His Asn Gly Arg Ile Leu Lys Trp Leu Trp
Pro Leu Cys Arg 145 150 155 160 Gly Glu Leu Lys Ala Asp Ile Gly Lys
Pro Gly Arg Met Pro Phe Asn 165 170 175 Phe Thr Val Thr Leu Phe Gln
Met Ala Ile Leu Leu Leu Tyr Asn Asp 180 185 190 Ala Asp Val Leu Thr
Leu Glu Asn Ile Gln Glu Gly Thr Ser Leu Thr 195 200 205 Ile Gln His
Ile Ala Ala Ala Met Val Pro Phe Ile Lys Phe Lys Leu 210 215 220 Ile
Gln Gln Val Pro Pro Gly Leu Asp Ala Leu Val Lys Pro Glu Thr 225 230
235 240 Gln Phe Lys Leu Ser Arg Pro Tyr Lys Ala Leu Lys Thr Asn Ile
Asn 245 250 255 Phe Ala Ser Gly Val Lys Asn Asp Ile Leu Gln Ser Leu
Ser Gly Gly 260 265 270 Gly His Asp Asn His Gly Asn Lys Leu Gly Asn
Lys Arg Leu Thr Glu 275 280 285 Asp Glu Arg Ile Glu Lys Glu Leu Asn
Thr Glu Arg Gln 290 295 300 27 272 PRT Saccharomyces cerevisiae 27
Asp Asp Leu Glu Ala Thr Trp Asn Phe Ile Glu Pro Gly Ile Asn Gln 1 5
10 15 Ile Leu Gly Asn Glu Lys Asn Gln Ala Ser Thr Ser Lys Arg Val
Tyr 20 25 30 Lys Ile Leu Ser Pro Thr Met Tyr Met Glu Val Tyr Thr
Ala Ile Tyr 35 40 45 Asn Tyr Cys Val Asn Lys Ser Arg Ser Ser Gly
His Phe Ser Thr Asp 50 55 60 Ser Arg Thr Gly Gln Ser Thr Ile Leu
Val Gly Ser Glu Ile Tyr Glu 65 70 75 80 Lys Leu Lys Asn Tyr Leu Lys
Asn Tyr Ile Leu Asn Phe Lys Gln Ser 85 90 95 Asn Ser Glu Thr Phe
Leu Gln Phe Tyr Val Lys Arg Trp Lys Arg Phe 100 105 110 Thr Ile Gly
Ala Ile Phe Leu Asn His Ala Phe Asp Tyr Met Asn Arg 115 120 125 Tyr
Trp Val Gln Lys Glu Arg Ser Asp Gly Lys Arg His Ile Phe Asp 130 135
140 Val Asn Thr Leu Cys Leu Met Thr Trp Lys Glu Val Met Phe Asp Pro
145 150 155 160 Ser Lys Asp Val Leu Ile Asn Glu Leu Leu Asp Gln Val
Thr Leu Gly 165 170 175 Arg Glu Gly Gln Ile Ile Gln Arg Ser Asn Ile
Ser Thr Ala Ile Lys 180 185 190 Ser Leu Val Ala Leu Gly Ile Asp Pro
Gln Asp Leu Lys Lys Leu Asn 195 200 205 Leu Asn Val Tyr Ile Gln Val
Phe Glu Lys Pro Phe Leu Lys Lys Thr 210 215 220 Gln Glu Tyr Tyr Thr
Gln Tyr Thr Asn Asp Tyr Leu Glu Lys His Ser 225 230 235 240 Val Thr
Glu Tyr Ile Phe Glu Ala His Glu Ile Ile Lys Arg Glu Glu 245 250 255
Lys Ala Met Thr Ile Tyr Trp Asp Asp His Thr Lys Lys Pro Leu Ser 260
265 270 28 194 PRT Saccharomyces cerevisiae 28 Met Val Thr Ser Asn
Val Val Leu Val Ser Gly Glu Gly Glu Arg Phe 1 5 10 15 Thr Val Asp
Lys Lys Ile Ala Glu Arg Ser Leu Leu Leu Lys Asn Tyr 20 25 30 Leu
