The Yeast Ubiquitin Protease, Ubp3p, Promotes Protein Stability

Corresponding author: Tim C. Huffaker, Biotechnology Bldg., Cornell University, Ithaca, NY 14853-2703., tch4{at}cornell.edu (E-mail)

Communicating editor: T. STEARNS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3 stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3 produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination.

IN yeast, the ubiquitin-proteasome pathway is responsible for degradation of short-lived and abnormal proteins, whereas the vacuole mediates degradation of long-lived proteins (JONES 1991 ; reviewed in FINLEY 1992 ; HOCHSTRASSER 1996 ; HERSHKO and CIECHANOVER 1998 ). Ubiquitin is covalently ligated to target proteins by a multienzymatic system consisting of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. A large number of E2 enzymes provide substrate specificity either alone or along with the E3 enzymes (HOCHSTRASSER 1996 ). Ubiquitinated proteins are targeted to the 26S proteasome, which consists of the 19S particle and the 20S proteasome (reviewed in GLICKMAN 2000 ). The 19S particle is the regulatory unit and consists of a polyubiquitin recognition site, ATPases that provide energy for unfolding proteins, and a deubiquitinating enzyme that recycles ubiquitin. The unfolded and extended polypeptides are then allowed to enter the rings of the 20S proteasome, the proteolytic core that contains multiple peptidase activities for protein degradation.

The deubiquitinating enzymes (Dubs) are a large family of cysteine proteases that cleave ubiquitin from conjugated protein substrates or precursor proteins (D'ANDREA and PELLMAN 1998 ; reviewed in CHUNG and BAEK 1999 ). These thiol proteases hydrolyze the amide bond between Gly76 of ubiquitin and a Lys residue of the substrate protein or preceding ubiquitin. There are two classes of Dub enzymes, the Ubp family (ubiquitin-specific proteases) and the Uch family (ubiquitin carboxy-terminal hydrolases). The Uch enzymes, for example, Yuh1p in yeast, cleave ubiquitin from peptides and small adducts (ROSE and WARMS 1983 ; PICKART and ROSE 1985 ; LIU et al. 1989 ; BAKER et al. 1992 ). Ubp enzymes cleave ubiquitin from a range of substrates.

It has been suggested that deubiquitination enzymes, like the ubiquitin-conjugating enzymes, have diverse roles in regulation of protein degradation or modification. The study of several of these enzymes suggests that they can have either a positive or an inhibitory effect on proteolysis. Positive regulation occurs when polyubiquitin chains are cleaved to produce free ubiquitin groups that are then available for attachment to new substrates targeted for degradation. For example, Ubp14p in yeast cleaves isopeptide-linked polyubiquitin chains that are unanchored to a substrate (AMERIK et al. 1997 ). This activity may also be necessary for the generation of free ubiquitin moieties from ubiquitin fusions that are encoded by UBI1, UBI2, UBI3, and UBI4 in yeast (OZKAYNAK et al. 1987 ). Doa4p/Ubp4p is required for cleavage of polyubiquitin chains from proteolytic intermediates, which provides free polyubiquitin chains (PAPA and HOCHSTRASSER 1993 ). Thus, Doa4p may promote proteolysis by increasing ubiquitin pools and removing proteolytic remnants that would otherwise inhibit proteasome activity by a negative feedback mechanism.

A negative effect on proteolysis could occur if substrates were diverted from proteasomal degradation by the reversal of ubiquitination. The Fat facets (FAF) protein is a Ubp enzyme in Drosophila that acts as a negative regulator of the ubiquitin system (HUANG et al. 1995 ). A proteasomal mutation was found to suppress the faf mutant phenotype, suggesting that FAF protein has activity that antagonizes proteasome function. It may have a proofreading function, reversing ubiquitination of substrate proteins to prevent or slow their degradation by the proteasome.

