The Yeast Ubiquitin Protease, Ubp3p, Promotes Protein Stability

Christine T. Brew1 and Tim C. Huffaker2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 Manuscript received February 18, 2002 Accepted for publication August 20, 2002

ABSTRACT 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.

N yeast, the ubiquitin-proteasome pathway is respon- classes of Dub enzymes, the Ubp family (ubiquitin-spe- I sible for degradation of short-lived and abnormal cific proteases) and the Uch family (ubiquitin carboxy- proteins, whereas the vacuole mediates degradation of terminal hydrolases). The Uch enzymes, for example, long-lived proteins (Jones 1991; reviewed in Finley 1992; Yuh1p in yeast, cleave ubiquitin from peptides and small Hochstrasser 1996; Hershko and Ciechanover 1998). adducts (Rose and Warms 1983; Pickart and Rose Ubiquitin is covalently ligated to target proteins by a 1985; Liu et al. 1989; Baker et al. 1992). Ubp enzymes multienzymatic system consisting of ubiquitin-activating cleave ubiquitin from a range of substrates. (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating It has been suggested that deubiquitination enzymes, (E3) enzymes. A large number of E2 enzymes provide like the ubiquitin-conjugating enzymes, have diverse substrate specificity either alone or along with the E3 roles in regulation of protein degradation or modifica- enzymes (Hochstrasser 1996). Ubiquitinated proteins tion. The study of several of these enzymes suggests that are targeted to the 26S proteasome, which consists of they can have either a positive or an inhibitory effect the 19S particle and the 20S proteasome (reviewed in on proteolysis. Positive regulation occurs when polyubi- Glickman 2000). The 19S particle is the regulatory unit quitin chains are cleaved to produce free ubiquitin and consists of a polyubiquitin recognition site, ATPases groups that are then available for attachment to new sub- that provide energy for unfolding proteins, and a deubi- strates targeted for degradation. For example, Ubp14p in quitinating enzyme that recycles ubiquitin. The un- yeast cleaves isopeptide-linked polyubiquitin chains that folded and extended polypeptides are then allowed to are unanchored to a substrate (Amerik et al. 1997). This enter the rings of the 20S proteasome, the proteolytic activity may also be necessary for the generation of free core that contains multiple peptidase activities for pro- ubiquitin moieties from ubiquitin fusions that are en- tein degradation. coded by UBI1, UBI2, UBI3, and UBI4 in yeast (Ozkay- The deubiquitinating enzymes (Dubs) are a large fam- nak et al. 1987). Doa4p/Ubp4p is required for cleavage ily of cysteine proteases that cleave ubiquitin from conju- of polyubiquitin chains from proteolytic intermediates, gated protein substrates or precursor proteins (D’An- which provides free polyubiquitin chains (Papa and drea and Pellman 1998; reviewed in Chung and Baek Hochstrasser 1993). Thus, Doa4p may promote prote- 1999). These thiol proteases hydrolyze the amide bond olysis by increasing ubiquitin pools and removing pro- between Gly76 of ubiquitin and a Lys residue of the teolytic remnants that would otherwise inhibit protea- substrate protein or preceding ubiquitin. There are two some 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) pro- 1Present address: 591 Life Sciences Addition, University of California, tein is a Ubp enzyme in Drosophila that acts as a negative Berkeley, CA 94720. regulator of the ubiquitin system (Huang et al. 1995). 2Corresponding author: Department of Molecular Biology and Genet- ics, Biotechnology Bldg., Cornell University, Ithaca, NY 14853-2703. A proteasomal mutation was found to suppress the faf E-mail: [email protected] mutant phenotype, suggesting that FAF protein has ac-

