From the
The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a
crucial role in the progression of the cell cycle from the G
The yeast ubiquitin conjugating enzyme CDC34 (UBC3) is one of a
class of eukaryotic proteins (referred to as E2s), that selectively
catalyze the covalent attachment of ubiquitin (Ub)
The CDC34 gene was
originally identified as a conditional cell-cycle mutation that
arrested cells after the ``Start'' regulatory step, but
before the initiation of S-phase
(6) . Cells arrested at the
CDC34-dependent step are unable to proceed with nuclear DNA
replication but remain competent to duplicate the spindle pole body and
periodically produce buds
(6) . Isolation and sequencing of the
CDC34 gene
(7) revealed amino acid similarity to a
previously characterized E2 encoded by the DNA repair gene, RAD6 (8) . The RAD6 and CDC34 proteins are two members of a
family of at least 10 Ub-conjugating enzymes that have been identified
in yeast
(4, 9) . The CDC34 protein consists of a highly
conserved catalytic domain common to all Ub-conjugating enzymes and a
unique carboxyl-terminal extension or tail
(7) . A segment of
the tail is essential for cell cycle function and can confer its
functional properties to RAD6, when appended to its carboxyl terminus
(10, 11) . Furthermore, genetic experiments suggest that
CDC34 can physically associate with itself or with RAD6 and that these
types of interactions are necessary for its function
(10) . In
recent work we have shown that the ability of CDC34 to self-associate
in vitro is dependent on the same region of the tail that is
required for its cell cycle function in vivo (12) .
Although in vitro evidence exists for the assembly of
Ub-conjugating enzyme homo-complexes
(13, 14, 15) , the idea that complex formation
is relevant to E2 function, as originally proposed in Silver et al. (10) , has gained strength from the recent observation that
the degradation of the MAT
In this study we show that increased Ub expression can
suppress the cell cycle defects associated with three structurally
unrelated mutant cdc34 alleles. These genetic results in
conjunction with Ub-CDC34 cross-linking studies, indicate that in
addition to the previously reported CDC34-CDC34 interaction
(10, 12) , CDC34 function also requires the correct
orientation of Ub on the E2 surface.
The CDC34
protein used in cross-linking experiments was prepared from
Escherichia coli cells containing the CDC34 overexpression plasmid pREGB6. This plasmid was constructed by
inserting a gene cassette encoding the wild-type CDC34 gene,
described above, in-frame between the SstI and KpnI
sites of a modified derivative of pET-3a
(20) . The construction
of the pET-3a derivative has been described in detail by Ptak et
al. (12) . Isolation of High-copy Suppressors of the cdc34-2 Mutation-A
genetic screen was developed to identify genes which when expressed at
high dosage could suppress the cdc34-2 ts mutation. In
this screen cdc34-2 mutant cells were transformed with a
genomic library on the high-copy vector PMA3A (Constructed by Mick
Tuite, Kent University). Suppressor plasmids were identified based on
their ability to allow mutant cells to form colonies on YEPD plates
(21) at the restrictive temperature of 32 °C. From
approximately 80,000 transformed cells, 28 colonies able to grow at 32
°C were identified. Plasmids were isolated from each
temperature-resistant yeast colony and used to transform E.
coli cells. The ability of a particular plasmid to suppress
the cdc34-2 mutation was then confirmed by
re-introducing each E. coli-purified plasmid back into the
cdc34-2 mutant strain.
The strategy used to measure the
suppression of the cdc34
In this study a genetic screen was employed to identify genes
of proteins that could interact with CDC34. The rationale of this
screen is based on the idea that overexpression of such a protein from
a high-copy plasmid would favor a CDC34-protein interaction thereby
stabilizing the ts CDC34 polypeptide resulting in an increased
resistance of the mutant to elevated temperature. From this screen 28
independent plasmids were isolated and grouped into three families on
the basis of Southern analysis. The first family had 16 members and was
shown to contain the wild-type CDC34 gene. The second plasmid
family, with 7 members, was found to contain a novel gene we have
called UBS1. Results concerning the UBS1 gene will be
presented elsewhere. The third family contained 5 members and was found
by sequence analysis to contain the poly-Ub gene UBI4 (29) .
