From the Department of Biochemistry and Molecular Biology, Indiana
University School of Medicine and the Walther Oncology Center,
Indianapolis, Indiana, 46202-5122
 |
INTRODUCTION |
Ubiquitin (Ub)1 is a
highly conserved 76-amino acid protein that is found as a
post-translational modification of other proteins. The best
characterized role of Ub modification is as a means of regulating
protein abundance. When a Ub chain forms on a particular substrate
protein, that protein is rapidly degraded by the proteasome (for
reviews, see Refs. 1-6). Poly-Ub chain formation on a substrate is
catalyzed by a member of the ubiquitin conjugating (Ubc) family of
proteins, also known as E2 enzymes. Prior to poly-Ub chain formation,
Ubc (E2) enzymes are charged with ubiquitin by a ubiquitin activating,
or E1, enzyme which has the ability to capture free Ub. In some cases,
the transfer of ubiquitin from an Ubc (E2) enzyme to a substrate
proceeds via a third protein known as a ubiquitin ligase, or E3 (7, 8).
Based on sequence similarity, there are 13 members of the Ubc (E2)
family in yeast and these proteins have diverse roles including cell
cycle progression, heavy metal resistance, peroxisome biogenesis, and
DNA repair. Members of this family have highly related catalytic
domains which receive ubiquitin from an E1 and pass it directly, or via
an E3, on to a substrate. The noncatalytic carboxyl termini of Ubc
enzymes are more variable and are found on only some members of the
family. The function of this region is unknown, however, it may be
required for substrate recognition or regulation of Ubc activity.
Cdc34p, a member of the Ubc family of proteins, is essential for cell
cycle progression and primarily acts at the G1 to S-phase transition (9). Cell cycle progression in yeast is coordinated by the
protein kinase, Cdc28p, and the activity of this kinase requires its
association with another protein termed cyclin (for review, see Refs.
10-12). It is thought that the cyclin will determine substrate
specificity for the kinase or direct the kinase to particular locations
in the cell. In yeast nine cyclins have been characterized for Cdc28p
and different cyclins associate with the Cdc28p kinase during different
phases of the cell cycle. For example, G1 progression is
coordinated by the cyclins Cln1p, Cln2p, and Cln3p that bind and
activate Cdc28p. To initiate DNA replication, or S-phase, Cdc28p
associates with cyclins Clb5p or Clb6p. Cyclin kinase activity can be
negatively regulated by the physical association of an inhibitor
(13-15). One such inhibitor in yeast is Sic1p and this protein
negatively regulates the activity of Cdc28p-Clb5p and Cdc28p-Clb6p
complexes (16-19). Only upon Sic1p degradation are these complexes
active and S-phase initiated.
Sic1p is one candidate substrate for Cdc34p-mediated ubiquitin
degradation (19). Cells that lack CDC34 activity are unable to remove Sic1p and thus arrest without initiating DNA replication. This phenotype is shared by cells that lack CDC4 and
CDC53 activity. Although their activity is required for
Sic1p ubiquitination (19), the precise function of these proteins is
unknown. CDC53 encodes a protein that defines a family of
evolutionary conserved proteins which may serve to recognize substrates
that have been targeted for ubiquitination (20-22). CDC4
encodes a protein containing 7 WD-40 repeats, a motif that has been
implicated in protein-protein interactions (23, 24). Furthermore, Cdc4p
also contains an F-Box, a domain that is the binding site for Skp1p
(25). Skp1p is required for the degradation of Sic1p, Cln2p, and Clb5p
(25). It has been speculated that a complex containing these proteins is required for the degradation of Sic1p and initiation of S-phase (26). Here, we define a region on Cdc34p that is necessary and sufficient for its binding Cdc4p and Cdc53p. We report that Cdc4p, Cdc34p, and Cdc53p form a high molecular weight complex and that this
association is required for Cdc34p function. We also show that these
three proteins are not only present throughout the cell cycle but that
Cdc34p is part of a complex during this process.