Genetics 162: 1079–1089 (November 2002) 1080 C. T. Brew and T. C. Huffaker tivity that antagonizes proteasome function. It may have nell University, Ithaca, NY). CUY1331 was created by transfor- a proofreading function, reversing ubiquitination of mation of a stu1-5::LEU2 integrating plasmid (pCK16) into substrate proteins to prevent or slow their degradation CUY1061 to generate stu1-5::LEU2::stu1-5. CUY1331 was then transformed with an ADE3 URA3 plasmid containing the STU1 by the proteasome. gene (pDP96) to create CUY1332. In yeast, 16 Ubp enzymes are predicted from se- YPD and SD media and plates were prepared as described quence analysis. They show little homology beyond six by Sherman (1991). Benomyl plates were prepared by adding conserved regions, three of which contain the enzyme an appropriate amount of 10 mg/ml stock to YPD plates. active site, and three that have unknown function but 5-FOA plates were made at a concentration of 1 mg/ml in may provide a ubiquitin binding site. The N-terminal SD. Geneticin (G418 sulfate, Stratagene, La Jolla, CA) was used at 0.2 mg/ml. regions are divergent and may provide substrate recog- Isolation of mutants that require STU1 for growth: The nition. Ubp enzymes have overlapping functions as sug- adenine red-white sectoring assay (Bender and Pringle 1991) gested by the normal growth rate of ubp1⌬ ubp2⌬ ubp3⌬ was used to identify mutations that are synthetically lethal with and the ubp8⌬-like growth rate of the ubp1⌬ ubp2⌬ stu1-5 (see results). Strain CUY1332 was mutagenized with ubp3⌬ ubp7⌬ ubp8⌬ quintuple mutants (Baker et al. methane sulfonic acid ethyl ester (EMS, Sigma, St. Louis) to viability (Guthrie and Fink 1991). For all genetic %45ف 1992; Amerik et al. 2000). In this article, we describe a crosses, we dissected at least 11 tetrads and defined genes as genetic interaction between UBP3 and STU1, a microtu- tightly linked if no recombinants were observed. bule-associated protein involved in formation of the Sac- Disruption of UBP5, UBP7, UBP8, UBP9, and UBP16: Dele- charomyces cerevisiae mitotic spindle (Pasqualone and tion strains doa4⌬, ubp10⌬, and ubp14⌬ and strains ubp1⌬, ubp2⌬, ⌬ ⌬ ⌬ ⌬ ⌬ Huffaker 1994). Stu1p is a member of the Stu1-MAST ubp6 , ubp11 , ubp12 , ubp13 , and ubp15 were kindly provided by Mark Hochstrasser (Yale University, New Haven, CT) family that includes S. cerevisiae Stu1p; Schizosaccharo- and Rohan Baker (Australian National University), respec- myces pombe Stu1p; and the more distantly related Dro- tively. The UBP3, UBP5, UBP7, UBP8, UBP9, and UBP16 genes sophila Mast, human CLASP1 and CLASP2, and three were completely disrupted by one-step gene replacement unknown open reading frames (ORFs) in Caenorhabditis (Baudin et al. 1993). PCR primers containing genomic DNA elegans (CeCO7H6.3, CeR107.6, and CeZC84.3; Pasqua- sequence 60 bp upstream and 60 bp downstream of each UBP lone and Huffaker 1994; Lemos et al. 2000; Akhma- gene were used to amplify pFA6a-His3MX6 (Wach et al. 1997). The resultant PCR products, UBP-flanking sequences on either nova et al. 2001). UBP3 was initially isolated in a screen side of the HIS5 gene, were transformed into CUY30. Histidine for yeast genes that, when coexpressed with Ub-␤-galac- prototrophs were selected, and correct integration was con- tosidase in Escherichia coli, resulted in removal of the firmed by PCR amplification of the respective UBP locus. ubiquitin moiety (Baker et al. 1992). UBP3 was also ubp1⌬–ubp16⌬ were then crossed with stu1-5, stu1-5::URA3, isolated as a high-copy suppressor of the temperature or stu1-5::LEU2 (CUY997, CUY1005, CUY1338, CUY1339, CUY1340) to create stu1-5 ubp⌬ double mutants. sensitivity of yeast cells lacking two molecular chaperone Flow cytometry: Haploid yeast cells were prepared for flow genes, SSA1 and SSA2 (Baxter and Craig 1998). The cytometry by the method of Hutter and Eipel (1978). The disruption of UBP3 resulted in the accumulation of ubi- DNA content of 10,000 cells was determined using a FACScan quitin-protein conjugates, suggesting that Ubp3p re- flow cytometer (Becton Dickinson). CELL QUEST software verses the ubiquitination of substrate proteins (Baxter was used to obtain and analyze data (BDIS, San Jose, CA). and Craig 1998). Ubp3p was also shown to bind to Immunoblot analysis: Strains were harvested by centrifugation; washed in breakage buffer (30 mm NaPO4, pH 7.0, 60 Sir4p and hypothesized to inhibit transcriptional silenc- mm B-glycerophosphate, 150 mm KCl, 6 mm EDTA, 6 mm ing (Moazed and Johnson 1996). In this study, we EGTA, 10% glycerol); resuspended in breakage buffer with 1 report the identification of UBP3 in a screen for muta- mm phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, and tions that are synthetically lethal with stu1-5. This genetic 10 ␮g/ml pepstatin; and flash frozen in liquid nitrogen. Fro- interaction is unique to ubp3⌬ in the Ubp family. Our zen pellets were ground with a mortar and pestle. Cell debris was removed by centrifugation. The quantity of protein in the data support the idea that Ubp3p deubiquitinates mis- extracts was determined by the Bradford assay (Bradford folded proteins, giving them an opportunity to refold 1976). and function in the cell. 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 MATERIALS AND METHODS membrane (Hybond-P, Amersham, Arlington Heights, IL). Membranes were incubated with anti-Stu1p polyclonal anti- Strains, plasmids, and media: The yeast strains and plasmids bodies (a gift from Liru You) in Tris-HCl, pH 8.0, 150 mm used in this study are listed in Tables 1 and 2, respectively. NaCl, 0.1% Tween-20 containing 5% nonfat dry milk, followed gim1⌬, gim4⌬, and gim5⌬ strains and plasmids containing by incubation with anti-rabbit IgG conjugated to alkaline phos- GIM1, GIM4, and GIM5, respectively, were provided by Elmar phatase (Amersham). The membranes were also blotted with Schiebel (The Beatson Institute for Cancer Research, Glasgow, anti-Act1p (a gift from Tony Bretscher) as a loading control. Scotland). Plasmids containing RAT1⌬NLS and kem1-E176G Antibody binding was detected using Western blotting ECF were provided by Arlen Johnson (University of Texas, Austin, reagents from Amersham. Levels of protein were quantified TX), and kem1-D206A, kem1-D208A, and kem1-D206A, D208A from data collected on a Storm 840 Phosphorimager (Molecu- were provided by Wolf-Dietrich Heyer (University of Califor- lar Dynamics, Sunnyvale, CA) and the use of ImageQuant nia, Davis, CA). ABY544 was provided by Tony Bretscher (Cor- software. The levels of Stu1p were normalized to Act1p levels. Ubp3p Promotes Protein Stability 1081