In yeast, 16 Ubp enzymes are predicted from sequence analysis. They show little homology beyond six conserved regions, three of which contain the enzyme active site, and three that have unknown function but may provide a ubiquitin binding site. The N-terminal regions are divergent and may provide substrate recognition. Ubp enzymes have overlapping functions as suggested by the normal growth rate of ubp1 ubp2 ubp3 and the ubp8-like growth rate of the ubp1 ubp2 ubp3 ubp7 ubp8 quintuple mutants (BAKER et al. 1992 ; AMERIK et al. 2000 ). In this article, we describe a genetic interaction between UBP3 and STU1, a microtubule-associated protein involved in formation of the Saccharomyces cerevisiae mitotic spindle (PASQUALONE and HUFFAKER 1994 ). Stu1p is a member of the Stu1-MAST family that includes S. cerevisiae Stu1p; Schizosaccharomyces pombe Stu1p; and the more distantly related Drosophila Mast, human CLASP1 and CLASP2, and three unknown open reading frames (ORFs) in Caenorhabditis elegans (CeCO7H6.3, CeR107.6, and CeZC84.3; PASQUALONE and HUFFAKER 1994 ; LEMOS et al. 2000 ; AKHMANOVA et al. 2001 ). UBP3 was initially isolated in a screen for yeast genes that, when coexpressed with Ub-ß-galactosidase in Escherichia coli, resulted in removal of the ubiquitin moiety (BAKER et al. 1992 ). UBP3 was also isolated as a high-copy suppressor of the temperature sensitivity of yeast cells lacking two molecular chaperone genes, SSA1 and SSA2 (BAXTER and CRAIG 1998 ). The disruption of UBP3 resulted in the accumulation of ubiquitin-protein conjugates, suggesting that Ubp3p reverses the ubiquitination of substrate proteins (BAXTER and CRAIG 1998 ). Ubp3p was also shown to bind to Sir4p and hypothesized to inhibit transcriptional silencing (MOAZED and JOHNSON 1996 ). In this study, we report the identification of UBP3 in a screen for mutations that are synthetically lethal with stu1-5. This genetic interaction is unique to ubp3 in the Ubp family. Our data support the idea that Ubp3p deubiquitinates misfolded proteins, giving them an opportunity to refold and function in the cell.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, plasmids, and media:
The yeast strains and plasmids used in this study are listed in Table 1 and Table 2, respectively. gim1, gim4, and gim5 strains and plasmids containing GIM1, GIM4, and GIM5, respectively, were provided by Elmar Schiebel (The Beatson Institute for Cancer Research, Glasgow, Scotland). Plasmids containing RAT1NLS and kem1-E176G were provided by Arlen Johnson (University of Texas, Austin, TX), and kem1-D206A, kem1-D208A, and kem1-D206A, D208A were provided by Wolf-Dietrich Heyer (University of California, Davis, CA). ABY544 was provided by Tony Bretscher (Cornell University, Ithaca, NY). CUY1331 was created by transformation of a stu1-5::LEU2 integrating plasmid (pCK16) into CUY1061 to generate stu1-5::LEU2::stu1-5. CUY1331 was then transformed with an ADE3 URA3 plasmid containing the STU1 gene (pDP96) to create CUY1332.

YPD and SD media and plates were prepared as described by SHERMAN 1991 . Benomyl plates were prepared by adding an appropriate amount of 10 mg/ml stock to YPD plates. 5-FOA plates were made at a concentration of 1 mg/ml in SD. Geneticin (G418 sulfate, Stratagene, La Jolla, CA) was used at 0.2 mg/ml.

Isolation of mutants that require STU1 for growth:
The adenine red-white sectoring assay (BENDER and PRINGLE 1991 ) was used to identify mutations that are synthetically lethal with stu1-5 (see RESULTS). Strain CUY1332 was mutagenized with methane sulfonic acid ethyl ester (EMS, Sigma, St. Louis) to 45% viability (GUTHRIE and FINK 1991 ). For all genetic crosses, we dissected at least 11 tetrads and defined genes as tightly linked if no recombinants were observed.

Disruption of UBP5, UBP7, UBP8, UBP9, and UBP16:
Deletion strains doa4, ubp10, and ubp14 and strains ubp1, ubp2, ubp6, ubp11, ubp12, ubp13, and ubp15 were kindly provided by Mark Hochstrasser (Yale University, New Haven, CT) and Rohan Baker (Australian National University), respectively. The UBP3, UBP5, UBP7, UBP8, UBP9, and UBP16 genes were completely disrupted by one-step gene replacement (BAUDIN et al. 1993 ). PCR primers containing genomic DNA sequence 60 bp upstream and 60 bp downstream of each UBP gene were used to amplify pFA6a-His3MX6 (WACH et al. 1997 ). The resultant PCR products, UBP-flanking sequences on either side of the HIS5 gene, were transformed into CUY30. Histidine prototrophs were selected, and correct integration was confirmed by PCR amplification of the respective UBP locus. ubp1–ubp16 were then crossed with stu1-5, stu1-5::URA3, or stu1-5::LEU2 (CUY997, CUY1005, CUY1338, CUY1339, CUY1340) to create stu1-5 ubp double mutants.

Flow cytometry:
Haploid yeast cells were prepared for flow cytometry by the method of HUTTER and EIPEL 1978 . The DNA content of 10,000 cells was determined using a FACScan flow cytometer (Becton Dickinson). CELL QUEST software was used to obtain and analyze data (BDIS, San Jose, CA).