The isolation of the UBI4 gene as a
suppressor of the cdc34-2 mutation suggested that Ub
overexpression could reduce the severity of this mutation. Consistent
with this view, we found that increased expression of a single Ub gene driven from the CUP1 promotor was also able to
suppress the cdc34-2 mutation. Thus, the suppression of
the cdc34-2 mutation results from increased Ub
expression.
The cdc34-2 mutation is one of several
available mutations for which the structural defect is known (Fig. 1).
Sequencing of the cdc34-2 allele revealed a Gly to Arg
codon substitution at position 58 (this work). Similarly, other work
has established that the ts cdc34-1 allele
(6) contains a Pro to Ser codon substitution at position 71
(30) . In addition to these two ts mutations, we have recently
found that deletion of the CDC34 tail codons from residue 201
to the carboxyl terminus results in a mutation
( cdc34
To date several amino acid residues have been
identified within Ub that play a defined and key role in Ub function
(Fig. 1). The carboxyl-terminal residues
Gly
The
expression of Ub having the Gly
The
suppression of only a specific subclass of mutations by expression of a
suppressor protein is frequently used as a genetic indicator of a
direct interaction between the mutant and suppressor proteins.
Therefore the ability of the UbR48 derivative to suppress only one of
the three cdc34 mutations tested, suggested that suppression resulted
from an elevated noncovalent interaction between Ub and cdc34 as a
consequence of Ub overexpression (see ``Discussion'').
The
existence of a specific noncovalent interaction between Ub and CDC34 is
illustrated by the in vitro chemical cross-linking experiment
described in Fig. 3. In this experiment, the amino specific
cross-linker BS
The results of the present study illustrate that three
different defects in CDC34 activity can each be suppressed by elevated
levels of Ub expression. The stabilization of temperature labile
proteins by increasing the concentration of interacting proteins is a
well documented phenomenon (see for example, Refs. 40 and 41). Two
pieces of evidence indicate that this suppression arises in part, from
a noncovalent interaction of Ub with the CDC34 polypeptide. First, an
elevated level of UbR48 expression suppresses only one of the
CDC34 mutations but has no effect on the other two. Allelic
specificity exhibited by high-copy suppression such as this, is
frequently used as a genetic argument that two proteins interact with
one another (see for example, Refs. 42 and 43). This argument is based
on the premise that suppression of a particular conditional protein
defect through protein-protein interaction at a specific surface is
expected to suppress only a subclass of the total range of possible
conditional protein defects, since defects that are distal to the
interacting surface need not be suppressed by this mechanism.
Conversely, suppression that does not rely on the physical association
of two proteins is expected to be generally insensitive to the nature
of the protein defect. Second, the concentration dependent interaction
between Ub and CDC34 in vivo that is inferred from the
specificity of UbR48 toward only one of three cdc34 alleles,
is strengthened by the specific, concentration-dependent noncovalent
interaction between Ub and CDC34 that we observe in vitro by
cross-linking analysis.
The dependence of suppression on Ub
concentration in vivo coupled with the dependence of Ub-CDC34
cross-linking on Ub concentration in vitro, indicates that the
noncovalent interaction between Ub and CDC34 is a weak one. This
conclusion is further borne out by our failure to detect interaction of
these components at concentrations exceeding 100 µ
M by gel
exclusion chromatography (results not shown). In view of the weak
nature of the Ub-CDC34 noncovalent interaction, the covalent attachment
of Ub to the active site of CDC34 through thiol ester formation would
be expected to enhance the suppressive effects of the noncovalent
interaction. This conclusion is supported by the observation that
deletion of Gly
A priori, there is no formal requirement for a specific
noncovalent interaction between Ub and Ub-conjugating enzyme in the
overall process of Ub conjugation since Ub binding pockets could
conceivably be provided by other E2 associated components. The results
of the present study, however, argue that a noncovalent Ub-E2
interaction represents an important facet of E2 function. Such an
interaction could participate in either or both of two steps in the Ub
conjugation mechanism. The correct positioning of Ub on the surface of
the E2 may, for example, be necessary for the transfer of Ub from the
ubiquitin activating enzyme E1 to the active site of the E2, a reaction
that is common to all E2s. Alternatively, the correct positioning of Ub
on the E2 surface may be a prerequisite for multi-Ub chain assembly, a
reaction that is common to many E2s. In either case, residues that
define the region of the E2 surface involved in Ub interaction can be
expected to be conserved between different E2s. The three-dimensional
distribution of highly conserved and identical surface residues for six
yeast E2s reveals that a disproportionate number of these positions are
situated on one of the two major E2 faces, particularly in a pocket
that surrounds the active site cysteine (Fig. 4). Significantly, the
amino acid substitutions that give rise to the cdc34-1 allele (Pro
The structural basis for the differential suppression by
UbR48 of the cdc34-2 allele relative to the
cdc34-1 allele is not known. It is possible that
Lys
It is also possible that Lys
Finally although previous work has demonstrated that
CDC34 can assemble Lys
Shown are the growth characteristics for three different
cdc34 mutants expressing different Ub derivatives (see Fig.