TABLE 1 Yeast strains used in this study

Strain Genotype Source CUY25 MATa ura3-52 his3-⌬200 leu2-3,112 ade2-101 This lab CUY30 MAT␣ ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This lab CUY997 MATa stu1-5::URA3::stu1⌬ ura3-52 his3-⌬200 leu2-3,112 ade2-101 This lab CUY999 MAT␣ stu1-5 ura3-52 his3-⌬200 leu2-3,112 This lab CUY1005 MAT␣ stu1-5 ura3-52 his3-⌬200 leu2-3,112 trp1-1 This lab CUY1061 MAT␣ stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 This lab CUY1070 MATa stu2-10::LEU2 ura3-52 his3-⌬200 leu2-3,112 This lab CUY1325 MAT␣ stu1-5 ubp3⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 trp1-1 This study CUY1326 MAT␣ ubp3⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study CUY1327 MAT␣ kem1-1 stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 [pDP96] This study CUY1328 MAT␣ ubp3-1 stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 [pDP96] This study CUY1329 MAT␣ gim3-1 stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 [pDP96] This study CUY1330 MAT␣ pac10-1 stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 [pDP96] This study CUY1331 MAT␣ stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 This study CUY1332 MAT␣ stu1-5::LEU2::stu1-5 ura3-52 his3-⌬200 leu2-3,112 ade2-101 ade3-24 [pDP96] This study CUY1333 MATa stu1-5 ACT1::HIS3 ura3-52 his3-⌬200 leu2-3,112 This study CUY1334 MAT␣ PAC10::LEU2 stu1-5 ura3-52 his3-⌬200 leu2-3,112 This study CUY1335 MATa UBP3::LEU2 stu1-5 ura3-52 his3-⌬200 leu2-3,112 This study CUY1336 MATa KEM1::LEU2 stu1-5 ura3-52 his3-⌬200 leu2-3,112 This study CUY1337 MATa GIM3::LEU2 stu1-5 ura3-52 his3-⌬200 leu2-3,112 This study CUY1338 MAT␣ stu1-5::URA3::stu1⌬ ACT1::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101 This study CUY1339 MAT␣ stu1-5::LEU2::stu1⌬ ACT1::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101 This study CUY1340 MATa stu1-5::LEU2::stu1⌬ ACT1::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101 This study CUY1341 MAT␣ ubp5⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study CUY1342 MAT␣ ubp7⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study CUY1343 MAT␣ ubp8⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study CUY1344 MAT␣ ubp9⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study CUY1345 MAT␣ ubp16⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 ade2-101, lys2-801 This study ABY544 MATa myo2-14::LEU2 ura3-52 his3-⌬200 leu2-3,112 ade2-101 lys2-801 A. Bretscher ⌬ ⌬ K7428 MATa cdc20 ::LEU2 PGAL-CDC20::TRP1 ura3-52 his3- 200 leu2-3,112 trp1-1 Lim et al. (1998) MHY623 MAT␣ doa4-⌬1::LEU2 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 M. Hochstrasser MHY840 MAT␣ ubp14⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 M. Hochstrasser MHY1228 MATa ubp10⌬::HIS3 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 M. Hochstrasser SGY101 MAT␣ gim1⌬::kanMX4 ura3-52 his3-⌬200 leu2-⌬1 ade2-101 lys2-801 trp1-⌬63 Geissler et al. (1998) SGY115 MATa gim4⌬::HIS3MX6 ura3-52 his3-⌬200 leu2-⌬1 ade2-101 lys2-801 trp1-⌬63 Geissler et al. (1998) SGY156 MAT␣ gim5⌬::kanMX4 ura3-52 his3-⌬200 leu2-⌬1 ade2-101 lys2-801 trp1-⌬63 Geissler et al. (1998) YRB113 MAT␣ ubp1-⌬1::URA3 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YRB174 MATa ubp2-⌬3::TRP1 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YRB205 MATa ubp6-⌬1::URA3 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YRB286 MATa ubp15-⌬1::LEU2 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YXW21 MATa ubp11-⌬1::URA3 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YXW37 MATa ubp12-⌬1::TRP1 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker YXW88 MAT␣ ubp13-⌬1::LEU2 ura3-52 his3-⌬200 leu2-3,112 lys2-801 trp1-1 R. Baker

The linear range of Stu1p was determined using serial dilu- performed using a strain that contained two copies of tions of yeast cell extracts. 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 RESULTS methods). At 30Њ, CUY1332 (stu1-5::LEU2::stu1-5 ade2 Screen for mutations that are synthetic lethal with ade3 ura3 [pDP96]) grows with a red colony color, which stu1-5: To identify genes whose products interact with sectors white upon spontaneous loss of pDP96 (STU1 Stu1p, we performed a genetic screen for extragenic ADE3 URA3). The presence of a second-site mutation mutations that are synthetically lethal with the tempera- that is lethal in combination with stu1-5 would render ture-sensitive stu1-5 allele. An initial screen produced the strain unable to lose pDP96 at the permissive tem- exclusively intragenic mutations so a second screen was perature and, therefore, unable to sector. 1082 C. T. Brew and T. C. Huffaker

