Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces Cerevisiae

MOLECULAR AND CELLULAR BIOLOGY, July 1996, p. 3255–3263 Vol. 16, No. 7 0270-7306/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces cerevisiae

HIDEKI YASHIRODA, TOMOKO OGUCHI, YUKO YASUDA, AKIO TOH-E, AND YOSHIKO KIKUCHI* Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Received 23 October 1995/Returned for modiﬁcation 27 November 1995/Accepted 26 March 1996

We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt؊ phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

Ubiquitination is a ubiquitous system for tagging proteins E6-AP (21). The E6 and E6-AP binary complex then binds to which is highly conserved in eukaryotes (6, 11, 17, 19, 27). Not a tumor suppressor protein, p53, which is ubiquitinated by only are aberrant proteins recognized and conjugated with E6-AP, and the tagged p53 is degraded by the 26S proteasome, ubiquitin molecules, but also many important proteins in var- leading to tumorigenesis (47). With an in vitro system, Schef- ious processes of the mitotic cell cycle, signal transduction, or fner et al. established that ubiquitins, ubiquitin-activating en- differentiation are known to be substrates of this system, such zyme (E1), ubiquitin-conjugating enzyme (E2), viral E6 pro- as cyclins and inhibitors of cyclin-dependent kinases (13, 25, tein, E6-AP (E3), and ATP are required for the ubiquitination 34, 48, 53). Subsequently, ATP-requiring proteolysis of those of p53 (45). The cysteine residue near the C terminus of E6-AP multiubiquitinated conjugates by the multicomponent protea- is essential for the formation of a thioester bond with a ubiq- some occurs (40). Since the proteolysis is an irreversible pro- uitin molecule (46). Ubiquitin molecules are transferred cess, the target proteins to be ubiquitinated should be recog- through an E1-E2-E3 enzyme thioester cascade to their target nized with a high degree of speciﬁcity and proper timing; thus, proteins (46). In this system, viral E6 protein is a connector strict and complicated regulatory systems are needed. Re- molecule between the enzyme and its substrate; it can recog- cently, large complexes were reported to be required for the nize the target protein and activate the ubiquitin ligase. ubiquitination of cyclin B in various systems (34, 53). Many proteins have been found to contain a 30-kDa hect In Saccharomyces cerevisiae, many genes are known to be domain at their C termini (20). In S. cerevisiae, at least three involved in the ubiquitination system. UBA1 encodes a ubiq- proteins carry this hect domain. One of them, Rsp5, was sug- uitin-activating enzyme (E1) (39). At least 10 UBC genes (E2) gested to be a ubiquitin ligase like E6-AP (20). rsp5 was iso- encoding ubiquitin-conjugating enzymes have been isolated so lated as a revertant of spt3 encoding a TFIID-binding protein far (27). These enzymes form thioester bonds with ubiquitin (8, 16). UFD4 (identical to YKL162) was found to be involved molecules and transfer them to substrate proteins. In some in the UFD (Ub fusion degradation) pathway (28). We are also cases, additional (E3) factors (18) are known to be required; characterizing another hect gene, TOM1, which is required for

UBR1, which encodes an E3␣ as a substrate-recognizing pro- the G2-M transition at a high temperature (56a). tein involved in N-end rule degradation, was isolated (1, 7). We have obtained temperature-sensitive (ts) mutants of S. Recently, another type of E3 enzyme, namely, a ubiquitin cerevisiae, as isolated previously (33), and one of them was ligase containing a hect (homologous to E6-AP C terminus) defective in the maintenance of minichromosomes at a high domain, was reported (20). temperature. We cloned a new gene, BUL1, and RSP5, whose Human E6-AP was the ﬁrst ubiquitin ligase described (45). product interacted physically with this new gene product. In When human cells are infected with the high-risk papilloma- this report, we describe these new components involved in the virus, the viral E6 protein binds to a host component, called ubiquitination pathway of S. cerevisiae.

MATERIALS AND METHODS * Corresponding author. Mailing address: Department of Biological Strains and genetic manipulations. Escherichia coli K-12 strain DH5␣ [supE44 Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, ⌬lacU169 (␾80lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used for Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3812-2111, ext. propagating plasmids. The following strains of S. cerevisiae were used: YPH499 4466. Fax: 81-3-5684-9420. Electronic mail address: [email protected] (MATa ade2 his3 leu2 trp1 ura3 lys2) (50); YPH4992, a homozygous diploid strain -tokyo.ac.jp. of YPH499; KA31 (MATa his3 leu2 ura3 trp1) (24); RAY-3A (MATa leu2 ura3

3255 3256 YASHIRODA ET AL. MOL.CELL.BIOL.