1). In the case of cdc34-1 and cdc34-2 mutants, cells were streaked on plates as in Fig. 3. Cells forming
colonies that were comparable in size and number to wild-type CDC34 cells were scored as +, whereas cells forming colonies that
were comparable in size and number to mutant cells lacking the Ub
plasmid were scored as -. Cells with either the cdc34-1 or cdc34-2 mutations were grown at the empirically
determined non-permissive temperature of 32 and 34.5 °C,
respectively (methods). In the case of
cdc34
We thank S. Smith for secretarial assistance and K.
Ellison for editorial assistance. We also thank M. Goebl for furnishing
the yeast strain YL10 and the CDC34 plasmid pGEM34H/S. We also thank D.
Gonda for supplying the yeast strain RS166.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
to S phase. In an effort to identify proteins that interact with
CDC34 we undertook a genetic screen to isolate genes whose increased
expression suppressed the cell cycle defect associated with the
cdc34-2 temperature-sensitive allele. From this screen,
the poly-Ub gene UBI4 was identified as a moderately strong
suppressor. The fact that the overexpression of a gene encoding a
single Ub protein could also suppress the cdc34-2 allele
indicated that suppression was related to the increased abundance of
Ub. Ub overexpression was found to suppress two other structurally
unrelated cdc34 mutations, in addition to the
cdc34-2 allele. In all three cases, suppression depended
on the expression of Ub with an intact carboxyl terminus. Only the
cdc34-2 allele, however, could be suppressed by Ub with
an amino acid substitution at lysine 48 which is known to be involved
in multi-Ub chain assembly. Genetic results showing allele specific
suppression of cdc34 mutations by various Ub derivatives
suggested a specific noncovalent interaction between Ub and CDC34.
Consistent with this prediction, we have shown by chemical
cross-linking the existence of a specific noncovalent Ub binding site
on CDC34. Together, these genetic and biochemical experiments indicate
that Ub suppression of these cdc34 mutations results from the
combined contributions of Ub-CDC34 thiol ester formation and a
noncovalent interaction between Ub and CDC34 and therefore suggest that
the correct positioning of Ub on a surface of the ubiquitin conjugating
enzyme is a requirement of enzyme function.
(
)
to other proteins thereby marking them, in many cases, for
degradation (for reviews, see Refs. 1-5). Mutational analyses of
E2 genes in yeast have revealed that these enzymes participate in a
broad range of cellular processes and probably play at least some role
in target protein recognition.
2 transcriptional repressor is strongly
correlated with the formation of a UBC6-UBC7 hetero-complex
(16) .
Plasmids and Yeast
Strains
cdc34is contained
on a high-copy TRP1-based plasmid. This plasmid is identical
to the CDC34- TRP1 plasmid previously described
(10) except that the region corresponding to the
carboxyl-terminal codons 201-295 of the CDC34 gene have
been deleted. Plasmids expressing the different yeast Ub derivatives
all contain a common previously described cassette
(17) consisting of the CUP1 promotor, Ub coding
sequence, and CYC1 transcriptional terminator. Cassettes
differ from one another only in the codon changes indicated in
Fig. 1
. High-copy LEU2-based plasmids containing these
cassettes are identical to YEp96
(18) except that the
ClaI/ HpaI fragment containing the TRP1 marker was replaced with a NarI/ HpaI fragment
containing the LEU2 gene from the YEp351 plasmid
(19) .
Ub mutants were constructed using polymerase chain
reaction-directed mutagenesis, the details of which are available upon
request. The CDC34-LEU2 plasmid is identical to the
CDC34-TRP1 plasmid previously described
(10) except
that the TRP1 marker was replaced with the LEU2 marker as described above for YEp96. The correct coding sequences
of all expression plasmids were confirmed by DNA sequencing on an
Applied Biosystems automated sequenator operated by the University of
Alberta DNA Sequencing and Synthesis Facility.