Immunoblot analysis:
Strains were harvested by centrifugation; washed in breakage buffer (30 mM NaPO₄, pH 7.0, 60 mM B-glycerophosphate, 150 mM KCl, 6 mM EDTA, 6 mM EGTA, 10% glycerol); resuspended in breakage buffer with 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin; and flash frozen in liquid nitrogen. Frozen pellets were ground with a mortar and pestle. Cell debris was removed by centrifugation. The quantity of protein in the extracts was determined by the Bradford assay (BRADFORD 1976 ).

For anti-Stu1 immunoblot analysis, cell extracts were boiled in Laemmli sample buffer and clarified by centrifugation. Extracts were separated by SDS-PAGE and transferred to PVDF membrane (Hybond-P, Amersham, Arlington Heights, IL). Membranes were incubated with anti-Stu1p polyclonal antibodies (a gift from Liru You) in Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20 containing 5% nonfat dry milk, followed by incubation with anti-rabbit IgG conjugated to alkaline phosphatase (Amersham). The membranes were also blotted with anti-Act1p (a gift from Tony Bretscher) as a loading control.

Antibody binding was detected using Western blotting ECF reagents from Amersham. Levels of protein were quantified from data collected on a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and the use of ImageQuant software. The levels of Stu1p were normalized to Act1p levels. The linear range of Stu1p was determined using serial dilutions of yeast cell extracts.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Screen for mutations that are synthetic lethal with stu1-5:
To identify genes whose products interact with Stu1p, we performed a genetic screen for extragenic mutations that are synthetically lethal with the temperature-sensitive stu1-5 allele. An initial screen produced exclusively intragenic mutations so a second screen was performed using a strain that contained two copies of stu1-5. The ssl (stu1-5 synthetic lethal) mutations were generated by EMS mutagenesis and identified by an adenine red-white sectoring assay (see MATERIALS AND METHODS). At 30°, CUY1332 (stu1-5::LEU2::stu1-5 ade2 ade3 ura3 [pDP96]) grows with a red colony color, which sectors white upon spontaneous loss of pDP96 (STU1 ADE3 URA3). The presence of a second-site mutation that is lethal in combination with stu1-5 would render the strain unable to lose pDP96 at the permissive temperature and, therefore, unable to sector.

A total of 31,500 colonies were screened for a nonsectoring phenotype at 30°. Of these, 185 (0.6%) nonsectoring colonies were further tested for a STU1 requirement by plating on 5-FOA, which selects against the URA3 gene. Ninety-one strains were 5-FOA sensitive indicating that their nonsectoring phenotype is due to their inability to lose the STU1 plasmid and not to reversion or conversion at the ade3 locus. Finally, to show that the nonsectoring and 5-FOA sensitivity phenotypes were due to the strains' requirement for STU1, a STU1 HIS3 plasmid (pCK17) was transformed into each strain, and 35 were found to sector and grow on 5-FOA. Thus, these 35 strains contain one or more mutations that are specifically lethal in combination with stu1-5.

Tetrad analysis was done to determine if the mutations responsible for synthetic lethality were in a single gene. Each stu1-5 ssl strain harboring pDP96 was mated with a stu1-5 strain (CUY1333), and the resultant diploid was sporulated. For 13 mutants, 5-FOA sensitivity segregated 2:2 in tetrads, indicating that synthetic lethality was due to a mutation in a single gene. One mutation was tightly linked to the TUB2 locus and none were linked to stu1-5.

Each of the 13 stu1-5/stu1-5 ssl/SSL diploids harboring pDP96 was 5-FOA resistant, indicating that all 13 ssl mutations are recessive. Complementation analysis was performed to determine the number of genes represented by these mutations. The stu1-5 ssl strains containing pDP96 were crossed in a pairwise fashion to one another and scored for growth on 5-FOA. The diploids that required pDP96 for growth contained alleles in the same complementation group. The 13 ssl mutations fall into 10 complementation groups, one with four members and nine with one member each (Table 3).

Identification of the genes:
Complementation groups I–IV were cloned by rescue of the synthetic lethality at 30°. stu1-5 ssl strains were transformed with a Ycp-based yeast genomic library (WANG and HUFFAKER 1997 ) and the transformants were screened for restoration of the sectoring phenotype. The rescuing plasmids were isolated and retransformed into the mutant to ensure plasmid dependence. The endpoints of the genomic insert were then sequenced to identify the genomic locus. Each chromosomal locus was marked by integration of an auxotrophic marker and shown to be linked to the corresponding synthetic lethal mutation in crosses. To identify the relevant ORF on the genomic fragment, individual ORFs were subcloned into LEU2 YCp plasmids, transformed into the parent strain, and analyzed for rescue of the synthetic lethality (Table 3).