 |
EXPERIMENTAL PROCEDURES |
Yeast Strains and Growth Conditions--
Yeast strains used in
this study are as follows; YL10-1 MATa cdc34-2
ura3-52 leu2
1 trp1
63 his3
(27) and
Y382 MAT
ade2 ade3 ura3 leu2 trp1 (kindly
provided by A. Bender). Standard rich (YPD) and defined minimal (SD)
were prepared as described previously (28). Transformations were
carried out as described previously (29). For plasmid selection, yeast
cells were grown in defined minimal medium supplemented with the
appropriate amino acids. For galactose induction, cells were first
grown to early logarithmic phase in minimal media containing sucrose
instead of dextrose and then transferred to minimal media containing
galactose as the sole carbon source and grown until late logarithmic
phase. For complementation experiments, patches derived from single
transformants were grown under permissive conditions of 23 °C and
then replica plated and incubated under nonpermissive conditions of
37 °C. For chemical arrests, cells in early logarithmic phase were
treated for 3-4 h with either 5 µM
-factor or 100 mM hydroxyurea or 10 µg/ml nocodazole. Arrest was
confirmed by microscopic examination. To obtain temperature
sensitive-dependent arrest, cells were grown to early
logarithmic phase at the permissive temperature of 23 °C and shifted
to the nonpermissive temperature of 37 °C for 3-4 h. Again, cell
arrest was confirmed by microscopic examination.
Plasmid Constructions--
Escherichia coli DH5
was used to propagate plasmids. Plasmid manipulations used standard
protocols (30). Plasmids pSJ4101, YEp34-1, and pYcDE53-1 have been
previously described (21). In pSJ4101, CDC4 is
expressed from the GAL10 promoter whereas pYcDE53-1
has CDC53 expressed from the ADH1 promoter.
The vector pEG(KG) was used for the expression of glutathione
S-transferase (GST) fusion proteins (31). Expression of the GST fusion proteins were from the GAL1-10 promoter. All
GST-CDC34 fusion constructs were created by cloning polymerase chain
reaction-generated DNA fragments using plasmid-borne CDC34
DNA as template using methods as described previously (27). The use of
the primers annealing at the 5'-end of CDC34 were designed
to incorporate a XbaI restriction site and the use of
primers annealing at the 3'-end of CDC34 were designed to
incorporate a SalI restriction site. The polymerase chain
reaction products were cleaved with these enzymes and ligated into
pEG(KG) that had previously been digested with the same enzymes. The
primers annealing to the 3'-end of CDC34 also contained two
successive stop codons. To generate full-length CDC34 we
used primers 5'-ATTCTAGACTCCATGAGTAGTCGCAAAAGCACCGC-3' and
5'-GGGGTCGACTTATTACTTTGAAACTCTTTCTACATCCTCCAC-3'. To generate a
fragment encoding residues 1-170 we used primers
5'-ATTCTAGACTCCATGAGTAGTCGCAAAAGCACCGC-3' and
5'-GGGGTCGACTTATTAATCTTGTTTCGATCTTTCCAC-3'. To generate a fragment
encoding residues 171-295 we used primers
5'-ATTCTAGACTCCGATATCCCTAAAGGTTTCATAATGC-3' and
5'-GGGGTCGACTTATTACTTTGAAACTCTTTCTACATCCTCCAC-3'. To generate a
fragment encoding residues 171-209 we used primers
5'-ATTCTAGACTCCGATATCCCTAAAGGTTTCATAATGC-3' and
5'-GGGGTCGACTTATTACAAATCACTATCATACCAAAAATTATCCG-3'. To generate a
fragment encoding residues 1-209 we used a combination of primers mentioned above.
Protein Preparation and Western Immunoblot Techniques--
Yeast
lysate preparations and Western immunoblot techniques were carried out
as described previously (21), except that cells were lysed in the
presence of 50 mM NaCl, 50 mM Tris-HCl (pH
7.5), 0.5 mM EDTA, 0.1% Triton X-100. Antibody production
against Cdc4p, Cdc34p, and Cdc53p and antibody purification have been
previously described (21, 37). Antibodies raised against GST were
purchased from Sigma.
Purification of GST-tagged Cdc34p--
Yeast cell lysates
containing 10 mg of protein were incubated for 1 h with 100 µl
of 50% slurry of glutathione-agarose beads (Sigma) at 4 °C that had
been previously equilibrated with lysis buffer. The beads were then
washed repeatedly with 1 ml of lysis buffer. The protein bound to the
beads was then subjected to Western analysis.
