©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Increased Ubiquitin Expression Suppresses the Cell Cycle Defect Associated with the Yeast Ubiquitin Conjugating Enzyme, CDC34 (UBC3)
EVIDENCE FOR A NONCOVALENT INTERACTION BETWEEN CDC34 AND UBIQUITIN (*)

John A. Prendergast , Christopher Ptak (§) , Terra G. Arnason (§) , Michael J. Ellison (¶)

From the (1) Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the Gto 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.


INTRODUCTION

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)() 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.

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 MAT2 transcriptional repressor is strongly correlated with the formation of a UBC6-UBC7 hetero-complex (16) .

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.


MATERIALS AND METHODS

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; leu21; trp1; ura3-52; cdc34-2) was constructed from a cross of the cdc34-2 mutant strain YL10 ( Mata; leu21; his3 trp163; 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 cdc34plasmid.

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.

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.

The strategy used to measure the suppression of the cdc34mutation by Ub differs from the above method in several respects. cdc34is 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- cdc34plasmid 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 10cells 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 BScross-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 Glyto Arg, respectively. These loci correspond to Proand Glyof 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.




RESULTS

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) 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 cdc34alleles could be suppressed by Ub overexpression, therefore the suppressor activity of Ub overexpression is not restricted to a particular class of cdc34 mutation.

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-Glyare required for Ub activation and conjugation (31, 32) . The Lysresidue 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 Lysand Lysare also involved in Ub chain formation and that Lysappears 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.

The expression of Ub having the Gly-Glyresidues 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 cdc34defects. 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 cdc34defects. Therefore, suppression is dependent on the types of Ub and cdc34 mutations placed in combination with each other.

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 BSwas 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 BSgives 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.


DISCUSSION

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-Glyfrom 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.

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 (Proto Ser) and the cdc34-2 allele (Glyto 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.()

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 Lysnormally makes a key interaction with CDC34 that is critical for stabilizing the cdc34-1 Proto Ser substitution but not the cdc34-2 Glyto Arg substitution owing to an orientation of Ub on the E2 surface that positions Lysclose to position 71 and distal to position 58. Substitution of Lysmight then negate the suppression of the cdc34-1 allele with little or no effect on the cdc34-2 allele.

It is also possible that Lysplays 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 Lyslinked Ub dimer (44) . Since Lysis 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.

Finally although previous work has demonstrated that CDC34 can assemble Lyslinked multi-Ub chains in vitro (45, 46) , the results of the present study suggest that Lyschain 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 Lystype 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 Lysand Lyscan 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 Lysthese other chain configurations are not essential to CDC34 function.

  
Table: Suppression of three cdc34 mutations by Ub

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, 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'').



FOOTNOTES

*
This work was supported in part by an operating grant from the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Funded by studentships from the Alberta Heritage Foundation for Medical Research.

Medical Council of Canada Research Scholar. To whom correspondence should be addressed. Tel.: 403-492-5839; Fax: 403-492-0886.

The abbreviations used are: Ub, ubiquitin; BSA, bovine serum albumin; BS, bis(sulfosuccinimidyl)suberate.

The cdc34 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.


ACKNOWLEDGEMENTS

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.


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