(Received for publication, June 14, 1995; and in revised form, August 22, 1995)
From the
The yeast Saccharomyces cerevisiae CDC34 gene encodes a
ubiquitin-conjugating enzyme that is required for the cell cycle
G/S transition. We show here that a dominant negative Cdc34
protein is generated by simultaneously replacing both Cys
and Leu
with Ser residues. Cys
is an
essential catalytic residue that forms a transient thiol ester with
ubiquitin during catalysis, and Leu
is highly conserved
among all known ubiquitin-conjugating enzymes. Mutants that encode
either an alanine or a serine at one or both of these two positions are
inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although
cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic
multibudded phenotype of cdc34 temperature-sensitive or null
mutants, this blockade is relieved by simultaneous overexpression of
wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to
inhibit in vitro ubiquitination of the Cdc34-specific
substrate, Cln2 protein. We suggest that Cdc34-C95S,L99S selectively
sequesters a subset of Cdc34 substrates or regulators. These findings
have implications for the structure/function relationships of
ubiquitin-conjugating enzymes, and suggest a general method for
identifying components and substrates of specific ubiquitination
pathways of eukaryotes.
The ubiquitin-conjugating enzymes (E2) ()constitute a family of conserved proteins that participate
either in an intermediate or in the final step of substrate
ubiquitination (Hershko and Ciechanover, 1992; Finley and Chau, 1991).
These enzymes form a thiol ester adduct with ubiquitin (Ub) in the
presence of ubiquitin-activating enzyme (E1) and ATP in the
following reactions: 1) E1
+ Ub + ATP
E1
+ AMP + PP
, and
2) E1
+ E2
E1
+ E2
.
Substrate proteins may be directly recognized by an individual E2 enzyme, resulting in the transfer of ubiquitin from an E2 to a lysine on substrate proteins.
Alternatively, substrate recognition may require the presence of
another group of proteins known as E3 or ubiquitin-protein
ligases (Reiss and Hershko, 1990; Bartel et al., 1990). A
requirement for a specific E3 protein had been shown for the
degradation of substrates in the N-end rule pathway (Bartel et
al., 1990) and for p53 (Scheffner et al., 1993). In the
N-end rule pathway, the Ubr1 (E3) protein contains separate
sites for Rad6 (E2) and substrate bindings and confers
specificity for one of the cellular Rad6-dependent ubiquitination
pathways (Varshavsky, 1992). In the p53 degradation pathway, it has
been further shown that ubiquitin from E2
is
transferred to a cysteine in the E3 protein (Scheffner et
al., 1995), leading to the formation of an E3
thiol ester.
CDC34 is one of 10 known
ubiquitin-conjugating enzyme-encoding genes in the yeast, S.
cerevisiae (Goebl et al., 1988; Jentsch, 1992). This gene
was initially identified on the basis of its requirement for cells to
undergo the cell cycle G to S transition (Byers and
Goetsch, 1973). Under nonpermissive conditions, temperature-sensitive
mutants of CDC34 develop numerous elongated buds, and the
spindle pole body duplicates but fails to undergo the separation
required for spindle formation (Byers and Goetsch, 1973). More recent
studies have established a direct role of this ubiquitin-conjugating
enzyme in targeting the degradation of specific regulators of the cell
cycle. Known Cdc34-specific substrates in this category include the
G
cyclins (Deshaies et al., 1995; Yaglom et
al., 1995) and the Cdc28 kinase inhibitor Sic1 (Schwob et
al., 1994). In addition, mutations in the CDC34 gene can
lead indirectly to the abnormal accumulation of other cell cycle
regulators such as the G
-specific B-type cyclins (Amon et al., 1994). Other than its cell cycle function, the
Cdc34-encoded ubiquitin-conjugating enzyme has also been shown to
target the degradation of the transcription factor GCN4 (Kornitzer et al., 1994), and it is likely that other functions of this
enzyme may be uncovered by the identification of additional substrate
proteins.
The 295-residue Cdc34 protein contains a 170-residue
N-terminal domain that is conserved among all E2 proteins.