Figure 1:
Mutations and constructs. Shown are the
primary protein structures for the various CDC34 and Ub derivatives
used in this study. Amino acid replacements or deletions are indicated
by position. Also indicated for CDC34 is the catalytic domain
( white box) and COOH-terminal extension ( black box).
cdc34-1 and cdc34-2 alleles are expressed
from the normal genomic locus. Other derivatives are expressed from
plasmids (``Materials and
Methods'').
Each Ub plasmid was
introduced into the three cdc34 mutant strains RS166, JP34G2,
and YES71. The temperature-sensitive (ts) cdc34-1 strain
RS166 ( Mata; his1-1; leu2-3,112; trp2; ura3-52;
cdc34-1) was obtained from D. Gonda (Yale University) and
maintained as described
(11) . The ts cdc34-2 mutant strain JP34G2 ( Mata; ade2-1; his3;
leu2
1; trp1; ura3-52; cdc34-2) was constructed
from a cross of the cdc34-2 mutant strain YL10
( Mata; leu2
1; his3
trp1
63; ura3-53;
cdc34-2) and the wild-type strain KMY15 ( Mata;
ade2-1; his3-832; trp1-289; ura3-52). YL10
was obtained from M. Goebl (University of Indiana) and KMY15 was
obtained from K. Madura (California Institute of Technology). The
nature of the cdc34-2 mutation was determined by
sequencing several copies of the cdc34-2 allele that had
been isolated from YL10 genomic DNA using the polymerase chain
reaction. The CDC34 disruption strain YES71 was created from
YL10 by disrupting the cdc34-2 allele with the
ApaI- EcoRI fragment from pGEM34H/S as described
previously
(7) . Viability of YES71 was maintained by the
CDC34- URA3 plasmid, described elsewhere
(10) .
In addition to carrying both the Ub plasmid and the CDC34 maintenance plasmid, YES71 cells also carried the
cdc34
plasmid.
Suppression of cdc34 Mutations by Ub
To
characterize the ability of Ub to suppress the cdc34-1 and cdc34-2 mutations, cells containing various Ub
derivatives were plated on YEPD medium and incubated at nonpermissive
temperatures specific for each strain. The incubation temperature for
each cdc34 mutant strain was chosen as the lowest
nonpermissive temperature at which mutant cells were unable to form
visible colonies after 3 days incubation. Wild-type cells will readily
form visible colonies under these conditions. This empirically
determined minimal nonpermissive temperature was found to be 34.5 and
32 °C for the cdc34-1 and cdc34-2 mutants, respectively. Cells bearing a specific cdc34 mutation in combination with various Ub plasmids were then
streaked on YEPD plates and incubated at the appropriate nonpermissive
temperatures for 3 days.
mutation
by Ub differs from the above method in several respects.
cdc34
is a non-conditional
mutation and therefore cannot support the growth of the cdc34 disruption strain YES71 in the absence of the CDC34 maintenance plasmid. To determine if Ub expression can reverse
this effect, YES71 cells containing the
TRP1- cdc34
plasmid in
combination with one of several LEU2-Ub expression plasmids
(described above) were tested for their ability to form colonies on
plates containing 5-fluoroorotic acid upon spontaneous loss of the
maintenance plasmid as described previously
(10) .
5-Fluoroorotic acid is a metabolic poison that kills cells retaining
the maintenance plasmid by virtue of the presence of the URA3 marker
(22) . As a negative control, the Ub plasmid was
substituted with the LEU2 high-copy plasmid YEP351
(19) . As a positive control, the Ub plasmid was replaced with
the CDC34-LEU2 plasmid (described above). Prior to plating,
these cells were grown under nonselective conditions at 25 °C in
YEPD broth. Approximately 3
10
cells for each
plasmid derivative were plated on S.D. minimal medium plates
(supplemented with 5-fluoroorotic acid and all nutrients except leucine
(21) ) and incubated for 4 days at 25 °C.