Asn Asp Met His Asp Ser Asn Leu Gln Asn Asn Ser Asp Ser Asp 35 40
45 Ser Asp Ser Asp Ser Glu Thr Asn His Lys Ser Lys Asp Asn Asn Asn
50 55 60 Gly Asp Asp Asp Asp Glu Asp Asp Asp Glu Ile Val Met Pro
Val Pro 65 70 75 80 Asn Val Arg Ser Ser Val Leu Gln Lys Val Ile Glu
Trp Ala Glu His 85 90 95 His Arg Asp Ser Asn Phe Pro Asp Glu Asp
Asp Asp Asp Ser Arg Lys 100 105 110 Ser Ala Pro Val Asp Ser Trp Asp
Arg Glu Phe Leu Lys Val Asp Gln 115 120 125 Glu Met Leu Tyr Glu Ile
Ile Leu Ala Ala Asn Tyr Leu Asn Ile Lys 130 135 140 Pro Leu Leu Asp
Ala Gly Cys Lys Val Val Ala Glu Met Ile Arg Gly 145 150 155 160 Arg
Ser Pro Glu Glu Ile Arg Arg Thr Phe Asn Ile Val Asn Asp Phe 165 170
175 Thr Pro Glu Glu Glu Ala Ala Ile Arg Arg Glu Asn Glu Trp Ala Glu
180 185 190 Asp Arg 29 779 PRT Saccharomyces cerevisiae 29 Met Gly
Ser Phe Pro Leu Ala Glu Phe Pro Leu Arg Asp Ile Pro Val 1 5 10 15
Pro Tyr Ser Tyr Arg Val Ser Gly Gly Ile Ala Ser Ser Gly Ser Val 20
25 30 Thr Ala Leu Val Thr Ala Ala Gly Thr His Arg Asn Ser Ser Thr
Ala 35 40 45 Lys Thr Val Glu Thr Glu Asp Gly Glu Glu Asp Ile Asp
Glu Tyr Gln 50 55 60 Arg Lys Arg Ala Ala Gly Ser Gly Glu Ser Thr
Pro Glu Arg Ser Asp 65 70 75 80 Phe Lys Arg Val Lys His Asp Asn His
Lys Thr Leu His Pro Val Asn 85 90 95 Leu Gln Asn Thr Gly Ala Ala
Ser Val Asp Asn Asp Gly Leu His Asn 100 105 110 Leu Thr Asp Ile Ser
Asn Asp Ala Glu Lys Leu Leu Met Ser Val Asp 115 120 125 Asp Gly Ser
Ala Ala Pro Ser Thr Leu Ser Val Asn Met Gly Val Ala 130 135 140 Ser
His Asn Val Ala Ala Pro Thr Thr Val Asn Ala Ala Thr Ile Thr 145 150
155 160 Gly Ser Asp Val Ser Asn Asn Val Asn Ser Ala Thr Ile Asn Asn
Pro 165 170 175 Met Glu Glu Gly Ala Leu Pro Leu Ser Pro Thr Ala Ser
Ser Pro Gly 180 185 190 Thr Thr Thr Pro Leu Ala Lys Thr Thr Lys Thr
Ile Asn Asn Asn Asn 195 200 205 Asn Ile Ala Asp Leu Ile Glu Ser Lys
Asp Ser Ile Ile Ser Pro Glu 210 215 220 Tyr Leu Ser Asp Glu Ile Phe
Ser Ala Ile Asn Asn Asn Leu Pro His 225 230 235 240 Ala Tyr Phe Lys
Asn Leu Leu Phe Arg Leu Val Ala Asn Met Asp Arg 245 250 255 Ser Glu
Leu Ser Asp Leu Gly Thr Leu Ile Lys Asp Asn Leu Lys Arg 260 265 270
Asp Leu Ile Thr Ser Leu Pro Phe Glu Ile Ser Leu Lys Ile Phe Asn 275
280 285 Tyr Leu Gln Phe Glu Asp Ile Ile Asn Ser Leu Gly Val Ser Gln
Asn 290 295 300 Trp Asn Lys Ile Ile Arg Lys Ser Thr Ser Leu Trp Lys