This conserved domain is apparently sufficient for E2 complex formation since the smallest E2 enzymes are
comprised almost exclusively of this domain (Jentsch, 1992). In Cdc34
this conserved domain also contains an extra 12-residue sequence near
the ubiquitin-accepting cysteine. This extra sequence is found only in
one other yeast E2 protein, Ubc7 (Jungmann et al.,
1993), and in both cases, the function of this extra sequence segment
remains undefined. In the present study, we report the effect of
mutations at the ubiquitin-accepting cysteine as well as at
Leu
, a residue that is adjacent to this 12-residue
segment. We show here that while both residues are essential for CDC34 functions, a unique dominant negative allele of this
gene, CDC34
, could be generated by simultaneously
substituting these two residues with serines. In addition to its
potential utility in genetic analysis, CDC34
can
be used to block Cdc34-dependent ubiquitination in vitro.
Figure 1:
Effect of CDC34-C95S,L99S expression in
yeast cells. Panel A, effect of CDC34-C95S,L99S expression in MGG15 (Table 2) cells carrying ts-cdc34-2 at the permissive temperature of 23 °C.
ABY210 (cdc34-2 + YEpGALCDC34), ABY214 (cdc34-2 + YEpGALcdc34-L99S), ABY216 (cdc34-2 + YEpGALcdc34-C95S), and ABY212 (cdc34-2 + YEp GALCDC34-C95S,L99S were
grown to an A of 0.2 in SR medium at 23 °C
and streaked in duplicate onto medium containing galactose. Panel
B, the GAL1-CDC34-C95S,L99S mutant was integrated into
wild type yeast strain 332 (ABY100). Similarly integrated
GAL1-CDC34 resulted in ABY200 (Table 2). Spot assays
were done to compare the viability of ABY100 and ABY200 cells upon
galactose-induced expression of CDC34 and CDC34-C95S,L99S alleles.
25,000 cells (grown in SR medium to an A
of 0.2, and counted by hemocytometer) were
spotted in the first spot on each panel. The next two spots in
each panel are two successive 10-fold dilutions. Plates were incubated
at 30 °C. ABY200 (URA3::GALCDC34) spots in
galactose medium show no deleterious effect or loss in viability,
whereas ABY100 (URA3::GALCDC34-C95S,L99S) spots in
galactose medium show lethal effect on wild type yeast cells by
expression of the CDC34-C95S,L99S allele.
To test whether the dominant effect of CDC34-C95S,L99S mutant is uniquely dependent on the cdc34-2 allele,
we also introduced this mutant gene into a yeast strain that carries a
wild type CDC34 allele. The ABY100 strain contains an
integrated copy of CDC34-C95S,L99S whose expression is
regulated by the P promoter (Table 2). These cells
also failed to grow on galactose medium (Fig. 1B) and
became inviable (data not shown). These results indicate that the
dominant effect of CDC34-C95S,L99S on cell growth and
viability is not restricted to the cdc34-2 strain.
Similar expression of the singly substituted mutants, cdc34-C95S and cdc34-L99S, did not alter cell viability (data not
shown).
Since the unique effect of the CDC34-C95S,L99S mutant may be due to its higher level of accumulation than those of the singly substituted mutants, cdc34-C95S and cdc34-L99S, we also used immunoblotting to assess the level of mutant proteins in cells expressing these various mutant genes. Immunoblots of Cdc34 proteins from cells before and after 2 or 8 h of induction in galactose indicated that the Cdc34-C95S,L99S protein did not accumulate to a higher level than the other mutants (data not shown). Thus, the unique effect of Cdc34-C95S,L99S on cell viability is not due simply to the overexpression of an inactive Cdc34 protein.
Figure 2:
Inhibition of Cln2 multiubiquitination by
Cdc34-C95S,L99S. [S]Cln2 was synthesized in
vitro in the reticulocyte lysate system and was incubated in 15
mg/ml yeast extract for 0 (lane 1) or 60 (remaining lanes) min. at 24 °C in the presence of the indicated
amounts of Cdc34 or Cdc34-C95S,L99S (see ``Experimental
Procedures''). Lane 2 has the reaction incubated without
exogenous Cdc34. Lane 3 has the reaction supplemented with 180
nM of wild type Cdc34. The reaction in lane 4 had 180
nM, and that in lane 5 had 540 nM of
Cdc34-C95S,L99S added. Cln2, unphosphorylated Cln2 with a
molecular mass of
66 kDa; PP-Cln2, hyperphosphorylated
Cln2 with a molecular mass of
84 kDa; Ub-Cln2,
multiubiquitinated Cln2.