CDC34 Expression and Purification
Expression of
the yeast CDC34 protein in E. coli and the subsequent
purification of the radiolabeled protein was performed as described
(12) . Briefly, the CDC34 overexpression plasmid pREGB6 was
co-transformed into the E. coli strain BL21 in combination
with the thermally induced T7 polymerase plasmid, pGP1-2
(23) . Cells were then grown in 6 ml of M9 media supplemented
with 1 m
M MgSO, 0.1 m
M CaCl
,
12 m
M glucose, 18 µg/ml thiamine, 50 µg/ml ampicillin,
75 µg/ml kanamycin, and all amino acids (40 µg/ml) except for
cysteine and methionine. Cultures were then induced at 42 °C for 20
min followed by the addition of rifampicin (300 µg/ml final). Cells
were then incubated for 10 min at 42 °C followed by a shift to 37
°C for 1 h. Trans-[
S]methionine (ICN) was
then added (25 µCi/ml) followed by incubation for 10 min at 37
°C. Cells were harvested by centrifugation, resuspended in 500
µl of 25% sucrose, 50 m
M Tris-Cl (pH 8.0) and lysed with
lysozyme as described previously
(24) . Labeled CDC34 protein
was purified using an FPLC system (Pharmacia) by passing clarified
supernatants over a MonoQ HR 5/10 ion exchange column (Pharmacia)
equilibrated with buffer A (50 m
M Tris-Cl, pH 7.5, 1 m
M EDTA, 1 m
M dithiothreitol) and eluted using an NaCl
gradient from 0 to 1
M (45) . Under these conditions,
CDC34 eluted as a major protein peak at 470 m
M NaCl. Peak
fractions were pooled, concentrated, and exchanged with buffer A by
Centricon (Amicon) filtration and stored at -80 °C in the
presence of 5% glycerol.
Protein Cross-linking
Chemical cross-linking of
radiolabeled CDC34 and unlabeled Ub (Sigma) or bovine serum albumin
(BSA) was performed using BS(bis(sulfosuccinimidyl)suberate (Pierce)). Prior to
cross-linking, purified CDC34, Ub, and BSA proteins were either
dialyzed or resuspended in cross-linking buffer (50 m
M HEPES
(pH 7.5), 150 m
M NaCl, 2 m
M dithiothreitol). The
final concentrations of CDC34, Ub, and BSA in respective cross-linking
reactions were adjusted to 1.0 µ
M for CDC34 and either 5.0
or 50 µ
M for Ub and BSA. Cross-linking reactions (40
µl) containing CDC34 alone or with Ub or BSA were preincubated on
ice for 5 min followed by the addition of an 0.1 volume of the BS
cross-linker, to a final concentration of 0.5 m
M.
Samples were incubated an additional 30 min on ice and the reaction was
quenched by the addition of 1
M Tris-HCl (pH 7.5) to a final
concentration of 50 m
M. Cross-linked species were separated by
SDS-polyacrylamide gel electrophoresis (10% acrylamide, 1.3%
bis-acrylamide) and detected by either autoradiography or
PhosphorImaging for quantitation. Molecular mass estimates of the
proteins and their cross-linked products were determined on the basis
of their migration on SDS-polyacrylamide gels relative to known
molecular mass standards. Protein standards (Bio-Rad) included: myosin,
205 kDa;
-galactosidase, 116.5 kDa; phosphorylase b, 106
kDa; BSA, 80 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa;
soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa.
Three-dimensional Analysis of E2 Conservation
Each
of the amino acid sequences for six yeast E2s (UBC1
(25) , UBC2
(8) , UBC3
(7) , UBC4
(26) , UBC6
(27) ,
UBC7
(28) ) were aligned in all possible pairwise combinations
using the alignment package provided in the IG suite of DNA programs.
Numerical values representing the similarity of amino acids for a given
position in the sequence of each pair were then summed for all pairs.
The resulting values at each amino position were then ranked into five
categories depending on magnitude: 1) unconserved; 2) poorly conserved;
3) moderately conserved; 4) strongly conserved; or 5) identical. Each
category was then assigned a shade of blue using the RGB numerical
color system provided in the Biosym Insight II molecular modeling
software package (unconserved, white; identical, dark blue). The CPK
image shown in Fig. 4was generated using this package on a
Silcon Graphics Iris Indigo work station.