Lys Leu Leu 305 310 315 320 Ile Ser Glu Asn Phe Val Ser Pro Lys Gly
Phe Asn Ser Leu Asn Leu 325 330 335 Lys Leu Ser Gln Lys Tyr Pro Lys
Leu Ser Gln Gln Asp Arg Leu Arg 340 345 350 Leu Ser Phe Leu Glu Asn
Ile Phe Ile Leu Lys Asn Trp Tyr Asn Pro 355 360 365 Lys Phe Val Pro
Gln Arg Thr Thr Leu Arg Gly His Met Thr Ser Val 370 375 380 Ile Thr
Cys Leu Gln Phe Glu Asp Asn Tyr Val Ile Thr Gly Ala Asp 385 390 395
400 Asp Lys Met Ile Arg Val Tyr Asp Ser Ile Asn Lys Lys Phe Leu Leu
405 410 415 Gln Leu Ser Gly His Asp Gly Gly Val Trp Ala Leu Lys Tyr
Ala His 420 425 430 Gly Gly Ile Leu Val Ser Gly Ser Thr Asp Arg Thr
Val Arg Val Trp 435 440 445 Asp Ile Lys Lys Gly Cys Cys Thr His Val
Phe Lys Gly His Asn Ser 450 455 460 Thr Val Arg Cys Leu Asp Ile Val
Glu Tyr Lys Asn Ile Lys Tyr Ile 465 470 475 480 Val Thr Gly Ser Arg
Asp Asn Thr Leu His Val Trp Lys Leu Pro Lys 485 490 495 Glu Ser Ser
Val Pro Asp His Gly Glu Glu His Asp Tyr Pro Leu Val 500 505 510 Phe
His Thr Pro Glu Glu Asn Pro Tyr Phe Val Gly Val Leu Arg Gly 515 520
525 His Met Ala Ser Val Arg Thr Val Ser Gly His Gly Asn Ile Val Val
530 535 540 Ser Gly Ser Tyr Asp Asn Thr Leu Ile Val Trp Asp Val Ala
Gln Met 545 550 555 560 Lys Cys Leu Tyr Ile Leu Ser Gly His Thr Asp
Arg Ile Tyr Ser Thr 565 570 575 Ile Tyr Asp His Glu Arg Lys Arg Cys
Ile Ser Ala Ser Met Asp Thr 580 585 590 Thr Ile Arg Ile Trp Asp Leu
Glu Asn Ile Trp Asn Asn Gly Glu Cys 595 600 605 Ser Tyr Ala Thr Asn
Ser Ala Ser Pro Cys Ala Lys Ile Leu Gly Ala 610 615 620 Met Tyr Thr
Leu Gln Gly His Thr Ala Leu Val Gly Leu Leu Arg Leu 625 630 635 640
Ser Asp Lys Phe Leu Val Ser Ala Ala Ala Asp Gly Ser Ile Arg Gly 645
650 655 Trp Asp Ala Asn Asp Tyr Ser Arg Lys Phe Ser Tyr His His Thr
Asn 660 665 670 Leu Ser Ala Ile Thr Thr Phe Tyr Val Ser Asp Asn Ile
Leu Val Ser 675 680 685 Gly Ser Glu Asn Gln Phe Asn Ile Tyr Asn Leu
Arg Ser Gly Lys Leu 690 695 700 Val His Ala Asn Ile Leu Lys Asp Ala
Asp Gln Ile Trp Ser Val Asn 705 710 715 720 Phe Lys Gly Lys Thr Leu
Val Ala Ala Val Glu Lys Asp Gly Gln Ser 725 730 735 Phe Leu Glu Ile
Leu Asp Phe Ser Lys Ala Ser Lys Ile Asn Tyr Val 740 745 750 Ser Asn
Pro Val Asn Ser Ser Ser Ser Ser Leu Glu Ser Ile Ser Thr 755 760 765
Ser Leu Gly Leu Thr Arg Thr Thr Ile Ile Pro 770 775 30 640 PRT