The inhibition of Cln2 protein ubiquitination is not due to general inactivation of ubiquitin conjugation pathways since the overexpression of this mutant protein did not affect ubiquitin conjugation to other endogenous proteins (Fig. 3A). We have also assayed the effect of Cdc34-C95S,L99S on purified ubiquitin-activating enzyme by monitoring the catalytic transfer of ubiquitin from the ubiquitin-activating enzyme to another yeast ubiquitin-conjugating enzyme, Rad6 (Fig. 3B). As shown in Fig. 3B, the level of ubiquitin-Rad6 thiol ester was not detectably affected by 2-20 µM of Cdc34-C95S,L99S. These results, taken together, indicate that Cdc34-C95S,L99S inhibits Cln2 ubiquitination via specific inhibition of the Cdc34-dependent pathway.
Figure 3:
Ubiquitin-activating enzyme is not
affected by overexpression of the CDC34-C95S,L99S. Panel A,
extracts were prepared from 10-h galactose-induced ABY200 (URA3::GALCDC34), and ABY100 (URA3::GALCDC34-C95S,L99S) in breakage buffer;
300 µg of total protein in 60 µl volume was supplemented
with 200 ng of
I-Ub and incubated at 30 °C for 30
min. 30 µl of 3
SDS-PAGE sample buffer was added, samples
were boiled for 3 min, and the reactions were analyzed by 14% Laemmli
gels. Lanes 1 and 3, ABY100 reaction at 10 and 30
min; lanes 2 and 4, ABY200 reaction at the same time
points. The radioactive protein ladder indicating ubiquitination of
yeast proteins is similar in intensity and pattern for extracts from
both of these cell types. The positions of Life Technologies, Inc.
prestained high molecular mass markers (myosin, 215 kDa; phosphorylase
B, 105 kDa; bovine serum albumin, 70 kDa; ovalbumin, 43 kDa; carbonic
anhydrase, 29 kDa;
-lactoglobulin, 18 kDa; and lysozyme, 14 kDa)
are marked on the left. Panel B, formation of
Rad6
thiol ester complexes was assayed by incubation
with 250 nM purified Rad6 protein and indicated amounts of
Cdc34-C95S,L99S protein with ubiquitin-activating enzyme (E1),
magnesium, ATP, and
I-ubiquitin for 20 min at 30 °C.
The reaction products were electrophoresed in a 14% SDS gel in the
absence of thiol-reducing agents, and radiolabeled proteins were
visualized by autoradiography.
While the above results indicated that CDC34-C95S,L99S exerts its effect by interfering with an essential CDC34-dependent process, cells overexpressing this mutant did not exhibit the morphological phenotype of previously characterized loss-of-function mutants. The cdc34 null mutant strain, as well as the temperature-sensitive cdc34-1 and cdc34-2 strains arrest as multibudded cells (Goebl et al., 1988). This morphology is absent in ABY100 cells that assume aberrant morphology after switching cells to a galactose-containing medium. A majority of these aberrant cells were found to have a single elongated bud (data not shown). The absence of multibudded cells is not due to a strain difference since MGG15 cells that are overexpressing Cdc34-C95S,L99S also took on, at both permissive and nonpermissive temperatures for cdc34-2, morphologies similar to those of ABY100 cells in galactose (data not shown). This difference in morphology is consistent with the notion that Cdc34-C95S,L99S does not simply inactivate endogenous wild type Cdc34 protein. Possible mechanisms of Cdc34-C95S,L99S action are described further under ``Discussion.''
The requirement for a serine
at the Cys residue may be due to the unique ability of a
serine to form a more stable linkage with ubiquitin (Sung et
al., 1991). In a normal ubiquitin-conjugating enzyme, the active
site cysteine forms a thiol ester linkage with the C-terminal carboxyl
of ubiquitin. This thiol ester-linked ubiquitin is subsequently
transferred either to a E3 protein or to substrates directly.