TABLE 2 Plasmids used in this study

Plasmid Relevant markers Source pCK16 stu1-5 LEU2 This study pCK17 STU1 CEN6 ARSH4 HIS3 This study pCK21 UBP3 CEN6 ARSH4 LEU2 This study pCK23 UBP3 LEU2 This study pCK27 PAC10 LEU2 This study pCK28 PAC10 CEN6 ARSH4 LEU2 This study pCK37 stu1-5 2␮ LEU2 This study pCK44 KEM1 CEN6 ARSH4 LEU2 This study pCK45 KEM1 LEU2 This study pCK48 GIM3 CEN6 ARSH4 LEU2 This study pCK49 GIM3 LEU2 This study pCT1 stu1-5 CEN6 ARSH4 LEU2 This lab pDP94 STU1 CEN6 ARSH4 LEU2 This lab pDP96 STU1 ADE3 CEN6 ARSH4 URA3 This lab pAJ1-7 kem1 (E176G) CEN6 ARSH4 LEU2 A. Johnson pAJ35 KEM1 CEN6 ARSH4 LEU2 A. Johnson pAJ203 RAT1 CEN6 ARSH4 LEU2 A. Johnson pAJ228 RAT1⌬NLS CEN6 ARSH4 LEU2 A. Johnson pFA6a-His3MX6 his5 Wach et al. (1997) pRS415 CEN6 ARSH4 LEU2 Sikorski and Hieter (1989) pSG55 GIM1 CEN6 ARSH4 LEU2 E. Schiebel pSI114 ADH-GIM5 CEN6 ARSH4 LEU2 E. Schiebel pSM480 3HA-GIM4 CEN6 ARSH4 LEU2 E. Schiebel pWDH241 kem1-D206A CEN4 ARS1 URA3 W.-D. Heyer pWDH242 kem1-D208A CEN4 ARS1 URA3 W.-D. Heyer pWDH243 kem1-D206A, D208A CEN4 ARS1 URA3 W.-D. Heyer

A total of 31,500 colonies were screened for a nonsec- sented by these mutations. The stu1-5 ssl strains con- toring phenotype at 30Њ. Of these, 185 (0.6%) nonsec- taining pDP96 were crossed in a pairwise fashion to one toring colonies were further tested for a STU1 require- another and scored for growth on 5-FOA. The diploids ment by plating on 5-FOA, which selects against the that required pDP96 for growth contained alleles in the URA3 gene. Ninety-one strains were 5-FOA sensitive in- same complementation group. The 13 ssl mutations fall dicating that their nonsectoring phenotype is due to into 10 complementation groups, one with four mem- their inability to lose the STU1 plasmid and not to rever- bers and nine with one member each (Table 3). sion or conversion at the ade3 locus. Finally, to show Identiﬁcation of the genes: Complementation groups that the nonsectoring and 5-FOA sensitivity phenotypes I–IV were cloned by rescue of the synthetic lethality at were due to the strains’ requirement for STU1,aSTU1 30Њ. stu1-5 ssl strains were transformed with a Ycp-based HIS3 plasmid (pCK17) was transformed into each strain, yeast genomic library (Wang and Huffaker 1997) and and 35 were found to sector and grow on 5-FOA. Thus, the transformants were screened for restoration of the these 35 strains contain one or more mutations that are sectoring phenotype. The rescuing plasmids were iso- speciﬁcally lethal in combination with stu1-5. lated and retransformed into the mutant to ensure plas- Tetrad analysis was done to determine if the muta- mid dependence. The endpoints of the genomic insert tions responsible for synthetic lethality were in a single were then sequenced to identify the genomic locus. gene. Each stu1-5 ssl strain harboring pDP96 was mated Each chromosomal locus was marked by integration of with a stu1-5 strain (CUY1333), and the resultant diploid an auxotrophic marker and shown to be linked to the was sporulated. For 13 mutants, 5-FOA sensitivity segre- corresponding synthetic lethal mutation in crosses. To gated 2:2 in tetrads, indicating that synthetic lethality identify the relevant ORF on the genomic fragment, was due to a mutation in a single gene. One mutation individual ORFs were subcloned into LEU2 YCp plas- was tightly linked to the TUB2 locus and none were mids, transformed into the parent strain, and analyzed linked to stu1-5. for rescue of the synthetic lethality (Table 3). Each of the 13 stu1-5/stu1-5 ssl/SSL diploids harboring Groups II and III consisted of two members of the pDP96 was 5-FOA resistant, indicating that all 13 ssl GimC complex, PAC10/GIM2 and GIM3. The GimC mutations are recessive. Complementation analysis was complex is homologous to the mammalian prefoldin performed to determine the number of genes repre- complex that binds to nascent polypeptides of tubulin Ubp3p Promotes Protein Stability 1083