Gel Filtration Chromatography--
Lysate from cells grown to
mid-logarithmic phase or arrested with nocodazole was prepared as
described previously (21). 0.5 mg of protein were applied at a flow
rate of 0.2 ml/min onto a Superose 12 HR10/30 column (FPLC, Pharmacia
Biotech Inc.) that had been previously equilibrated in 50 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1% Triton X-100. Protein from 400-µl fractions was precipitated with 10% trichloroacetic acid. The precipitate was washed with 80% acetone, resolubilized in 25 µl of
0.1 M NaOH and 5 µl of SDS-PAGE sample buffer solubilized
in 30 µl of SDS sample buffer, separated by SDS-PAGE, and subjected to Western immunoblot analysis. The column had been previously calibrated and a standard curve was produced. The proteins used to
calibrate the column were thyroglobulin (669 kDa), apoferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), chymotrypsin (25 kDa), and ribonuclease (13.7 kDa).
 |
RESULTS |
The Carboxyl Terminus of Cdc34p Is Required for Its in Vivo
Activity and Binding to Cdc4p and Cdc53p--
We previously
demonstrated that CDC4, CDC34, and
CDC53 genetically interact and that Cdc34p physically
associates with Cdc4p and Cdc53p (21). We set out to determine a
binding domain in Cdc34p required for its association with Cdc4p and
Cdc53p. The Ubc proteins each contain a highly conserved catalytic
domain. However, the catalytic domain of Cdc34p contains features that define a subclass of ubiquitin-conjugating catalytic domains (27). In
some Ubc proteins a carboxyl-terminal extension exists and is distinct
among the different members of the family. The highly acidic
carboxyl-terminal extension of Cdc34p is also unique in its sequence
composition and in that its length is greater than in other members of
the family. To determine whether the catalytic domain or the
carboxyl-terminal extension on Cdc34p is required for its association
with Cdc4p and Cdc53p, GST fusions were constructed encoding either
full-length Cdc34p or domains of Cdc34p composed of the catalytic
domain (amino acid residues 1-170) and the carboxyl terminus (amino
acid residues 171-295) (Fig.
1A). To assess the function of
these fusion proteins, and fusion proteins subsequently used in this
study, each construct as well as the empty GST vector was transformed
into strain YL10-1 which contains the temperature-sensitive cdc34-2 allele. Isolated transformants were transferred onto
medium lacking uracil, which selected for the presence of the plasmid, and then incubated at 23 °C, the permissive temperature for cells containing cdc34-2. These patches were replica plated to the
same media except that galactose was included to induce expression of
each GST fusion and were incubated at either 36 °C. The ability of
each GST fusion to rescue the temperature-sensitive phenotype of YL10-1
was assessed by the growth of each patch at 36 °C (Fig. 1B). The plasmid encoding full-length Cdc34p fused to GST
was able to complement the cdc34-2 mutation and allowed
YL10-1 cells to grow at the nonpermissive temperature. The tagged
Cdc34p had to be overproduced to complement the cdc34-2
allele, however, indicating that the NH2-terminal tag may
affect Cdc34p function. Low level expression of this construct,
achieved when cells are grown in the presence of dextrose, did not
complement the cdc34-2 allele whereas low level expression
of untagged Cdc34p is able to complement the cdc34-2 allele
(Ref. 27 and data not shown). Neither GST alone nor the GST fusion
constructs containing Cdc34p residues 1-170 or 171-295 were able to
complement the cdc34-2 mutation and failed to allow growth
of YL10-1 cells at the nonpermissive temperature. Microscopic
examination showed that these cells displayed the multielongated bud
phenotype typical of loss of CDC34 activity. These results
are in agreement with previous studies which have demonstrated that
Cdc34p residues 1-170 are necessary but not sufficient to maintain
growth in a cdc34 temperature-sensitive strain at 36 °C
(32-34).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Domains of Cdc34p. A, schematic
diagram illustrating the GST fusion constructs used in this study.
B, YL10-1 cells containing the cdc34-2
temperature-sensitive mutation were transformed with a plasmid that
allowed the expression of the indicated GST fusion in the presence of
galactose. Replica patches were then made and cells incubated at the
indicated temperature in the presence of galactose for 3 days.
|
|
To assess whether the GST fusion proteins could associate with Cdc4p
and Cdc53p in vivo, lysate prepared from cells expressing each construct was incubated with glutathione-Sepharose beads. Proteins
bound to beads were analyzed by SDS-PAGE followed by Western immunoblot
analysis with antibodies raised against GST, Cdc4p, or Cdc53p (Fig.