Saccharomyces cerevisiae 30 Met Arg Arg Glu Arg Gln Arg Met Met Ser
Phe Glu Asp Lys Asp Lys 1 5 10 15 Asp Asp Leu Asp Asn Ser Asn Ser
Asn Asn Ser Ser Glu Met Thr Asp 20 25 30 Thr Ala Met Met Pro Pro
Leu Lys Arg Leu Leu Ile Thr Gly Ser Ser 35 40 45 Asp Asp Leu Ala
Gln Gly Ser Ser Gly Lys Lys Lys Met Thr Met Ala 50 55 60 Thr Arg
Ser Pro Ser Ser Ser Pro Asp Leu Ala Thr Asn Asp Ser Gly 65 70 75 80
Thr Arg Val Gln Pro Leu Pro Glu Tyr Asn Phe Thr Lys Phe Cys Tyr 85
90 95 Arg His Asn Pro Asp Ile Gln Phe Ser Pro Thr His Thr Ala Cys
Tyr 100 105 110 Lys Gln Asp Leu Lys Arg Thr Gln Glu Ile Asn Ala Asn
Ile Ala Lys 115 120 125 Leu Pro Leu Gln Glu Gln Ser Asp Ile His His
Ile Ile Ser Lys Tyr 130 135 140 Ser Asn Ser Asn Asp Lys Ile Arg Lys
Leu Ile Leu Asp Gly Ile Leu 145 150 155 160 Ser Thr Ser Cys Phe Pro
Gln Leu Ser Tyr Ile Ser Ser Leu Val Thr 165 170 175 His Met Ile Lys
Ile Asp Phe Ile Ser Ile Leu Pro Gln Glu Leu Ser 180 185 190 Leu Lys
Ile Leu Ser Tyr Leu Asp Cys Gln Ser Leu Cys Asn Ala Thr 195 200 205
Arg Val Cys Arg Lys Trp Gln Lys Leu Ala Asp Asp Asp Arg Val Trp 210
215 220 Tyr His Met Cys Glu Gln His Ile Asp Arg Lys Cys Pro Asn Cys
Gly 225 230 235 240 Trp Gly Leu Pro Leu Leu His Met Lys Arg Ala Arg
Ile Gln Gln Asn 245 250 255 Ser Thr Gly Ser Ser Ser Asn Ala Asp Ile
Gln Thr Gln Thr Thr Arg 260 265 270 Pro Trp Lys Val Ile Tyr Arg Glu
Arg Phe Lys Val Glu Ser Asn Trp 275 280 285 Arg Lys Gly His Cys Arg
Ile Gln Glu Phe Lys Gly His Met Asp Gly 290 295 300 Val Leu Thr Leu
Gln Phe Asn Tyr Arg Leu Leu Phe Thr Gly Ser Tyr 305 310 315 320 Asp
Ser Thr Ile Gly Ile Trp Asp Leu Phe Thr Gly Lys Leu Ile Arg 325 330
335 Arg Leu Ser Gly His Ser Asp Gly Val Lys Thr Leu Tyr Phe Asp Asp
340 345 350 Arg Lys Leu Ile Thr Gly Ser Leu Asp Lys Thr Ile Arg Val
Trp Asn 355 360 365 Tyr Ile Thr Gly Glu Cys Ile Ser Thr Tyr Arg Gly
His Ser Asp Ser 370 375 380 Val Leu Ser Val Asp Ser Tyr Gln Lys Val
Ile Val Ser Gly Ser Ala 385 390 395 400 Asp Lys Thr Val Lys Val Trp
His Val Glu Ser Arg Thr Cys Tyr Thr 405 410 415 Leu Arg Gly His Thr
Glu Trp Val Asn Cys Val Lys Leu His Pro Lys 420 425 430 Ser Phe Ser
Cys Phe Ser Cys Ser Asp Asp Thr Thr Ile Arg Met Trp 435 440 445 Asp
Ile Arg Thr Asn Ser Cys Leu Lys Val Phe Arg Gly His Val Gly 450 455
460 Gln Val Gln Lys Ile Ile Pro Leu Thr Ile Lys Asp Val Glu Asn Leu