TABLE 3 lethality (data not shown), suggesting that the exo- ssl mutants nuclease function of Rat1p is sufficient to restore the growth of stu1-5 kem1. We also tested several KEM1 exo- Complementation nuclease point mutations for their ability to comple- group Alleles Gene ment stu1-5 kem1 synthetic lethality. If the synthetic lethality is due solely to overlapping Stu1p and Kem1p I1UBP3 II 1 PAC10 function in a microtubule-associated role, then the exo- III 1 GIM3 nuclease activity of Kem1p should not be needed to IV 1 KEM1 complement the double mutant. We tested four kem1 V1TUB2 mutations (kem1-E176G, kem1-D206A, kem1-D208A, and VI 1 Not yet identified kem1-D206A, D208A) that specifically eliminate KEM1 VII 1 Not yet identified exonuclease activity (Page et al. 1998; Solinger et al. VIII 1 Not yet identified 1999). None of these mutant alleles rescued the growth IX 1 Not yet identified X 4 Not yet identified 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⌬: Com- and actin during translation and, when synthesis is complementation group I was identified as UBP3. Ubp3p is plete, transfers these proteins to the cytosolic chaper- a member of a large family of ubiquitin proteases. To onin (Cowan 1998; Hansen et al. 1999). Because we determine if other family members are functionally re- identified two of the SSL genes as PAC10 and GIM3,we lated to UBP3, we tested whether stu1-5 was synthetically tested the remaining ssl mutations for rescue by other lethal with deletions of the other 15 UBP genes. stu1-5 GimC complex members, GIM1, GIM4,orGIM5, but ubp3⌬ strains do grow at 26Њ, but grow only poorly at none were rescued. We also tested gim1⌬, gim4⌬, and 30Њ and not at all at 33Њ, a temperature that is permissive gim5⌬ for synthetic lethality with stu1-5, and found that for stu1-5 growth. All of the other stu1-5 ubp⌬ double all family members were lethal in combination with mutants grew well at these temperatures, with the excep- stu1-5 (data not shown). The synthetic lethality may tion of stu1-5 ubp5⌬, which grew somewhat more slowly result from the combined effects of tubulin misfolding than wild-type at 30Њ and 33Њ (Figure 1). Conversely, and defects in microtubule assembly caused by the doa4⌬ and ubp6⌬ partially rescued stu1-5 temperature stu1-5 mutation. Alternatively, the GimC complex may sensitivity at 37Њ. play a direct role in Stu1p folding. stu1-5 and ubp3⌬ are benomyl sensitive: We examined Group IV was identified as KEM1/XRN1/SEP1. Kem1p the benomyl sensitivity of stu1-5 and ubp⌬ alleles because is a nonessential cytoplasmic 5Ј–3Ј RNA exonuclease many mutants with altered microtubule function are that is responsible for turnover of mRNA and rRNA sensitive to this microtubule-depolymerizing drug. Wild- (Stevens et al. 1991; Larimer et al. 1992; Muhlrad et type haploid cells grow well on 15 ␮g/ml of benomyl al. 1994; Caponigro and Parker 1996). In addition, but fail to grow on 30 ␮g/ml (Figure 2). stu1-5 cells are several studies suggest that Kem1p may play a role in more sensitive than wild-type cells, growing very poorly microtubule function. kem1 mutations cause hypersensi- on 15 ␮g/ml benomyl. However, the sensitivity can be tivity to benomyl, karyogamy defects, increased chromo- fully rescued by providing stu1-5 on a low-copy YCp some loss frequencies, impaired spindle pole body or high-copy YEp plasmid. This indicates that benomyl (SPB) separation, and defective nuclear migration and sensitivity results from a low quantity of protein rather show genetic interactions with tubulin genes (Kim et al. than from a protein that is inactive at the restrictive 1990; Interthal et al. 1995). These latter phenotypes temperature. and the synthetic lethality with stu1-5 may be secondary Several of the ubp mutations also alter the benomyl to defects in RNA turnover or indicate an independent sensitivity of cells (Figure 2), but only ubp3⌬ is as beno- role for Kem1p in microtubule function. To answer this myl sensitive as stu1-5. ubp7⌬ and ubp16⌬ are somewhat question with regard to stu1-5 synthetic lethality, we more sensitive than wild-type cells, and ubp6⌬ and used two types of mutants. RAT1 encodes a nuclear ubp10⌬ are more resistant to benomyl than are wild- protein that shares considerable homology with Xrn1p type cells. and is required for RNA degradation and numerous Stu1p levels are constant through the cell cycle: The RNA 5Ј processing reactions important for ribosome role of ubiquitination in cell-cycle regulation is well biogenesis (Henry et al. 1994; Petfalski et al. 1998). documented. The APC activates the ubiquitination and There is no evidence for a microtubule-associated role degradation of Pds1p, B-type cyclins, and spindle-associ- for Rat1p thus far. Mutations in the NLS of Rat1p mislo- ated Ase1p for cells to undergo sister chromatid separa- calize the protein to the cytoplasm and complement tion and exit from mitosis (Cohen-Fix et al. 1996; Juang kem1 mutant phenotypes (Johnson 1997). We found et al. 1997; Ciosk et al. 1998). Because Stu1p is required that rat1⌬NLS (pAJ228) rescues stu1-5 kem1 synthetic for mitosis, we looked at the levels of Stu1p through 1084 C. T. Brew and T. C. Huffaker