2A). The fusion of GST with
full-length Cdc34p allowed co-purification of both Cdc4p and Cdc53p
whereas GST alone did not. The fusion of GST with the Cdc34p catalytic domain (residues 1-170) did not co-purify with either Cdc4p or Cdc53p,
whereas the fusion of GST with the Cdc34p carboxyl-terminal extension
(residues 171-295) retained the ability to co-purify with both Cdc4p
and Cdc53p. Thus the binding domain required for Cdc34p to interact
with Cdc4p and Cdc53p in vivo is located within the
carboxyl-terminal portion of the protein. Since the region delineated
by residues 171-295 is required for the cell cycle function of Cdc34p
(Fig. 1B), our results suggest that the association of
Cdc4p/Cdc34p/Cdc53p is essential for Cdc34p activity.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2.
Cdc4p and Cdc53p both associate with cell
cycle determining residues 171-209 of Cdc34p. Lysate prepared
from cells producing the indicated GST-Cdc34p fusions was incubated in
the presence of glutathione beads. Proteins immobilized on the beads were subjected to Western immunoblot analysis with antibodies raised
against GST, Cdc4p, and Cdc53p, as indicated. A, Cdc4p and
Cdc53p both associate with the carboxyl terminus of Cdc34p. B, Cdc34p residues 1-209 and C, Cdc34p residues
171-209 bind Cdc4p and Cdc53p.
|
|
Residues 171-209 of Cdc34p Are Necessary and Sufficient for
Binding Both Cdc4p and Cdc53p--
We next set out to determine more
precisely the binding domain on Cdc34p required for its association
with Cdc4p and Cdc53p. Previous work has demonstrated that the portion
of CDC34 that is necessary and sufficient for its cell cycle
function is that portion encoding the catalytic domain and a domain of
the carboxyl-terminal extension, residues 1-209 (34). We wanted to
test whether these Cdc34p residues are sufficient to bind Cdc4p and
Cdc53p. We therefore constructed a GST fusion protein that contained
residues 1-209 of the Cdc34p protein (Fig. 1A). When this
protein is overproduced in YL10-1 cells, it is able to complement the
cdc34-2 allele (Fig. 1B) and allows growth of
YL10-1 cells at 36 °C, demonstrating that residues 1-209 are
sufficient for Cdc34p activity in vivo. Furthermore, the
GST-Cdc34(1-209)p fusion was able to co-precipitate Cdc4p and Cdc53p
(Fig. 2B). Therefore, the addition of 39 residues to the
catalytic domain of Cdc34p (residues 1-170), creating Cdc34(1-209)p, restores in vivo activity and the ability to associate with
Cdc4p and Cdc53p.
As described above, we have demonstrated that residues 1-170 of Cdc34p
have no in vivo activity and cannot bind Cdc4p and Cdc53p.
We wanted to test whether the Cdc34p residues 171-209 are sufficient
by themselves to bind Cdc4p and Cdc53p. A GST fusion construct encoding
these residues was made (Fig. 1A) and as shown in Fig.
2C this GST fusion protein is sufficient to bind Cdc4p and
Cdc53p selectively in vivo. Thus we have defined a 39-amino acid region on Cdc34p that is necessary for CDC34 activity
and sufficient for associating with Cdc4p and Cdc53p.
Cdc4p, Cdc34p, and Cdc53p Are Present within a High Molecular
Weight Species--
The above data and our previous work demonstrating
genetic interactions between CDC4, CDC34, and
CDC53 strongly suggest that Cdc34p forms a complex with
Cdc4p and Cdc53p in vivo. To test this model and to obtain
approximate size for this complex, yeast lysate was subjected to gel
filtration column chromatography. To allow separation of protein
complexes with high molecular mass, lysate was applied to a Superose-12
column that was calibrated up to 670 kDa. Fractions were collected and
the presence of Cdc4p, Cdc34p, and Cdc53p was detected by Western
blotting with the appropriate antibody. Densitometric scanning of these
results revealed that Cdc4p, Cdc34p, and Cdc53p were absent in
fractions corresponding to their monomeric size of 88, 40, and 96 kDa,
respectively. Instead, the detectable cellular pool of these proteins
was present in fractions corresponding to high molecular masses,
indicating that these proteins are part of multiprotein complexes (Fig.