Figure 4:
Structural defects associated with the
cdc34-1 and cdc34-2 alleles are situated
on the conserved E2 surface. Shown is a space-filling three-dimensional
model of the yeast UBC4 E2 (47) used here a general representation of
an E2 catalytic domain. Both images differ from each other by a
180° rotation. The conservation of each amino acid position within
the structure is indicated by varying shades of blue as described under
``Materials and Methods'': white, unconserved; faint blue,
poorly conserved; light blue, moderately conserved; medium blue, highly
conserved; dark blue, identical. Amino acid positions that are
substituted in the cdc34-1 and cdc34-2 proteins are
indicated in red. The cdc34-1 and cdc34-2 mutations result in Proto Ser and Gly
to Arg, respectively. These loci correspond to Pro
and Gly
of the UBC4 protein. The COOH-terminal
residue ( C) denotes the boundary that separates the catalytic
domain of CDC34 from its tail. The catalytic cysteine is indicated in
yellow.
) that can fully rescue
either of the ts mutations described above, but which fails to support
growth of a cdc34 disruption strain
(12) . In view of
the different positions of each of these mutations, it was of interest
to see if the suppression by Ub overexpression observed for the
cdc34-2 allele applied to these other two alleles. As
seen in Table I, both the cdc34-1 and
cdc34
alleles could be suppressed
by Ub overexpression, therefore the suppressor activity of Ub
overexpression is not restricted to a particular class of cdc34 mutation.
-Gly
are required for Ub activation and
conjugation
(31, 32) . The Lys
residue is
targeted for multi-Ub chain assembly
(33) , an apparent
requirement for efficient proteolysis
(33, 34, 35, 36, 37, 38) .
In addition, we have recently found that Lys
and
Lys
are also involved in Ub chain formation and that
Lys
appears to play an important role in the yeast stress
response
(39) . Based on the functional significance of these
residues, we examined whether or not the overexpression of Ub carrying
mutations at these positions affected the suppression of the three
cdc34 mutants described above (). Fig. 2 visually
illustrates the influence of various Ub derivatives on the colony
forming ability of one cdc34 mutant strain
( cdc34-1) at the nonpermissive temperature.
-Gly
residues
deleted ( Ub
) failed to suppress the growth defect
associated with any of the three cdc34 mutations. These
results indicate that only Ub molecules that can be covalently attached
to the active site cysteine of CDC34 can function as suppressors.
Interestingly, Ub with a Lys to Arg mutation at position 48
( UbR48), fully suppressed the defect associated with the
cdc34-2 allele, but failed to suppress either the
cdc34-1 or cdc34
defects. Similarly, Lys to Arg mutations at positions 29, 48, and
63 (UbR
) also fully suppressed the cdc34-2 defect, but failed to suppress the cdc34-1 or
cdc34
defects. Therefore,
suppression is dependent on the types of Ub and cdc34 mutations placed in combination with each other.
was used in combination with purified
radiolabeled CDC34 in the presence or absence of a molar excess of
either Ub or BSA (as a nonspecific negative control). CDC34 in the
presence of BS
gives rise to a series of cross-linked CDC34
multimers indicating a weak but specific interaction of CDC34 with
itself as described previously
(10) . Under these
electrophoretic conditions, monomeric CDC34 migrates with an apparent
molecular mass of 42 kDa, therefore the lowest CDC34 multimer band
which migrates at 90 kDa corresponds to a CDC34 cross-linked dimer. The
relative migration of the other two multimer bands indicated in
Fig. 3
fall well outside of the normal logarithmic relationship
between mobility and molecular mass observed for molecular mass
standards under these gel conditions, therefore although their
molecular masses have been roughly estimated to be 170 and 190 kDa,
their stoichiometry is uncertain.
Figure 3:
Cross-linking of Ub to CDC34. Radiolabeled
CDC34 was incubated in the presence (+) or absence (-) of
the cross-linker BSand in combination with either a 5-fold
(5
) or 50-fold (50
) molar excess of Ub or BSA relative
to CDC34 (1.0 µ
M). Following cross-linking, each reaction
was electrophoresed on a 10% SDS-polyacrylamide gel followed by
autoradiography. Indicated are the positions of monomeric CDC34
(CDC34), the Ub-CDC34 cross-linked product, and three cross-linked
CDC34 multimers. Dots denote the position of two minor
co-purifying non-CDC34 contaminants. Apparent molecular masses are as
follows: CDC34, 42 kDa; Ub-CDC34, 50 kDa; CDC34 multimers, 90, 170, and
190 kDa.