465 470 475 480 Ala Thr Asp Asn Thr Ser Asp Gly Ser Ser Pro Gln Asp
Asp Pro Thr 485 490 495 Met Thr Asp Gly Ala Asp Glu Ser Asp Thr Pro
Ser Asn Glu Gln Glu 500 505 510 Thr Val Leu Asp Glu Asn Ile Pro Tyr
Pro Thr His Leu Leu Ser Cys 515 520 525 Gly Leu Asp Asn Thr Ile Lys
Leu Trp Asp Val Lys Thr Gly Lys Cys 530 535 540 Ile Arg Thr Gln Phe
Gly His Val Glu Gly Val Trp Asp Ile Ala Ala 545 550 555 560 Asp Asn
Phe Arg Ile Ile Ser Gly Ser His Asp Gly Ser Ile Lys Val 565 570 575
Trp Asp Leu Gln Ser Gly Lys Cys Met His Thr Phe Asn Gly Arg Arg 580
585 590 Leu Gln Arg Glu Thr Gln His Thr Gln Thr Gln Ser Leu Gly Asp
Lys 595 600 605 Val Ala Pro Ile Ala Cys Val Cys Ile Gly Asp Ser Glu
Cys Phe Ser 610 615 620 Gly Asp Glu Phe Gly Cys Val Lys Met Tyr Lys
Phe Asp Leu Asn Asp 625 630 635 640 31 1151 PRT Saccharomyces
cerevisiae 31 Met Asp Gln Asp Asn Asn Asn His Asn Asp Ser Asn Arg
Leu His Pro 1 5 10 15 Pro Asp Ile His Pro Asn Leu Gly Pro Gln Leu
Trp Leu Asn Ser Ser 20 25 30 Gly Asp Phe Asp
Asp Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn 35 40 45 Asn Ser
Thr Arg Pro Gln Met Pro Ser Arg Thr Arg Glu Thr Ala Thr 50 55 60
Ser Glu Arg Asn Ala Ser Glu Val Arg Asp Ala Thr Leu Asn Asn Ile 65
70 75 80 Phe Arg Phe Asp Ser Ile Gln Arg Glu Thr Leu Leu Pro Thr
Asn Asn 85 90 95 Gly Gln Pro Leu Asn Gln Asn Phe Ser Leu Thr Phe
Gln Pro Gln Gln 100 105 110 Gln Thr Asn Ala Leu Asn Gly Ile Asp Ile
Asn Thr Val Asn Thr Asn 115 120 125 Leu Met Asn Gly Val Asn Val Gln
Ile Asp Gln Leu Asn Arg Leu Leu 130 135 140 Pro Asn Leu Pro Glu Glu
Glu Arg Lys Gln Ile His Glu Phe Lys Leu 145 150 155 160 Ile Val Gly
Lys Lys Ile Gln Glu Phe Leu Val Val Ile Glu Lys Arg 165 170 175 Arg
Lys Lys Ile Leu Asn Glu Ile Glu Leu Asp Asn Leu Lys Leu Lys 180 185
190 Glu Leu Arg Ile Asp Asn Ser Pro Gln Ala Ile Ser Tyr Leu His Lys
195 200 205 Leu Gln Arg Met Arg Leu Arg Ala Leu Glu Thr Glu Asn Met
Glu Ile 210 215 220 Arg Asn Leu Arg Leu Lys Ile Leu Thr Ile Ile Glu
Glu Tyr Lys Lys 225 230 235 240 Ser Leu Tyr Ala Tyr Cys His Ser Lys
Leu Arg Gly Gln Gln Val Glu 245 250 255 Asn Pro Thr Asp Asn Phe Ile
Ile Trp Ile Asn Ser Ile Asp Thr Thr 260 265 270 Glu Ser Ser Asp Leu
Lys Glu Gly Leu Gln Asp Leu Ser Arg Tyr Ser 275 280 285 Arg Gln Phe
Ile Asn Asn Val Leu Ser Asn Pro Ser Asn Gln Asn Ile 290 295 300 Cys