Groups II and III consisted of two members of the GimC complex, PAC10/GIM2 and GIM3. The GimC complex is homologous to the mammalian prefoldin complex that binds to nascent polypeptides of tubulin and actin during translation and, when synthesis is complete, transfers these proteins to the cytosolic chaperonin (COWAN 1998 ; HANSEN et al. 1999 ). Because we identified two of the SSL genes as PAC10 and GIM3, we tested the remaining ssl mutations for rescue by other GimC complex members, GIM1, GIM4, or GIM5, but none were rescued. We also tested gim1, gim4, and gim5 for synthetic lethality with stu1-5, and found that all family members were lethal in combination with stu1-5 (data not shown). The synthetic lethality may result from the combined effects of tubulin misfolding and defects in microtubule assembly caused by the stu1-5 mutation. Alternatively, the GimC complex may play a direct role in Stu1p folding.

Group IV was identified as KEM1/XRN1/SEP1. Kem1p is a nonessential cytoplasmic 5'–3' RNA exonuclease that is responsible for turnover of mRNA and rRNA (STEVENS et al. 1991 ; LARIMER et al. 1992 ; MUHLRAD et al. 1994 ; CAPONIGRO and PARKER 1996 ). In addition, several studies suggest that Kem1p may play a role in microtubule function. kem1 mutations cause hypersensitivity to benomyl, karyogamy defects, increased chromosome loss frequencies, impaired spindle pole body (SPB) separation, and defective nuclear migration and show genetic interactions with tubulin genes (KIM et al. 1990 ; INTERTHAL et al. 1995 ). These latter phenotypes and the synthetic lethality with stu1-5 may be secondary to defects in RNA turnover or indicate an independent role for Kem1p in microtubule function. To answer this question with regard to stu1-5 synthetic lethality, we used two types of mutants. RAT1 encodes a nuclear protein that shares considerable homology with Xrn1p and is required for RNA degradation and numerous RNA 5' processing reactions important for ribosome biogenesis (HENRY et al. 1994 ; PETFALSKI et al. 1998 ). There is no evidence for a microtubule-associated role for Rat1p thus far. Mutations in the NLS of Rat1p mislocalize the protein to the cytoplasm and complement kem1 mutant phenotypes (JOHNSON 1997 ). We found that rat1NLS (pAJ228) rescues stu1-5 kem1 synthetic lethality (data not shown), suggesting that the exonuclease function of Rat1p is sufficient to restore the growth of stu1-5 kem1. We also tested several KEM1 exonuclease point mutations for their ability to complement stu1-5 kem1 synthetic lethality. If the synthetic lethality is due solely to overlapping Stu1p and Kem1p function in a microtubule-associated role, then the exonuclease activity of Kem1p should not be needed to complement the double mutant. We tested four kem1 mutations (kem1-E176G, kem1-D206A, kem1-D208A, and kem1-D206A, D208A) that specifically eliminate KEM1 exonuclease activity (PAGE et al. 1998 ; SOLINGER et al. 1999 ). None of these mutant alleles rescued the growth defect of stu1-5 kem1. Thus, the lack of exonuclease activity is likely to be responsible for the lethality between stu1-5 and kem1.

stu1-5 is uniquely synthetically lethal with ubp3:
Complementation group I was identified as UBP3. Ubp3p is a member of a large family of ubiquitin proteases. To determine if other family members are functionally related to UBP3, we tested whether stu1-5 was synthetically lethal with deletions of the other 15 UBP genes. stu1-5 ubp3 strains do grow at 26°, but grow only poorly at 30° and not at all at 33°, a temperature that is permissive for stu1-5 growth. All of the other stu1-5 ubp double mutants grew well at these temperatures, with the exception of stu1-5 ubp5, which grew somewhat more slowly than wild-type at 30° and 33° (Fig 1). Conversely, doa4 and ubp6 partially rescued stu1-5 temperature sensitivity at 37°.

View larger version (30K): In this window In a new window Download PPT slide   Figure 1. stu1-5 is uniquely synthetically lethal with ubp3. STU1 (CUY25), stu1-5 (CUY999), and stu1-5 ubp1 through ubp16 were plated onto YPD media and assayed for growth at the indicated temperature.

stu1-5 and ubp3 are benomyl sensitive:
We examined the benomyl sensitivity of stu1-5 and ubp alleles because many mutants with altered microtubule function are sensitive to this microtubule-depolymerizing drug. Wild-type haploid cells grow well on 15 µg/ml of benomyl but fail to grow on 30 µg/ml (Fig 2). stu1-5 cells are more sensitive than wild-type cells, growing very poorly on 15 µg/ml benomyl. However, the sensitivity can be fully rescued by providing stu1-5 on a low-copy YCp or high-copy YEp plasmid. This indicates that benomyl sensitivity results from a low quantity of protein rather than from a protein that is inactive at the restrictive temperature.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. stu1-5 and ubp3 are benomyl sensitive. STU1 (CUY25), stu1-5 (CUY999), stu1-5 transformed with YEp stu1-5 (pCK37) or YCp stu1-5 (pCT1), and ubp1 through ubp16 (see Table 1) were plated onto various concentrations of benomyl and assayed for growth at 30°.