3). However, Cdc4p, Cdc34p, and Cdc53p
did not co-elute precisely into the same fractions. Instead we observed
that Cdc4p, Cdc34p, and Cdc53p exist as overlapping families of high
molecular weight complexes and, by comparison to protein standards,
these fractions corresponded to molecular sizes of between 220 and 670 kDa peaking at about 400 kDa. Approximately 10% of the total amount of
Cdc4p and Cdc53 were detected in fractions that correspond to a
apparent molecular size of approximately 1,200 kDa. Since this value
was obtained by linear extrapolation from the size markers, 1,200 kDa
is an underestimate of its size. Furthermore, about 30% of Cdc4p was detected alone in fractions that correspond to a molecular size of
approximately 180 kDa. These data suggest that Cdc4p and Cdc53p associate with other as yet uncharacterized proteins.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Cdc4p, Cdc34p, and Cdc53p occur within
overlapping high molecular weight complexes upon Superose 12 gel-filtration column chromatography. Densitometric scanning
profiles of immunoblots of Cdc4p, Cdc34p, and Cdc53p obtained after
fractionation of yeast whole cell lysate on a Superose 12 column. The
subsequent fractions were concentrated and then subjected to Western
immunoblot analysis using antibodies raised against Cdc4p, Cdc34p and
Cdc53p. Size estimates, in kDa, were calculated based on the standard
curve produced from the molecular markers described under
"Experimental Procedures."
|
|
Cellular Cdc4p Levels Are Increased by Increased Amounts of
Cdc34p--
A number of studies have shown that the steady state level
of a protein that is a component of a multiprotein complex may be
enhanced by increasing the abundance of other members of the complex
(35, 36). We wanted to observe whether the steady state levels of
Cdc4p, Cdc34p, or Cdc53p were enhanced by increasing the level of one
of the other proteins. Plasmids that overexpress CDC4,
CDC34, and CDC53 have been previously described
(21). Overexpression of CDC4 was achieved by replacing its
own promoter with a galactose inducible promoter; the promoter of
CDC53 was replaced by the ADH promoter and
finally CDC34 overexpression was obtained from a high copy
plasmid. Y382 and Y382 transformants containing these plasmids were
grown under conditions to allow for CDC4 induction, protein
lysates were prepared and subjected to Western immunoblot analysis
using antibodies raised against Cdc4p, Cdc34p, or Cdc53p. Although
overproduction of each protein was achieved, the overproduction of one
protein did not result in an increase in the amount of the other
proteins (data not shown).
We next carried out a similar experiment but overproduced Cdc4p,
Cdc34p, and Cdc53p in different pairwise combinations and also all
three proteins together. We could control the timing of CDC4
overexpression since this gene was under the control of a
galactose-inducible promoter and is only expressed when cells are grown
in the presence of galactose. This allowed us to follow Cdc4p
accumulation as a time course with galactose being added at time point
0 and cells were harvested every 30 min. The abundance of Cdc34p and
Cdc53p was unaffected when these proteins were co-overproduced with
either of the other two proteins or all three proteins co-overproduced together (data not shown). However, we observed that Cdc4p abundance was increased when CDC4 was co-overexpressed with
CDC34 and increased even further when CDC4 was
co-overexpressed with both CDC34 and CDC53 (Fig.
4). The levels of Cdc4p were
significantly greater under these conditions than that observed when
CDC4 was either overexpressed alone or co-overexpressed with
CDC53. One interpretation of these results is that Cdc4p may
directly associate with Cdc34p and this association allows greater
stability of Cdc4p than when Cdc4p is not associated with Cdc34p. The
Cdc4p·Cdc34p heterodimer may associate with Cdc53p and this complex
results in the greatest stability of Cdc4p. This data also suggests
that perhaps Cdc4p does not associate with Cdc53p in the absence of
Cdc34p since we observed no increase in Cdc4p stability when
CDC4 was co-overexpressed with CDC53.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Steady state levels of Cdc4p are increased
when CDC4 is co-overexpressed with CDC34 and
CDC53. Y382 cells were transformed with different
combinations of plasmids that were capable of overexpressing CDC4, CDC34, and CDC53, as indicated.
Plasmid borne expression of CDC4 was induced in the presence
of galactose. Lysate prepared from cells at the times indicated were
subjected to Western immunoblot analysis using anti-Cdc4p antibodies.