Figure 2.—stu1-5 and ubp3⌬ are benomyl sensitive. STU1 Figure 1.—stu1-5 is uniquely synthetically lethal with ubp3⌬. (CUY25), stu1-5 (CUY999), stu1-5 transformed with YEp STU1 (CUY25), stu1-5 (CUY999), and stu1-5 ubp1⌬ through stu1-5 (pCK37) or YCp stu1-5 (pCT1), and ubp1⌬ through ubp16⌬ were plated onto YPD media and assayed for growth ubp16⌬ (see Table 1) were plated onto various concentrations at the indicated temperature. of benomyl and assayed for growth at 30Њ. the cell cycle to determine if it is regulated in a similar quitin system also targets misfolded proteins for degra- manner. dation, so we examined the possibility that stu1-5 ubp3⌬ An effective approach for synchronizing yeast cells is to synthetic lethality may result from a decrease in the arrest them in M phase by depletion of Cdc20p and then stability of Stu1-5p in the absence of Ubp3p. A STU1 release them from this block by inducing Cdc20p (Lim et strain (CUY25) grows at temperatures up to 37Њ (Figure ⌬ Њ al. 1998). Strain K7428 (cdc20 PGAL-CDC20::TRP1) was 4A). The stu1-5 strain (CUY999) grows at 33 but does grown at 26Њ to mid-log phase in galactose media. The not grow at 35Њ. The stu1-5 ubp3⌬ double mutant cells were washed and grown in glucose media for 3 hr, (CUY1325) grows poorly at 30Њ and is inviable at 33Њ. which arrested the cells in M phase. The culture was We examined the levels of Stu1p in STU1, stu1-5, and shifted back to galactose media and aliquots of cells stu1-5 ubp3⌬ strains grown at 26Њ,33Њ, and 35Њ to deter- were examined at 5- to 20-min intervals after release mine if the progressive decrease in permissive tempera- from the cdc20⌬ block. The samples were processed for ture was correlated with lower protein levels (Figure 4, flow cytometry and immunoblotting. B and C). Compared to STU1 cells at 26Њ, stu1-5 cells FACS analysis showed that the cdc20⌬ cells arrested contained 3-fold less protein at 26Њ and 5-fold less after uniformly in early M phase with 2C DNA content. After 6hrat33Њ. The stu1-5 ubp3⌬ strain contained 4-fold release from the block, cells progressed through the less protein at 26Њ and 10-fold less at 33Њ. Similar results were obtained when the strains were shifted to 35Њ (not 102ف cell cycle in synchrony, reaching M phase again by min (Figure 3A). The levels of Stu1p remained fairly shown). Overall, there is a good correlation between constant through the cell cycle (Figure 3B). The modest Stu1p levels and cell viability. The ubp3⌬ causes a 2-fold min is likely due to decrease in Stu1-5p levels at 33Њ, indicating that Ubp3p 30ف increase in protein from 0 to the change of carbon source from glucose to galactose. plays a role in stabilizing Stu1-5p at this temperature. Because Stu1p does not show significant fluctuation To further examine the effect of stu1-5 levels on viabil- through the cell cycle, this protein is apparently not the ity, we constructed strains containing extra copies of target of ubiquitin-mediated cell-cycle regulation. Thus, stu1-5. We transformed stu1-5 (CUY999) with low-copy the synthetic lethality between stu1-5 and ubp3⌬ must YCp vectors carrying STU1 (pDP94), stu1-5 (pCK1), and involve some other process. no STU1 gene (pRS415) and a high-copy YEp vector Overexpression of stu1-5 suppresses both stu1-5 heat carrying stu1-5 (pCT37). stu1-5 YEp rescued the growth sensitivity and synthetic lethality with ubp3⌬: The ubi- of stu1-5 completely at 35Њ and partially at 37Њ. stu1-5 Ubp3p Promotes Protein Stability 1085

Figure 4.—Viability and Stu1p levels are decreased in Figure 3.—Stu1p levels are constant through the cell cycle. stu1-5 and stu1-5 ubp3. (A) STU1 (CUY25), stu1-5 (CUY999), ⌬ and stu1-5 ubp3⌬ (CUY1325) strains were plated onto YPD cdc20 PGAL-CDC20::TRP1 (CUY1348) was grown to mid-log phase in galactose medium. The culture was shifted to glucose media and assayed for growth at the indicated temperature. Њ medium for 3 hr, which arrested the cells in M phase. The (B) The same yeast strains were grown at 26 to mid-log phase Њ culture was shifted back to galactose medium at time zero. and then shifted to 33 for the indicated time. Cell extracts (A) The DNA content of the cells at each time point was were analyzed by immunoblotting with an anti-Stu1p antibody. determined by ﬂow cytometry. (B) Cell extracts were analyzed (C) The quantitation of the data from three independent by immunoblotting with anti-Stu1p and anti-Act1p antibodies experiments is shown. Stu1p levels were normalized to Act1p and Stu1p levels were normalized to Act1p levels. The range levels. of data from two independent experiments is indicated. The amount of Stu1p in unsynchronized cultures grown in galactose medium is indicated by the arrow. Stu1-5p should be able to rescue this lethality. Both stu1-5 YEp and stu1-5 YCp plasmids rescued the synthetic Њ YCp partially rescued growth at 35Њ and not at all at 37Њ lethality of the double mutant at 33 (Figure 6A). How- (Figure 5A). We measured Stu1p levels in all of these ever, neither plasmid restored the growth of stu1-5 ⌬ Њ Њ strains at 26Њ and 35Њ (Figure 5, B and C). At 35Њ, the ubp3 at 35 as well as it did for stu1-5.At33, the Stu1p ⌬ Stu1p level in the stu1-5 [stu1-5 YEp] strain was Ͼ10- level in the stu1-5 ubp3 [stu1-5 YEp] strain was fold higher than that in the stu1-5 [YCp] strain and 10-fold higher than that in the stu1-5 [YCp] strain and equal to the level in stu1-5 [STU1 YCp]. Thus, a level equivalent to the level in stu1-5 [STU1 YCp]. Thus, in of Stu1-5p equivalent to the amount of wild-type Stu1p the presence of the ubp3⌬, a level of Stu1-5p equivalent produced from a YCp plasmid allows growth at the re- to the amount of wild-type Stu1p produced from a YCp strictive temperature, although growth is slower at 37Њ. plasmid allows growth at 35Њ but not at 37Њ. The Stu1- The Stu1-5p level in stu1-5 [stu1-5 YCp] was 5-fold higher 5p level in stu1-5 ubp3⌬ [stu1-5 YCp] was 3-fold higher fold less than that-3ف fold less than that than that in stu1-5 [YCp] but still-3ف than that in stu1-5 [YCp] but still in stu1-5 [STU1 YCp] at 35Њ. Thus, there appears to be in stu1-5 [STU1 YCp] at 33Њ. This indicates the presence a threshold of Stu1-5p, near the level in stu1-5 [stu1-5 of a threshold, somewhere between the protein level of YCp] cells, that will allow growth at 35Њ. The threshold stu1-5 ubp3⌬ [stu1-5 YCp] and stu1-5 ubp3⌬ [YCp], that for growth is lower at 26Њ where stu1-5 [YCp] is viable is necessary for the growth of stu1-5 ubp3⌬ strains at 33Њ. despite even lower protein levels. Overall, these results are consistent with the idea that If the synthetic lethality of stu1-5 ubp3⌬ is due solely temperature sensitivity of stu1-5 strains is due primarily to diminished levels of Stu1p, then overexpression of to the instability of the mutant protein. Loss of Ubp3p 1086 C. T. Brew and T. C. Huffaker