Several of the ubp mutations also alter the benomyl sensitivity of cells (Fig 2), but only ubp3 is as benomyl sensitive as stu1-5. ubp7 and ubp16 are somewhat more sensitive than wild-type cells, and ubp6 and ubp10 are more resistant to benomyl than are wild-type cells.

Stu1p levels are constant through the cell cycle:
The role of ubiquitination in cell-cycle regulation is well documented. The APC activates the ubiquitination and degradation of Pds1p, B-type cyclins, and spindle-associated Ase1p for cells to undergo sister chromatid separation and exit from mitosis (COHEN-FIX et al. 1996 ; JUANG et al. 1997 ; CIOSK et al. 1998 ). Because Stu1p is required for mitosis, we looked at the levels of Stu1p through the cell cycle to determine if it is regulated in a similar manner.

An effective approach for synchronizing yeast cells is to arrest them in M phase by depletion of Cdc20p and then release them from this block by inducing Cdc20p (LIM et al. 1998 ). Strain K7428 (cdc20 P_GAL-CDC20::TRP1) was grown at 26° to mid-log phase in galactose media. The cells were washed and grown in glucose media for 3 hr, which arrested the cells in M phase. The culture was shifted back to galactose media and aliquots of cells were examined at 5- to 20-min intervals after release from the cdc20 block. The samples were processed for flow cytometry and immunoblotting.

FACS analysis showed that the cdc20 cells arrested uniformly in early M phase with 2C DNA content. After release from the block, cells progressed through the cell cycle in synchrony, reaching M phase again by 102 min (Fig 3A). The levels of Stu1p remained fairly constant through the cell cycle (Fig 3B). The modest increase in protein from 0 to 30 min is likely due to the change of carbon source from glucose to galactose. Because Stu1p does not show significant fluctuation through the cell cycle, this protein is apparently not the target of ubiquitin-mediated cell-cycle regulation. Thus, the synthetic lethality between stu1-5 and ubp3 must involve some other process.

View larger version (23K): In this window In a new window Download PPT slide   Figure 3. Stu1p levels are constant through the cell cycle. cdc20 PGAL-CDC20::TRP1 (CUY1348) was grown to mid-log phase in galactose medium. The culture was shifted to glucose medium for 3 hr, which arrested the cells in M phase. The culture was shifted back to galactose medium at time zero. (A) The DNA content of the cells at each time point was determined by flow cytometry. (B) Cell extracts were analyzed by immunoblotting with anti-Stu1p and anti-Act1p antibodies and Stu1p levels were normalized to Act1p levels. The range of data from two independent experiments is indicated. The amount of Stu1p in unsynchronized cultures grown in galactose medium is indicated by the arrow.

Overexpression of stu1-5 suppresses both stu1-5 heat sensitivity and synthetic lethality with ubp3:
The ubiquitin system also targets misfolded proteins for degradation, so we examined the possibility that stu1-5 ubp3 synthetic lethality may result from a decrease in the stability of Stu1-5p in the absence of Ubp3p. A STU1 strain (CUY25) grows at temperatures up to 37° (Fig 4A). The stu1-5 strain (CUY999) grows at 33° but does not grow at 35°. The stu1-5 ubp3 double mutant (CUY1325) grows poorly at 30° and is inviable at 33°. We examined the levels of Stu1p in STU1, stu1-5, and stu1-5 ubp3 strains grown at 26°, 33°, and 35° to determine if the progressive decrease in permissive temperature was correlated with lower protein levels (Fig 4B and Fig C). Compared to STU1 cells at 26°, stu1-5 cells contained 3-fold less protein at 26° and 5-fold less after 6 hr at 33°. The stu1-5 ubp3 strain contained 4-fold less protein at 26° and 10-fold less at 33°. Similar results were obtained when the strains were shifted to 35° (not shown). Overall, there is a good correlation between Stu1p levels and cell viability. The ubp3 causes a 2-fold decrease in Stu1-5p levels at 33°, indicating that Ubp3p plays a role in stabilizing Stu1-5p at this temperature.

View larger version (40K): In this window In a new window Download PPT slide   Figure 4. Viability and Stu1p levels are decreased in stu1-5 and stu1-5 ubp3. (A) STU1 (CUY25), stu1-5 (CUY999), and stu1-5 ubp3 (CUY1325) strains were plated onto YPD media and assayed for growth at the indicated temperature. (B) The same yeast strains were grown at 26° to mid-log phase and then shifted to 33° for the indicated time. Cell extracts were analyzed by immunoblotting with an anti-Stu1p antibody. (C) The quantitation of the data from three independent experiments is shown. Stu1p levels were normalized to Act1p levels.