Substitution of the active site cysteine by a serine has been shown to
inactivate other ubiquitin-conjugating enzymes (Sung et al.,
1991; Seufert et al., 1995) presumably because the more stable
oxygen ester-linked ubiquitin is not further transferred. In the case
of Cdc34, we have previously shown that the thiol ester-linked
ubiquitin can also be transferred to a lysine within this enzyme in an
intramolecular reaction to form a Lys
-specific
multiubiquitin chain (Banerjee et al., 1993). This
autoubiquitination reaction was used here to test the effect of
Cys
substitution. For these experiments, the cdc34 mutants were expressed in E. coli and assayed for their
ability to accept [
I]ubiquitin in the presence
of added ubiquitin-activating enzyme (Fig. 4). Under our assay
conditions, the reaction with wild type Cdc34 leads to products that
migrated as a set of discrete bands on SDS gels (Fig. 4A, lane 1). These discrete bands are
due to the linkage of multiple ubiquitin groups, in the form of a
ubiquitin chain, to a lysine residue in Cdc34 (Banerjee et
al., 1993). In contrast, only a single ubiquitin-Cdc34 adduct was
detected with the mutants Cdc34-C95S, Cdc34-C95S,L99A, and
Cdc34-C95S,L99S. This single adduct was not present in any of the
C95A-substituted mutants, consistent with the notion that this single
adduct is due to the linkage of ubiquitin to the substituted serine at
position 95. The absence of additional adducts of lower electrophoretic
mobilities in these C95S-substituted mutants indicates that
autoubiquitination did not occur with these mutants. These results
indicate that the C95S-substituted cdc34 mutants can indeed
form a more stable oxygen ester with ubiquitin and raise the
possibility that the effect of CDC34-C95S,L99S on cell
viability may require the linkage of ubiquitin to Ser
.
Figure 4:
Formation of Ub-Cdc34 complexes by the
mutant Cdc34 proteins. Panel A, Cdc34 or its mutants were
expressed in E. coli harboring the appropriate M13
mp19/18-CDC34 constructs (see ``Experimental
Procedures''). Amounts of the recombinant protein in the bacterial
extracts were normalized for the assays. Thiol-insensitive Ub-Cdc34
complexes were assayed by incubating E. coli extracts with
100 nM overexpressed Cdc34 or its mutant proteins with
ubiquitin-activating enzyme, magnesium, ATP, and
I-ubiquitin for 45 min. Samples were adjusted to contain
5%
-mercaptoethanol and heated at 90 °C for 3 min prior to
SDS-PAGE. The uppermost band corresponds to ubiquitin linked to a
lysine on E1. The ladder bands with the wild type Cdc34 are
due to the linkage of a multiubiquitin chain to a lysine on Cdc34
(Banerjee et al., 1993). The single band with the C95S mutants
is presumably due to the formation of a Ub-Cdc34 oxygen-ester at
Cdc34-Ser
(position indicated by arrow on the right). Panel B, formation of Cdc34
thiol ester complexes was assayed by incubating E. coli extracts with
100 nM overexpressed Cdc34 or its
mutant proteins with ubiquitin-activating enzyme, magnesium, ATP, and
[
I]ubiquitin as in panel A, except the
reaction time was reduced to 15 min. The reaction products were
electrophoresed in a 14% SDS gel in the absence of thiol-reducing
agents, and radiolabeled proteins were visualized by autoradiography. Open arrow indicates Ub-Cdc34 complex; closed arrow indicates E1
complex.
We have also monitored the formation of ubiquitin-Cdc34 thiol ester
with shorter reaction time (15 min), using a nonreducing SDS gel (Fig. 4B, lanes 1-3). Both Cdc34-L99S
and Cdc34-L99A were found to retain partial activity as indicated by
the presence of a 42-kDa radiolabeled band that corresponds to
ubiquitin-Cdc34 thiol ester. This result indicated that unlike
mutations at Cys, mutants of Leu
are inactive
at a step subsequent to ubiquitin-thiol ester formation. Thus,
mutations at Leu
appear to interfere with Cdc34
interactions with either substrate or E3 protein(s).
Interestingly, a higher activity was found for the L99S-substituted
mutant than the L99A mutant. Similarly, the shorter reaction time also
revealed a faster ubiquitin linkage to Ser
in
Cdc34-C95S,L99S as compared with Cdc34-C95S and Cdc34-C95S,L99A (Fig. 4B, lanes 4, 9, and 5,
respectively). Whether this difference in reactivity could account for
the stringent requirement for the L99S substitution remains to be
determined.