Thr Ser Val Thr Arg Arg Ser Pro Val Phe Ala Leu Asn Met Leu 305 310
315 320 Pro Ser Glu Ile Leu His Leu Ile Leu Asp Lys Leu Asn Gln Lys
Tyr 325 330 335 Asp Ile Val Lys Phe Leu Thr Val Ser Lys Leu Trp Ala
Glu Ile Ile 340 345 350 Val Lys Ile Leu Tyr Tyr Arg Pro His Ile Asn
Lys Lys Ser Gln Leu 355 360 365 Asp Leu Phe Leu Arg Thr Met Lys Leu
Thr Ser Glu Glu Thr Val Phe 370 375 380 Asn Tyr Arg Leu Met Ile Lys
Arg Leu Asn Phe Ser Phe Val Gly Asp 385 390 395 400 Tyr Met His Asp
Thr Glu Leu Asn Tyr Phe Val Gly Cys Lys Asn Leu 405 410 415 Glu Arg
Leu Thr Leu Val Phe Cys Lys His Ile Thr Ser Val Pro Ile 420 425 430
Ser Ala Val Leu Arg Gly Cys Lys Phe Leu Gln Ser Val Asp Ile Thr 435
440 445 Gly Ile Arg Asp Val Ser Asp Asp Val Phe Asp Thr Leu Ala Thr
Tyr 450 455 460 Cys Pro Arg Val Gln Gly Phe Tyr Val Pro Gln Ala Arg
Asn Val Thr 465 470 475 480 Phe Asp Ser Leu Arg Asn Phe Ile Val His
Ser Pro Met Leu Lys Arg 485 490 495 Ile Lys Ile Thr Ala Asn Asn Asn
Met Asn Asp Glu Leu Val Glu Leu 500 505 510 Leu Ala Asn Lys Cys Pro
Leu Leu Val Glu Val Asp Ile Thr Leu Ser 515 520 525 Pro Asn Val Thr
Asp Ser Ser Leu Leu Lys Leu Leu Thr Arg Leu Val 530 535 540 Gln Leu
Arg Glu Phe Arg Ile Thr His Asn Thr Asn Ile Thr Asp Asn 545 550 555
560 Leu Phe Gln Glu Leu Ser Lys Val Val Asp Asp Met Pro Ser Leu Arg
565 570 575 Leu Ile Asp Leu Ser Gly Cys Glu Asn Ile Thr Asp Lys Thr
Ile Glu 580 585 590 Ser Ile Val Asn Leu Ala Pro Lys Leu Arg Asn Val
Phe Leu Gly Lys 595 600 605 Cys Ser Arg Ile Thr Asp Ala Ser Leu Phe
Gln Leu Ser Lys Leu Gly 610 615 620 Lys Asn Leu Gln Thr Val His Phe
Gly His Cys Phe Asn Ile Thr Asp 625 630 635 640 Asn Gly Val Arg Ala
Leu Phe His Ser Cys Thr Arg Ile Gln Tyr Val 645 650 655 Asp Phe Ala
Cys Cys Thr Asn Leu Thr Asn Arg Thr Leu Tyr Glu Leu 660 665 670 Ala
Asp Leu Pro Lys Leu Lys Arg Ile Gly Leu Val Lys Cys Thr Gln 675 680
685 Met Thr Asp Glu Gly Leu Leu Asn Met Val Ser Leu Arg Gly Arg Asn
690 695 700 Asp Thr Leu Glu Arg Val His Leu Ser Tyr Cys Ser Asn Leu
Thr Ile 705 710 715 720 Tyr Pro Ile Tyr Glu Leu Leu Met Ser Cys Pro
Arg Leu Ser His Leu 725 730 735 Ser Leu Thr Ala Val Pro Ser Phe Leu
Arg Pro Asp Ile Thr Met Tyr 740 745 750 Cys Arg Pro Ala Pro Ser Asp
Phe Ser Glu Asn Gln Arg Gln Ile Phe 755 760 765 Cys Val Phe Ser Gly