The loading control is a protein that cross-reacts with the anti-Cdc4p
antibodies and its abundance is not affect by overexpressing
CDC4, CDC34, or CDC53.
|
|
Cdc4p, Cdc34p, and Cdc53p Complexes Are Present throughout the Cell
Cycle--
A feature of some cell cycle regulators is that their
abundance oscillates during cell cycle progression. For example, the cyclins Cln1p and Cln2p are present late in G1 and the CDK
inhibitor Sic1p is present throughout G1, yet both cyclins
and Sic1p are absent for the remainder of the cell cycle (11). Since
these proteins are strong candidates for Cdc34p substrates, we wanted to test whether Cdc34p containing complexes are present only during G1 or throughout the cell cycle.
First we set out to determine whether the levels of Cdc4p, Cdc34p, or
Cdc53p fluctuate throughout the cell cycle. Lysate from cells that had
been arrested at specific points of the cell cycle was prepared. We
employed the use of common cell cycle inhibitors for this process.
Logarithmically growing Y382 cells were incubated in the presence of
either
-mating factor, which arrests cells during G1;
hydroxyurea, which arrests cells during S-phase; or nocodazole which
arrests cells during mitosis. The presence of Cdc4p, Cdc34p, and Cdc53p
was tested by Western immunoblot analysis which showed that the level
of each protein was invariant throughout the cell cycle (Fig.
5). Both Cdc34p and Cdc53p are modified
by ubiquitin (22, 37) and in the case of Cdc53p, both the modified and
unmodified species are equally prevalent throughout the cell cycle
(Fig. 5). However, Cdc34p ubiquitination, which has been mapped to the
carboxyl-terminal (residues 254-295 (27)), appeared to be cell
cycle-specific (Fig. 5). Cdc34p is least ubiquitinated in cells
arrested with
-factor and most ubiquitinated in cells arrested with
nocadozole, although this modification appears to affect only a small
portion of the Cdc34p population.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 5.
Cdc4p, Cdc34p, and Cdc53p are present
throughout the cell cycle. Lysate prepared from cells either in
logarithmic phase or arrested with the indicated chemical were
subjected to Western immunoblot analysis with the indicated
antibody.
|
|
To test whether Cdc34p containing complexes are present throughout the
cell cycle, lysate prepared from cells arrested with nocodazole was
applied to the same gel filtration procedure as described above. The
resulting fractions were analyzed using antibodies raised against
Cdc34p. The profile obtained from cells arrested with nocodazole was
similar to that for unarrested cells (Fig. 6) for the majority of the Cdc34p. A
small amount of Cdc34p, approximately 10%, did appear to be of very
high molecular mass, in excess of 669 kDa. A similar analysis of Cdc53p
also demonstrated that the gel filtration profiles for this protein was
unchanged (data not shown). These data suggest that Cdc34p is present
as a high molecular weight complex throughout the cell cycle.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Cdc34p is present within a high molecular
weight complex throughout the cell cycle. Densitometric scanning
profiles of immunoblots of Cdc34p obtained after fractionation of yeast whole cell lysate on a Superose 12 column. Lysate prepared from cells
either in logarithmic phase (log) or arrested with
nocodazole (No) were subjected to Western immunoblot
analysis with anti-Cdc34p antibody. Size markers are shown in
kDa.
|
|
 |
DISCUSSION |
One essential function of the ubiquitin-conjugating enzyme Cdc34p
is to attach Ub onto the cyclin-dependent kinase inhibitor Sic1p and thus signal Sic1p for proteolysis. The precise mechanism by
which Cdc34p transfers Ub onto Sic1p is not known, however, this
process is dependent on at least two other proteins, Cdc4p and Cdc53p.
In this report we map a Cdc4p- and Cdc53p-binding domain on Cdc34p and
show the importance of in vivo interactions between these
three proteins, strengthening the hypothesis that Sic1p ubiquitination
is mediated by a multi-protein complex.