The ability of Cdc34-C95S,L99S to inhibit the cell cycle
function of CDC34 is indicated by its in vivo effect
on cell viability and by its in vitro effect on Cln2
ubiquitination. Proteins that are known to be targeted by Cdc34 for
ubiquitin-mediated proteolysis include G cyclins (Deshaies et al., 1995, Yaglom et al., 1995), the yeast
transcription factor GCN4 (Kornitzer et al., 1994) and the
Cdc28 kinase inhibitor Sic1 (Schwob et al., 1994). Since
overexpression of Cln2 does not lead to cell inviability (Lew and Reed,
1993), it is likely that Cdc34-C95S,L99S also inhibits the degradation
of other Cdc34-dependent substrates. One likely candidate is Sic1,
which is normally degraded prior to cell entry into the S phase (Schwob et al., 1994), and a moderate overexpression of this protein
has previously been shown to produce cellular morphology (Nugroho and
Mendenhall, 1994) similar to those found for cells overexpressing
Cdc34-C95S,L99S. Consistent with this notion is the recent
demonstration that the human homolog of Cdc34-C95S,L99S could also
inhibit Cdc34-dependent degradation of the cyclin-dependent kinase
inhibitor, p27 (Pagano et al., 1995). The ability of
Cdc34-C95S,L99S to inhibit the in vitro ubiquitination and/or
degradation of two dissimilar substrates in two different species
raises the possibility that this mutant may be used in analogous manner
to establish the identity of additional Cdc34-specific substrates.
Cells expressing Cdc34-C95S,L99S exhibit a morphology that differs significantly from the multibudded morphology of previously characterized loss-of-function cdc34 mutants. A significant proportion of these cells contain a single, elongated bud, while multibudded cells are conspicuously absent. As the mechanism for multibudding in the cdc34 null mutant has not been defined, the morphological difference here could not be readily addressed. Nonetheless, this difference suggests that the effect of Cdc34-C95S,L99S is not equivalent to a straightforward loss of CDC34 functions. Previous studies have indicated that Cdc34 is capable of self association, and this process requires a region in the sequence that is apparently essential for its cell cycle function (Ptak et al., 1994). Conceivably, Cdc34-C95S,L99S could exert its effect by sequestering endogenous Cdc34. However, this mechanism is incompatible with the absence of multibudded cells. Furthermore, the effect of Cdc34-C95S,L99S could not be obtained with the other seven inactive cdc34 mutants that contain the same determinant for self-association. In addition, we have obtained preliminary results indicating that purified Cdc34-C95S,L99S does not inhibit a previously characterized in vitro autoubiquitination of Cdc34 (Banerjee et al., 1993) or the conjugation of ubiquitin to histone proteins (Haas et al., 1991). Thus, it is unlikely that the effect of Cdc34-C95S,L99S is due to the sequestration of endogenous Cdc34.
Since Cdc34-C95S,L99S encodes an inactive ubiquitin-conjugating enzyme, the mechanism of inhibition is likely to reside in a binding step where this mutant could compete effectively with Cdc34. A key unanswered question here is whether substrate recognition in this pathway also requires E3 proteins. A requirement for E3 has been shown for several other ubiquitination pathways. For example, the Ubr1 protein is required in the N terminus rule pathway (Bartel et al., 1990), and a protein known as E6AP is required for the ubiquitination of p53 (Scheffner et al., 1993). The ubiquitination of mitotic cyclins appears to require a large protein complex consisting of several distinct proteins (Sudakin et al.(1995); reviewed in Murray(1995)). Thus, the effect of Cdc34-C95S,L99S could result from the sequestration of Cdc34-specific substrates or the required E3 protein(s).
Cys and Leu
are
located within a sequence region that is highly conserved among
ubiquitin-conjugating enzymes. This conserved sequence region has been
termed the catalytic core domain and is conserved in tertiary folding
as shown by the crystal structures of Arabidopsis thaliana Ubc1 and Saccharomyces cerevisiae Ubc4 (Cook et
al., 1993). A structural model of the Cdc34 catalytic core could
be constructed by aligning residues 10-100 of Cdc34 with the
N-terminal 91 residues of Ubc4. Both Cys
and Leu
could be placed within this structural model at positions that
are occupied by identical amino acids in Ubc4. In this model,
substitution of Cys
by either alanine or serine would not
introduce other structural perturbations. Thus, the difference between
Cdc34-C95S,L99S and Cdc34-C95A,L99S is unlikely to be structural but
rather in the ability of Ser
to form a stable oxygen ester
with ubiquitin. This suggests that the inhibitory effect of
Cdc34-C95S,L99S may require prior formation of the
ubiquitin-Cdc34-C95S,L99S ester.
A model that could account for the
inhibitory effect of Cdc34-C95S,L99S is depicted in Fig. 5A.