Lys Gly Val His Lys Leu Arg His Tyr Leu Val 770 775 780 Asn Leu Thr
Ser Pro Ala Phe Gly Pro His Val Asp Val Asn Asp Val 785 790 795 800
Leu Thr Lys Tyr Ile Arg Ser Lys Asn Leu Ile Phe Asn Gly Glu Thr 805
810 815 Leu Glu Asp Ala Leu Arg Arg Ile Ile Thr Asp Leu Asn Gln Asp
Ser 820 825 830 Ala Ala Ile Ile Ala Ala Thr Gly Leu Asn Gln Ile Asn
Gly Leu Asn 835 840 845 Asn Asp Phe Leu Phe Gln Asn Ile Asn Phe Glu
Arg Ile Asp Glu Val 850 855 860 Phe Ser Trp Tyr Leu Asn Thr Phe Asp
Gly Ile Arg Met Ser Ser Glu 865 870 875 880 Glu Val Asn Ser Leu Leu
Leu Gln Val Asn Lys Thr Phe Cys Glu Asp 885 890 895 Pro Phe Ser Asp
Val Asp Asp Asp Gln Asp Tyr Val Val Ala Pro Gly 900 905 910 Val Asn
Arg Glu Ile Asn Ser Glu Met Cys His Ile Val Arg Lys Phe 915 920 925
His Glu Leu Asn Asp His Ile Asp Asp Phe Glu Val Asn Val Ala Ser 930
935 940 Leu Val Arg Val Gln Phe Gln Phe Thr Gly Phe Leu Leu His Glu
Met 945 950 955 960 Thr Gln Thr Tyr Met Gln Met Ile Glu Leu Asn Arg
Gln Ile Cys Leu 965 970 975 Val Gln Lys Thr Val Gln Glu Ser Gly Asn
Ile Asp Tyr Gln Lys Gly 980 985 990 Leu Leu Ile Trp Arg Leu Leu Phe
Ile Asp Lys Phe Ile Met Val Val 995 1000 1005 Gln Lys Tyr Lys Leu
Ser Thr Val Val Leu Arg Leu Tyr Leu Lys Asp 1010 1015 1020 Asn Ile
Thr Leu Leu Thr Arg Gln Arg Glu Leu Leu Ile Ala His Gln 1025 1030
1035 1040 Arg Ser Ala Trp Asn Asn Asn Asn Asp Asn Asp Ala Asn Arg
Asn Ala 1045 1050 1055 Asn Asn Ile Val Asn Ile Val Ser Asp Ala Gly
Ala Asn Asp Thr Ser 1060 1065 1070 Asn Asn Glu Thr Asn Asn Gly Asn
Asp Asp Asn Glu Thr Glu Asn Pro 1075 1080 1085 Asn Phe Trp Arg Gln
Phe Gly Asn Arg Met Gln Ile Ser Pro Asp Gln 1090 1095 1100 Met Arg
Asn Leu Gln Met Gly Leu Arg Asn Gln Asn Met Val Arg Asn 1105 1110
1115 1120 Asn Asn Asn Asn Thr Ile Asp Glu Ser Met Pro Asp Thr Ala
Ile Asp 1125 1130 1135 Ser Gln Met Asp Glu Ala Ser Gly Thr Pro Asp
Glu Asp Met Leu 1140 1145 1150 32 22 PRT Saccharomyces cerevisiae
32 Ile Leu Ser Pro Thr Met Tyr Met Glu Val Tyr Thr Ala Ile Tyr Asn
1 5 10 15 Tyr Cys Val Asn Lys Ser 20 33 22 PRT Saccharomyces
cerevisiae 33 Asn Met Ala Pro Lys Asp Tyr Met Thr Leu Tyr Thr Ser
Val Tyr Asp 1 5 10 15 Tyr Cys Thr Ser Ile Thr 20 34 22 PRT
Saccharomyces cerevisiae 34 His Met Ser Lys Lys Tyr Tyr Met Met Leu
Tyr Asp Ala Val Tyr Asn 1 5 10 15 Ile Cys Thr Thr Thr Thr 20 35 22