To further examine the effect of stu1-5 levels on viability, we constructed strains containing extra copies of stu1-5. We transformed stu1-5 (CUY999) with low-copy YCp vectors carrying STU1 (pDP94), stu1-5 (pCK1), and no STU1 gene (pRS415) and a high-copy YEp vector carrying stu1-5 (pCT37). stu1-5 YEp rescued the growth of stu1-5 completely at 35° and partially at 37°. stu1-5 YCp partially rescued growth at 35° and not at all at 37° (Fig 5A). We measured Stu1p levels in all of these strains at 26° and 35° (Fig 5B and Fig C). At 35°, the Stu1p level in the stu1-5 [stu1-5 YEp] strain was >10-fold higher than that in the stu1-5 [YCp] strain and equal to the level in stu1-5 [STU1 YCp]. Thus, a level of Stu1-5p equivalent to the amount of wild-type Stu1p produced from a YCp plasmid allows growth at the restrictive temperature, although growth is slower at 37°. The Stu1-5p level in stu1-5 [stu1-5 YCp] was 5-fold higher than that in stu1-5 [YCp] but still 3-fold less than that in stu1-5 [STU1 YCp] at 35°. Thus, there appears to be a threshold of Stu1-5p, near the level in stu1-5 [stu1-5 YCp] cells, that will allow growth at 35°. The threshold for growth is lower at 26° where stu1-5 [YCp] is viable despite even lower protein levels.

View larger version (40K): In this window In a new window Download PPT slide   Figure 5. Overexpression of stu1-5 suppresses stu1-5 heat sensitivity and restores Stu1p levels in stu1-5 cells. stu1-5 (CUY999) was transformed with YCp STU1 (pDP94), YEp stu1-5 (pCK37), YCp stu1-5 (pCT1), or YCp (pRS415). (A) Transformants were plated onto SD-Leu media and assayed for growth at the indicated temperature. (B) The strains were grown at 26° to mid-log phase and then shifted to 35° for the indicated time. Cell extracts were analyzed by immunoblotting with an anti-Stu1p polyclonal antibody. (C) The quantitation of the data from two independent experiments is shown. Stu1p levels were normalized to Act1p levels.

If the synthetic lethality of stu1-5 ubp3 is due solely to diminished levels of Stu1p, then overexpression of Stu1-5p should be able to rescue this lethality. Both stu1-5 YEp and stu1-5 YCp plasmids rescued the synthetic lethality of the double mutant at 33° (Fig 6A). However, neither plasmid restored the growth of stu1-5 ubp3 at 35° as well as it did for stu1-5. At 33°, the Stu1p level in the stu1-5 ubp3 [stu1-5 YEp] strain was 10-fold higher than that in the stu1-5 [YCp] strain and equivalent to the level in stu1-5 [STU1 YCp]. Thus, in the presence of the ubp3, a level of Stu1-5p equivalent to the amount of wild-type Stu1p produced from a YCp plasmid allows growth at 35° but not at 37°. The Stu1-5p level in stu1-5 ubp3 [stu1-5 YCp] was 3-fold higher than that in stu1-5 [YCp] but still 3-fold less than that in stu1-5 [STU1 YCp] at 33°. This indicates the presence of a threshold, somewhere between the protein level of stu1-5 ubp3 [stu1-5 YCp] and stu1-5 ubp3 [YCp], that is necessary for the growth of stu1-5 ubp3 strains at 33°. Overall, these results are consistent with the idea that temperature sensitivity of stu1-5 strains is due primarily to the instability of the mutant protein. Loss of Ubp3p exacerbates this defect, lowering the restrictive temperature.

View larger version (53K): In this window In a new window Download PPT slide   Figure 6. Overexpression of stu1-5 suppresses stu1-5 ubp3 synthetic lethality and restores Stu1p levels in stu1-5 ubp3 cells. stu1-5 ubp3 (CUY1325) was transformed with YCp STU1 (pCU435), YEp stu1-5 (pCK37), YCp stu1-5 (pCT1), or YCp (pRS415). (A) Transformants were plated onto SD-Leu media and assayed for growth at the indicated temperature. (B) The strains were grown at 26° to mid-log phase and then shifted to 33° for the indicated time. Cell extracts were analyzed by immunoblotting with an anti-Stu1p polyclonal antibody. (C) The quantitation of the data from two independent experiments is shown. Stu1p levels were normalized to Act1p levels.