In this model, ubiquitin contributes partly to the energetics of the
ternary complex formation between the ubiquitin-E2 thiol ester and E3. Once ubiquitin has been transferred to E3, the
ubiquitin-conjugating enzyme would presumably bind less tightly since
it is no longer linked to ubiquitin. The reduced affinity may then
facilitate the dissociation of the ubiquitin-conjugating enzyme, which
could be recharged with ubiquitin by the ubiquitin-activating enzyme (E1). The existence of a ubiquitin binding site on E3
is supported by studies on a reticulocyte E3 in the N-end rule
pathway (Reiss and Hershko, 1990). This model makes the prediction that E2 mutants containing a stably linked ubiquitin would be
better inhibitors than inactive enzymes that cannot be linked with
ubiquitin and explains the unique requirement for the C95S mutation.
The requirement for the L99S mutation could be explained by the
observation that this mutation causes ubiquitin to be linked to
Ser at a faster rate (Fig. 4). A structural basis
of this effect could not be readily assessed using the two known
structures of E2 enzymes since Cdc34 contains an extra
12-residue segment beginning at residue 101, and this extra segment
could not be accommodated in a structural model. A similar sequence
segment is also present in the yeast Ubc7 protein (Fig. 5B), and the crystal structure of this
ubiquitin-conjugating enzyme has recently been determined, (
)and work is in progress to determine the mutational effect
of the corresponding leucine residue in this enzyme.
Figure 5:
Panel A, model of the ternary complex
formation between E3 and E2 ubiquitin-E2 thiol ester complex docks on an E3
by noncovalent interactions to form II. Both ubiquitin and E2
contribute to the stability of II. Transthiolation (Scheffner et
al., 1995) leads to the attachment of ubiquitin to a cysteine in E3, and ubiquitin no longer contributes to the retention of E2 in the ternary complex III. In the case of
Ub-Cdc34-C95S,L99S oxygen ester, ubiquitin is not transferred to E3, leading to the sequestration of E3 in an inactive
complex. Panel B, alignment of the Cdc34 catalytic site
sequence with other ubiquitin-conjugating enzyme sequences. The yeast
Cdc34 (Goebl et al., 1988) sequence is aligned with those of
human Cdc34 (Plon et al., 1993), yeast Ubc7 (Jungmann et
al., 1993), Rad6 (Jentsch et al., 1987), and Ubc4
(Seufert and Jentsch, 1990) to show the positioning of the
12/13-residue segment in Cdc34 and Ubc7. Positions of mutant residues
in Cdc34-C95S,L99S are indicated below by closed
circles. The starting and end residue numbers for each sequence in
the alignment are given in parentheses.
Dominant
negative mutants in other yeast genes have proved useful for the
identification of interacting proteins via suppresser analyses. If the
action of Cdc34-C95S,L99S depends on its interaction with E3
proteins, one class of suppressers is expected to be comprised of these
proteins. The ability of purified Cdc34-C95S,L99S to block the in
vitro ubiquitination of Cln2 suggests a further utility of this
mutant for the identification of additional Cdc34-specific substrates.
Although substrates in a specific ubiquitin-dependent pathway could
usually be identified in yeast by showing their increased stability in
a specific ubc mutant, this approach may be insufficient to
provide unambiguous identification of CDC34-specific
substrates. For example, while the G-specific B-type
cyclins are stabilized in CDC34 mutants (Amon et al.,
1994), degradation of these cyclins is apparently mediated by UBC9 (Seufert et al., 1995), and the effect of cdc34 in this case could be attributed to the abnormal accumulation of
Cln proteins in cdc34 mutants. Thus, an unambiguous
identification of a CDC34-specific substrate may also require
the use of in vitro approaches to demonstrate a direct
requirement of this ubiquitin-conjugating enzyme. One such approach may
be by using the Cdc34-C99S,L99S mutant protein to inhibit the
ubiquitination or the degradation of a candidate substrate protein in a
cell-free system. This approach has been used recently to help in
establishing a role of Cdc34 in degradation of the human
cyclin-dependent kinase inhibitor, p27. Dominant negative mutants of
yeast UBC genes could be readily identified by genetic
screens. The creation of an analogous dominant negative mutant of human
Cdc34 with mutations identified in the yeast enzyme points to the
important possibility that other dominant negative mutants of mammalian
ubiquitin-conjugating enzymes could be obtained by a similar approach.
Such mutants could then be used for exploring substrates and/or
regulators of protein ubiquitination in mammalian systems.