Significantly, the addition of a
50-fold molar excess of BSA as a control for nonspecific cross-linking
does not appreciably lower the yield of cross-linked CDC34 multimers or
result in detectable levels of BSA-CDC34 cross-linked products. Thus,
the cross-linking of CDC34 to itself reflects a specific set of
interactions between CDC34 monomers. By comparison with BSA addition, a
5-fold molar excess of Ub is sufficient to produce a major cross-linked
product containing radiolabeled CDC34 with an apparent molecular mass
of 50 kDa, precisely that predicted for a cross-linked Ub-CDC34
heterodimer (42 kDa for the CDC34 monomer plus 8 kDa for the Ub
monomer). The abundance of this species increases from 7% of the CDC34
monomer at a 5-fold excess of Ub, to 22% at a 50-fold excess of Ub,
therefore the formation of this species is strongly dependent on Ub
concentration. Based on these findings it can be concluded that Ub
forms a weak but specific noncovalent interaction with CDC34.
-Gly
from the carboxyl
terminus of Ub eliminates the ability of Ub to suppress the defects
associated with each of the three cdc34 mutations. Based on
these findings, we believe that Ub stabilizes each of the three defects
in the cdc34 polypeptide through its direct noncovalent interaction
with CDC34 and that this interaction in turn is stabilized by formation
of the Ub-CDC34 thiol ester intermediate.
to Ser) and the cdc34-2 allele (Gly
to Arg) both occur at highly conserved
positions that are situated on this side of the E2. Based on these
observations we speculate that Ub interacts with the conserved side of
the E2, thereby stabilizing the thermolabile defects associated with
the cdc34-1 and cdc34-2 alleles by virtue
of their proximity to the site of interaction.
(
)
normally makes a key interaction with CDC34 that is
critical for stabilizing the cdc34-1 Pro
to Ser
substitution but not the cdc34-2 Gly
to Arg
substitution owing to an orientation of Ub on the E2 surface that
positions Lys
close to position 71 and distal to position
58. Substitution of Lys
might then negate the suppression
of the cdc34-1 allele with little or no effect on the
cdc34-2 allele.
plays a role in stabilizing the interaction of CDC34 with itself,
an interaction that has previously been correlated with CDC34 function
(10, 12) . This argument is based on the idea that the
stability of two interacting CDC34 monomers each coupled to a Ub
molecule through thiol ester formation is governed in part by the
noncovalent interaction between each Ub molecule in a manner that
facilitates the covalent linkage of one to the other at
Lys
. Such an interaction has been suggested from the
recently determined crystal structure of the Lys
linked Ub
dimer
(44) . Since Lys
is situated at the Ub dimer
interface, its substitution might be expected to weaken the Ub-Ub
interaction to the detriment of one cdc34 allele but not the
other. In this example, Ub binding pockets on each E2 monomer might
participate in the correct orientation of Ub monomers for multi-Ub
chain assembly.
linked multi-Ub chains in vitro (45, 46) , the results of the present study suggest
that Lys
chain assembly on the putative target(s) of CDC34
may not be obligatory for CDC34's cell cycle function. This
conclusion is based on the fact that the levels of UbR48 suppression
obtained for cdc34-2 were indistinguishable when
compared to the expression of wild-type Ub. Since Ub suppression
requires an intact carboxyl terminus, it is only reasonable to assume
that the Ub pool which participates in suppression is also being
transferred to the target of CDC34. The fact that UbR48 is fully
functional as a suppressor of the cdc34-2 allele
suggests that CDC34 need not target a Lys
type multi-Ub
chain to its target to maintain its function and that the transfer of a
single Ub may be sufficient for target inactivation. Recently we have
found that Lys
and Lys
can also serve as
linkage sites for multi-Ub chain assembly in vivo (39) . The observation that mutation of any of these
linkage positions has no effect on cdc34-2 suppression
suggests that, like Lys
these other chain configurations
are not essential to CDC34 function.
Table: Suppression of three cdc34 mutations by Ub
, growth was assessed as the
ability to form colonies on 5-fluoroorotic acid plates as a consequence
of the loss of the CDC34 maintenance plasmid (``Materials
and Methods'').
,
bis(sulfosuccinimidyl)suberate.
tail deletion mutant conveys no additional
information in this regard since the structure and position of the tail
with respect to the catalytic domain have not yet been determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.