ubp3 is synthetically lethal with myo2-14 and stu2-10:
To determine whether the effect of ubp3 is specific to stu1-5, we checked two other mutations for synthetic lethality with ubp3. Myo2p is an essential type V myosin implicated in vesicular transport and polarized growth, as well as nuclear migration (GOVINDAN et al. 1995 ; YIN et al. 2000 ). Stu2p is an essential microtubule-binding protein that regulates microtubule dynamics and is required for nuclear migration and spindle elongation (KOSCO et al. 2001 ; SEVERIN et al. 2001 ). myo2-14 and stu2-10 are temperature-sensitive alleles that cause decreased levels of these proteins. Myo2p levels are decreased by 8-fold in the myo2-14 mutant (SCHOTT 2000 ), and Stu2p levels are decreased by 2.5-fold in the stu2-10 mutant (KOSCO 2002 ) compared to wild type. myo2-14 ubp3 and stu2-10 ubp3 were made by crossing myo2-14 (ABY544) and stu2-10 (CUY1070) strains to a ubp3::HIS3 strain (CUY1326). In both cases, the double mutants were more temperature sensitive than the single mutants (Fig 7). These data support the notion that Ubp3p plays a general role in stabilizing proteins.

View larger version (30K): In this window In a new window Download PPT slide   Figure 7. Genetic interactions of ubp3 with stu1-5, myo2-14, and stu2-10. ubp3 (CUY1326) was crossed to myo2-14 (ABY544) and stu2-10 (CUY1070), and the diploids were sporulated. Spores were plated onto YPD media and assayed for growth at the indicated temperature.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Stu1p is an essential microtubule-associated protein that is involved in spindle assembly. We have identified four genes whose loss is lethal in stu1-5 cells at temperatures that are permissive for the stu1-5 cells. The first is UBP3, which encodes a ubiquitin protease. The second and third, PAC10/GIM2 and GIM3, are both genes that encode members of the GIM family in yeast and are important for folding of tubulin and actin. The fourth gene, KEM1/XRN1/SEP1, encodes an RNA exonuclease that also has been reported to influence microtubule function.

Stu1p and Ubp3p:
Ubp3p is known to be one of a large group of deubiquitination enzymes in yeast. These proteins are believed to act as either positive or negative regulators of proteolysis, but there are few data on what specific function these proteases perform. The 16 Ubp enzymes likely have overlapping functions as suggested by the normal growth rate of multiple deletion mutants. Doa4p, Ubp14p, and Ubp3p are the only enzymes of this group that have been examined in detail. Doa4p cleaves ubiquitin chains from proteolytic intermediates (PAPA and HOCHSTRASSER 1993 ), while Ubp14p is responsible for the cleavage of the free polyubiquitin chains (WILKINSON et al. 1995 ; AMERIK et al. 1997 ). These steps provide free ubiquitin monomer that can be attached to new targets for degradation. Ubp3p has been hypothesized to cleave ubiquitin chains from substrate proteins because its disruption leads to the accumulation of ubiquitin-protein intermediates (BAXTER and CRAIG 1998 ). We have found that stu1-5 creates a situation in which UBP3 is essential. Thus, understanding the stu1-5 defect may shed more light on the role of Ubp3p.

We considered two possible explanations for the stu1-5 ubp3 synthetic lethality. First, we examined the possibility that Ubp3p plays a role in the cell-cycle regulation of Stu1p. Several mitotic proteins are subject to cell-cycle regulation involving ubiquitin-mediated protein degradation. However, the levels of Stu1p are relatively constant throughout the cell cycle, indicating that it is not a substrate for this specific ubiquitin-dependent pathway.

Next, we considered the possibility that mutant Stu1-5p is an unstable protein that is targeted for ubiquitin-mediated degradation. We showed that the levels of Stu1p in stu1-5 cells are several-fold lower than the levels in wild-type cells. In addition, the temperature sensitivity of stu1-5 can be suppressed by producing wild-type levels of the mutant protein. These results indicate that the temperature sensitivity is caused by reduced levels of Stu1p and not by reduced function. Interestingly, the critical threshold of Stu1-5p protein level appears to be somewhat higher at higher temperatures.