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 Figure 5.—Overexpression of stu1-5 suppresses stu1-5 heat and assayed for growth at the indicated temperature. (B) The sensitivity and restores Stu1p levels in stu1-5 cells. stu1-5 (CUY999) strains were grown at 26Њ to mid-log phase and then shifted was transformed with YCp STU1 (pDP94), YEp stu1-5 (pCK37), to 33Њ for the indicated time. Cell extracts were analyzed by YCp stu1-5 (pCT1), or YCp (pRS415). (A) Transformants were immunoblotting with an anti-Stu1p polyclonal antibody. (C) plated onto SD-Leu media and assayed for growth at the indi- Њ The quantitation of the data from two independent experi- cated temperature. (B) The strains were grown at 26 to mid- ments is shown. Stu1p levels were normalized to Act1p levels. log phase and then shifted to 35Њ 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 nor- (ABY544) and stu2-10 (CUY1070) strains to a ubp3 ::HIS3 malized to Act1p levels. strain (CUY1326). In both cases, the double mutants were more temperature sensitive than the single mutants (Fig- ure 7). These data support the notion that Ubp3p plays exacerbates this defect, lowering the restrictive tempera- a general role in stabilizing proteins. ture. ubp3⌬ is synthetically lethal with myo2-14 and stu2-10: DISCUSSION To determine whether the effect of ubp3⌬ is specific to stu1-5, we checked two other mutations for synthetic Stu1p is an essential microtubule-associated protein lethality with ubp3⌬. Myo2p is an essential type V myosin that is involved in spindle assembly. We have identified implicated in vesicular transport and polarized growth, four genes whose loss is lethal in stu1-5 cells at tempera- as well as nuclear migration (Govindan et al. 1995; Yin tures that are permissive for the stu1-5 cells. The first is et al. 2000). Stu2p is an essential microtubule-binding UBP3, which encodes a ubiquitin protease. The second protein that regulates microtubule dynamics and is re- and third, PAC10/GIM2 and GIM3, are both genes that quired for nuclear migration and spindle elongation encode members of the GIM family in yeast and are (Kosco et al. 2001; Severin et al. 2001). myo2-14 and important for folding of tubulin and actin. The fourth stu2-10 are temperature-sensitive alleles that cause de- gene, KEM1/XRN1/SEP1, encodes an RNA exonuclease creased levels of these proteins. Myo2p levels are de- that also has been reported to influence microtubule creased by 8-fold in the myo2-14 mutant (Schott 2000), function. and Stu2p levels are decreased by 2.5-fold in the stu2-10 Stu1p and Ubp3p: Ubp3p is known to be one of a mutant (Kosco 2002) compared to wild type. myo2-14 large group of deubiquitination enzymes in yeast. These ubp3⌬ and stu2-10 ubp3⌬ were made by crossing myo2-14 proteins are believed to act as either positive or negative Ubp3p Promotes Protein Stability 1087