The Association of Cdc34p with Cdc4p and Cdc53p Is Essential for
Cdc34p Activity--
We have defined a Cdc4p/Cdc53p-binding domain on
Cdc34p (Fig. 7). This region is not found
in other Ubc proteins which suggests that these interactions are unique
to Cdc34p. However, the efficiency of Cdc4p and Cdc53p binding to a
fusion containing Cdc34p residues 1-209 or residues 171-209 is less
than that of a fusion containing full-length Cdc34p. Therefore the
remainder of the Cdc34p protein may act to stabilize its association
with Cdc4p and Cdc53p. Ptak et al. (34) have shown that this
region is required for Cdc34p dimerization in vitro and it
is possible that Cdc4p and Cdc53p bind to a Cdc34p dimer in
vivo. Absence of this binding region results in loss of Cdc34p
activity and cells are prevented from entering S-phase due to failure
to degrade the CDK inhibitor Sic1p. Thus the association of Cdc34p with
Cdc4p and Cdc53p is essential for Sic1p ubiquitination. What role does
Cdc4p and Cdc53p play in Sic1p ubiquitination? Cdc34p residues 1-170
contain the catalytic domain of Cdc34p and its active site cysteine
(Fig. 7). This region has been shown to be charged with ubiquitin by
the ubiquitin-activating enzyme, E1 (9), an event that does not require
Cdc4p or Cdc53p association. Therefore Cdc4p and Cdc53p most likely
play a role in transferring Ub from Cdc34p onto its substrates either
by substrate recognition or by having a Ub-ligase (E3) activity.
Ub-ligases (E3s) have been described (7, 8) yet the sequence comparison of these proteins do not resemble either Cdc4p or Cdc53p. Possibly Cdc53p is required to present the substrate to Cdc34p. Cdc53p has been
found to associate with Cln2p, a G1 cyclin that is another candidate Cdc34p substrate (22).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Functional domains on Cdc34p. A
schematic diagram illustrating different domains on Cdc34p.
Numbers indicate amino acid residues. The catalytic domain,
residues 1-170, contains the active site cysteine, *, at residue 95. The Cdc4p/Cdc53p-binding domain is residues 171-209. Residues 254-295
constitute a region that is ubiquitinated (37).
|
|
Cdc4p, Cdc34p, and Cdc53p Are Members of a Multiprotein
Complex--
Gel filtration analysis shows that Cdc4p, Cdc34p, and
Cdc53p are not found as monomers in yeast lysate and take part in
associations resulting in the formation of high molecular mass
complexes. Overlap of the three proteins' distribution suggests that
the Cdc4p·Cdc34p·Cdc53p complex is approximately 400 kDa. This size
is greater than that predicted for a complex containing one of each
protein (218 kDa), that have a predicted molecular mass of 88, 34, and
96 kDa for Cdc4p, Cdc34p, and Cdc53p, respectively. Therefore either
these proteins may act as multimers or additional proteins are part of
this complex. Skp1p is required for the ubiquitination of Sic1p and has
been shown to bind to Cdc4p through a Cdc4p domain called the F-box
(25). However, Skp1p is only 22 kDa and perhaps other such proteins are
required for Cdc34p to interact with Cdc4p and Cdc53p. Significant
amounts of Cdc4p and Cdc34p co-fractionate in the absence of Cdc53p and
together with our data demonstrating the stabilization of Cdc4p when
co-overproduced with Cdc34p suggest that these two proteins form a
heterodimer. Again, this association, of approximately 200 kDa may
occur in the presence of other proteins. Therefore it appears that
components of the Cdc4p·Cdc34p·Cdc53p complex may be separated and
be redistributed for distinct ubiquitinating activities.
The Cdc34p Complex Is Present throughout the Cell
Cycle--
Finally we determined whether Cdc34p existed within a
complex throughout the cell cycle. Many cell cycle regulators are
themselves only present during specific times of the cell cycle. We
have shown here that Cdc4p, Cdc34p, and Cdc53p are present throughout the cell cycle and that Cdc34p appears to reside as part of a complex
throughout the cell cycle. Potentially, Cdc34p activity may be required
throughout the cell cycle and not just at the G1/S
transition in spite of the fact that some of its substrates are
degraded in a cell cycle specific manner. Phosphorylation of Sic1p and
Cln2p is a prerequisite for these substrates to be ubiquitinated by
Cdc34p and Cdc34p activity may be solely regulated by recognizing
phosphorylated substrates (38, 39). Other candidate Cdc34p substrates
include Cdc6p (40), Far1p (41), and Gcn4p (42). Both Cdc6p and Far1p
are degraded in a cell cycle specific manner but outside the
G1/S transition and therefore the Cdc34p complex must be
active throughout the cell cycle.
We thank Dr. Ron Wek for invaluable comments
during the course of this work and Dr. Peter Roach for reading the
manuscript.