Given that the primary effect of the stu1-5 mutation is to lower the levels of Stu1p, the synthetic lethality caused by ubp3 might result from a further lowering of protein levels. This is what ubp3 does, decreasing the protein level by nearly half. This decrease is evidently enough to put the Stu1p level below the threshold required for viability at 33°. Unfortunately, we have not been able to directly measure the effect of ubp3 on the stability of Stu1-5p because the low levels of Stu1-5p make it difficult to detect in a pulse-chase experiment. Nonetheless, our data indicate that Ubp3p plays a role in stabilizing Stu1-5p. This is supported by additional genetic evidence: myo2-14 and stu2-10 decrease the levels of Myo2p and Stu2p, respectively, and in both cases ubp3 lowered their restrictive temperature. Thus, Ubp3p may have a general role in the deubiquitination of misfolded proteins. Previous data indicate this role for Ubp3p. First, there is an accumulation of ubiquitin-protein conjugates in ubp3 (BAXTER and CRAIG 1998 ; AMERIK et al. 2000 ). In addition, overexpression of UBP3 suppresses the heat-shock mutant ssa1 ssa2, in contrast to UBI4 and UBC4, two genes that encode promoters of proteolysis. Overall, Ubp3p appears to act as a proofreading enzyme, reversing the ubiquitination of misfolded, temperature-sensitive proteins and allowing them to refold.

ubp3 is unique among ubp's when judged by the severity of its synthetic lethality with stu1-5. Only ubp5 shows a similar synthetic effect, although to a lesser degree (Fig 1). Thus, Ubp5p may also be an inhibitor of proteolysis whose function partially overlaps Ubp3p. In contrast, we found that doa4 and ubp6 suppressed stu1-5 temperature sensitivity at 37°. This is consistent with the idea that Doa4p promotes proteolysis and that, in its absence, Stu1-5p is not degraded as rapidly. The fact that ubp6 also suppresses the temperature-sensitive phenotype suggests that it too may promote proteolysis. An overlap in function between these two enzymes was previously suggested because loss of Doa4p or Ubp6p results in lower levels of ubiquitin and amino acid analogue hypersensitivity (AMERIK et al. 2000 ). These results suggest that the deubiquitinating enzymes perform diverse functions or have a high degree of substrate specificity.

Stu1p and the Gim proteins:
We also isolated gim3 and pac10/gim2 alleles as synthetically lethal with stu1-5. GIM1-GIM5 were previously identified in a synthetic lethal screen with tub4-1, and their protein products were shown to associate in common complexes (GEISSLER et al. 1998 ). We tested gim1, gim4, and gim5 and found that these mutations were also lethal in combination with stu1-5. The gim's were shown to be benomyl supersensitive, due to reduced levels of -tubulin, but not ß- or -tubulin (GEISSLER et al. 1998 ). A distinct Gim function is related to -tubulin, based on experiments that show the Gim proteins bind to Tub4p and genetic interactions between gim1 and alleles of SPC98 and SPC97 (GEISSLER et al. 1998 ). In addition, tubulin immunofluorescence of gim's showed that microtubule attachment to the SPB was intact, but microtubule stability was impaired. Thus, stu1-5 gim1-5 synthetic lethality may result from the combined effects of tubulin misfolding due to loss of the Gim1-5 and spindle assembly defects caused by stu1-5.

Alternatively, the synthetic lethality between stu1-5 and the gim's may result because the GimC complex promotes formation of functional Stu1p. GimC functions with chaperonin during translation and folding of tubulin or actin (GEISSLER et al. 1998 ; SIEGERS et al. 1999 ). Stu1p is a large protein (174 kD) that may require chaperonin to fold. Stu1-5p may be particularly susceptible to loss of GimC because it encodes a protein that is less stable than wild type.

Stu1p and Kem1p:
Kem1p is a 5'–3' cytoplasmic exonuclease, conserved from yeast to mammals, whose primary role is degradation of decapped mRNA (LARIMER et al. 1992 ; HSU and STEVENS 1993 ). Kem1p may also have a role in the microtubule cytoskeleton, based on the analysis of kem1 mutant phenotypes and its ability to influence tubulin polymerization and cosediment with microtubules in vitro (KIM et al. 1990 ; INTERTHAL et al. 1995 ). We considered the possibility that the synthetic lethality we observed between stu1-5 and kem1 was revealing a specific microtubule-related defect in Kem1p. However, our results showed that cytoplasmic localization of the exonuclease Rat1p could suppress synthetic lethality. In addition, amino acid substitutions in Kem1p that specifically alter its exonuclease active site were unable to suppress synthetic lethality. Both of these experiments indicate that the exonuclease activity, and not some other independent microtubule-related function, of Kem1p is compromised in the kem1 allele that is synthetically lethal with stu1-5.

	FOOTNOTES

¹ Present address: 591 Life Sciences Addition, University of California, Berkeley, CA 94720.

	ACKNOWLEDGMENTS

We thank Priya Sharma for help in cloning GIM3 and Rohan Baker, Tony Bretscher, Wolf-Dietrich Heyer, Mark Hochstrasser, Arlen Johnson, and Elmar Schiebel for gifts of strains and reagents. This work was supported by a grant from the National Institutes of Health (GM-40479).

Manuscript received February 18, 2002; Accepted for publication August 20, 2002.

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