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 33Њ. 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 Figure 7.—Genetic interactions of ubp3⌬ with stu1-5, myo2-14, in stabilizing Stu1-5p. This is supported by additional and stu2-10. ubp3⌬ (CUY1326) was crossed to myo2-14 (ABY544) genetic evidence: myo2-14 and stu2-10 decrease the levels and stu2-10 (CUY1070), and the diploids were sporulated. of Myo2p and Stu2p, respectively, and in both cases Spores were plated onto YPD media and assayed for growth ubp3⌬ lowered their restrictive temperature. Thus, at the indicated temperature. 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- regulators of proteolysis, but there are few data on what protein conjugates in ubp3⌬ (Baxter and Craig 1998; specific function these proteases perform. The 16 Ubp Amerik et al. 2000). In addition, overexpression of UBP3 enzymes likely have overlapping functions as suggested suppresses the heat-shock mutant ssa1 ssa2, in contrast by the normal growth rate of multiple deletion mutants. to UBI4 and UBC4, two genes that encode promoters Doa4p, Ubp14p, and Ubp3p are the only enzymes of of proteolysis. Overall, Ubp3p appears to act as a proof- this group that have been examined in detail. Doa4p reading enzyme, reversing the ubiquitination of mis- cleaves ubiquitin chains from proteolytic intermediates folded, temperature-sensitive proteins and allowing them (Papa and Hochstrasser 1993), while Ubp14p is re- to refold. sponsible for the cleavage of the free polyubiquitin ubp3⌬ is unique among ubp⌬’s when judged by the chains (Wilkinson et al. 1995; Amerik et al. 1997). severity of its synthetic lethality with stu1-5. Only ubp5⌬ These steps provide free ubiquitin monomer that can shows a similar synthetic effect, although to a lesser be attached to new targets for degradation. Ubp3p has degree (Figure 1). Thus, Ubp5p may also be an inhibitor been hypothesized to cleave ubiquitin chains from sub- of proteolysis whose function partially overlaps Ubp3p. strate proteins because its disruption leads to the accu- In contrast, we found that doa4⌬ and ubp6⌬ suppressed mulation of ubiquitin-protein intermediates (Baxter stu1-5 temperature sensitivity at 37Њ. This is consistent and Craig 1998). We have found that stu1-5 creates a with the idea that Doa4p promotes proteolysis and that, situation in which UBP3 is essential. Thus, understand- in its absence, Stu1-5p is not degraded as rapidly. The ing the stu1-5 defect may shed more light on the role fact that ubp6⌬ also suppresses the temperature-sensitive of Ubp3p. phenotype suggests that it too may promote proteolysis. We considered two possible explanations for the stu1-5 An overlap in function between these two enzymes was ubp3⌬ synthetic lethality. First, we examined the possibil- previously suggested because loss of Doa4p or Ubp6p ity that Ubp3p plays a role in the cell-cycle regulation results in lower levels of ubiquitin and amino acid ana- of Stu1p. Several mitotic proteins are subject to cell- logue hypersensitivity (Amerik et al. 2000). These results cycle regulation involving ubiquitin-mediated protein suggest that the deubiquitinating enzymes perform di- degradation. However, the levels of Stu1p are relatively verse functions or have a high degree of substrate speci- constant throughout the cell cycle, indicating that it ficity. is not a substrate for this specific ubiquitin-dependent Stu1p and the Gim proteins: We also isolated gim3 pathway. and pac10/gim2 alleles as synthetically lethal with stu1-5. Next, we considered the possibility that mutant Stu1- GIM1-GIM5 were previously identified in a synthetic le- 5p is an unstable protein that is targeted for ubiquitin- thal screen with tub4-1, and their protein products were mediated degradation. We showed that the levels of shown to associate in common complexes (Geissler et Stu1p in stu1-5 cells are several-fold lower than the levels al. 1998). We tested gim1⌬, gim4⌬, and gim5⌬ and found in wild-type cells. In addition, the temperature sensitivity that these mutations were also lethal in combination of stu1-5 can be suppressed by producing wild-type levels with stu1-5. The gim⌬’s were shown to be benomyl super- of the mutant protein. These results indicate that the sensitive, due to reduced levels of ␣-tubulin, but not temperature sensitivity is caused by reduced levels of ␤-or␥-tubulin (Geissler et al. 1998). A distinct Gim Stu1p and not by reduced function. Interestingly, the function is related to ␥-tubulin, based on experiments critical threshold of Stu1-5p protein level appears to be that show the Gim proteins bind to Tub4p and genetic somewhat higher at higher temperatures. interactions between gim1⌬ and alleles of SPC98 and Given that the primary effect of the stu1-5 mutation SPC97 (Geissler et al. 1998). In addition, tubulin immu- 1088 C. T. Brew and T. C. Huffaker nofluorescence of gim⌬’s showed that microtubule at- Baxter, B. K., and E. A. Craig, 1998 Isolation of UBP3, encoding tachment to the SPB was intact, but microtubule stability a de-ubiquitinating enzyme, as a multicopy suppressor of a heat- ⌬ shock mutant strain of S. cerevisiae. Curr. Genet. 33: 412–419. was impaired. Thus, stu1-5 gim1-5 synthetic lethality Bender, A., and J. R. Pringle, 1991 Use of a screen for synthetic may result from the combined effects of tubulin misfold- lethal and multicopy suppressee mutants to identify two new ing due to loss of the Gim1-5 and spindle assembly genes involved in morphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 1295–1305. defects caused by stu1-5. Bradford, M. M., 1976 A rapid and sensitive method for the quanti- Alternatively, the synthetic lethality between stu1-5 tation of microgram quantities of protein utilizing the principle and the gim⌬’s may result because the GimC complex of protein-dye binding. Anal. Biochem. 72: 248–254. Caponigro, G., and R. Parker, 1996 Mechanisms and control of promotes formation of functional Stu1p. GimC func- mRNA turnover in Saccharomyces cerevisiae. Microbiol. Rev. 60: tions with chaperonin during translation and folding 233–249. of tubulin or actin (Geissler et al. 1998; Siegers et al. Chung, C. H., and S. H. 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