PRT Saccharomyces cerevisiae 35 Ser Leu Thr Arg Ser Gln Tyr Met Arg
Phe Tyr Thr His Val Tyr Asp 1 5 10 15 Tyr Cys Thr Ser Val Ser 20 36
22 PRT Saccharomyces cerevisiae 36 Ser Met Ala Lys Ser Arg Tyr Met
Glu Leu Tyr Thr His Val Tyr Asn 1 5 10 15 Tyr Cys Thr Ser Val His
20 37 22 PRT Saccharomyces cerevisiae 37 Ala Phe Asp Ser Glu Gln
Tyr Met Met Leu Tyr Thr Thr Ile Tyr Asn 1 5 10 15 Met Cys Thr Gln
Lys Pro 20 38 22 PRT Saccharomyces cerevisiae 38 Gly Met Thr Ile
Thr Lys Tyr Met Glu Leu Tyr Thr Ala Ile His Asn 1 5 10 15 Tyr Cys
Ala Asp Ala Ser 20 39 22 PRT Saccharomyces cerevisiae 39 Leu Gly
Leu Lys Thr Gly Tyr Gln Glu Leu Tyr Ser Gly Val Glu Asn 1 5 10 15
Leu Thr Arg Ala Asp Gln 20 40 22 PRT Saccharomyces cerevisiae 40
Pro Ile Thr Asn Val Gln Trp His His Lys Phe Ser Asp Val Tyr Asp 1 5
10 15 Ile Cys Val Ser Ile Pro 20 41 22 PRT Saccharomyces cerevisiae
41 Tyr Val Glu Arg Ala Thr Trp Asn Asp Arg Phe Ser Asp Ile Tyr Ala
1 5 10 15 Leu Cys Val Ala Tyr Pro 20 42 22 PRT Saccharomyces
cerevisiae 42 Gln Tyr Val Thr Gln Thr Trp Glu Leu Leu Lys Arg Ala
Ile Gln Glu 1 5 10 15 Ile Gln Arg Lys Asn Asn 20 43 22 PRT
Saccharomyces cerevisiae 43 Gly Ser Val Gly Arg Asp Trp Ala Val Leu
Ser Asp Asn Val Phe Ala 1 5 10 15 Ile Leu Glu Asp Arg Lys 20 44 22
PRT Saccharomyces cerevisiae 44 Ser Val Thr Pro Ala Ala Trp Gln Asp
Leu Phe Tyr His Val Tyr Lys 1 5 10 15 Ile Thr Ser Trp Val Asp 20 45
22 PRT Saccharomyces cerevisiae 45 Ser Val Thr Lys Gln Gln Trp Phe
Asp Leu Phe Ser Asp Val His Ala 1 5 10 15 Val Cys Leu Trp Asp Asp
20 46 21 PRT Saccharomyces cerevisiae 46 Thr Ser Gln Leu Ser Phe
Glu Glu Leu Tyr Arg Asn Ala Tyr Ile Leu 1 5 10 15 Val Leu His Lys
Tyr 20 47 21 PRT Saccharomyces cerevisiae 47 Met Ala Asp Leu Ser
Phe Glu Gln Val Tyr Lys Thr Ile Tyr Thr Ile 1 5 10 15 Val Leu Asn
Lys Lys 20 48 14 PRT Saccharomyces cerevisiae UNSURE (1) Xaa = one
to ten amino acids 48 Xaa Tyr Met Xaa Xaa Tyr Xaa Xaa Xaa Tyr Xaa
Xaa Cys Xaa 1 5 10 49 19 PRT Saccharomyces cerevisiae UNSURE (1)
Xaa = Ile, Asn, His, Ser or Ala 49 Xaa Xaa Xaa Xaa Xaa Xaa Tyr Met
Xaa Xaa Tyr Xaa Xaa Xaa Tyr Xaa 1 5 10 15 Xaa Cys Xaa 50 9 PRT
Saccharomyces cerevisiae UNSURE (3) Xaa = Met Arg Thr or Glu 50 Tyr
Met Xaa Xaa Tyr Xaa Xaa Xaa Tyr 1 5
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