(this laboratory). For pHY16, the PstI site inside RSP5 was fused to the downstream region of the myc epitope tag (amino acid sequence EQKLISEEDL) of the pKT10-mycN vector (constructed by K. Fujimura) so that the fusion gene encoding the myc tag at the N terminus of Rsp5 was expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter (55). pHY17 is YEplac112 containing the 5-kb HindIII fragment of RSP5. pHY18 is pW1 carrying the 4-kb EcoRI-SacI fragment of BUL1. pHY19 is YEplac112 containing both the 5-kb HindIII fragment of RSP5 and the 4-kb EcoRI-SacI fragment of BUL1. pHY20 is YEplac181 bearing BUL1-HA from pHY15. The Ub-X-LacZ plasmids (7) were generously given by A. Varshavsky via K. Tanaka of Tokushima University. For E. coli transformation, the cells were prepared as described by Inoue et al. (23). Yeast transformation was performed with lithium acetate (26). Gene re- placement was carried out as described by Rothstein (42). For BUL1 gene disruption, the plasmid pHY05 (Fig. 1A) was digested with SacI and SalI and used to transform the YPH4992 cells for tryptophan prototrophy. For RSP5 gene disruption, plasmid pTOP232 (Fig. 1B) was digested with HindIII and introduced into strain YPH4992 to select uracil prototrophy. Rolling method. Genomic DNA of strain YHY002, in which plasmid pYH03 was integrated at the BUL1 locus, was isolated, purified by phenol extraction, and digested with restriction enzyme EcoRI. The DNA circularized with T4 DNA ligase (Bethesda Research Laboratories) was introduced into an E. coli strain to select ampicillin-resistant colonies. The recovered plasmid contained the entire BUL1 gene, and the 4-kb EcoRI-SacI DNA fragment was inserted into either pRS316 (pHY06) or YEUp3 (pHY66). FIG. 1. Restriction maps of BUL1 and RSP5 and structures of various plas- DNA sequencing. Nucleotide sequences were determined by the dideoxy mids. (A) Map of BUL1 and its neighboring regions, constructed from two chain-termination method (44), using double-stranded DNAs as templates and a plasmids, pHY01 and pHY06. Only inserts of various plasmids are given (lines). Dye Deoxy Cycle sequencing kit (Applied Biosystems). After PCRs, the samples YEp, multicopy plasmid; YCp, single-copy plasmid; and Ylp, integration plas- were analyzed by using 6% polyacrylamide gels and an ABI model 370 se- mid. The complementation activity of each plasmid is indicated on the right. quencer. Plasmid pHY05 was used to disrupt BUL1 (see text), and pHY07, which contains DNA isolation and Southern hybridization. Yeast DNA was isolated (43), a gene encoding Bul1 fused to the LexA DNA binding domain, was used for digested with appropriate restriction enzymes, and run on 1% agarose gels. screening plasmids by the two-hybrid system. (B) Map of RSP5 and structure of During the treatment of the gel with 0.4 N NaOH for 4 h, the denatured DNA the plasmids. Plasmid pHY09 was isolated by the two-hybrid system; the C- was blotted onto Hybond Nϩ membrane filters (Amersham). Southern hybrid- terminal part of RSP5 was fused in frame to the Gal4 activation domain on ization was performed by using the enhanced chemiluminescence direct nucleic plasmid pGAD. Plasmid pTOP232 was used for disrupting RSP5. Restriction acid labeling system, according to the procedure recommended by the manufac- sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; Pv, PvuII; turer (Amersham). For the evaluation of the BUL1 gene disruption, the 3-kb RV, EcoRV; Sc, SacI; Sl, SalI; and X, XhoI. BamHI-SacI fragment of pHY01 was used as a probe. For physical mapping of BUL1, we obtained a membrane filter from the American Type Culture Collec- tion, on which contiguous lambda phage DNAs containing the S. cerevisiae genome were spotted. To verify the RSP5 gene disruption, the 1.1-kb XhoI-PvuI trp1 his3) (56); YHY001 (MAT␣ bul1-1 ura3 trp1 lys2 his3) (33); YHY002, an fragment of pTOP223 was used as a probe. integrant of YHY001 with the plasmid pHY03; YHY003 (bul1::TRP1 in the Cell lysis assay. Yeast cells were streaked as patches on yeast-peptone-dex- YPH499 background); YAT2-1C (MAT␣ his3 leu2 trp1 ura3 rsp5-101) (this trose (YPD) plates, which were incubated at 37ЊC overnight. Then the patches laboratory); and L40 (MATa his3 leu2 trp1 URA3::lexA-lacZ LYS2::lexA-HIS3) were covered with a mixture of 2 ml of 1% melted agar, 2.5 ml of 100 mM (57). The media and methods for mating, sporulation, and tetrad analysis were glycine-HCl (pH 9.5), and 434 ␮l of BCIP (5-bromo-4-chloro-3-indolylphosphate described previously (49). toluidinium) (Promega). After incubation at 37ЊC for 1 h, the patches of lysed Plasmids and transformation. The following multicopy plasmid vectors were cells should turn blue. used in this study: YEp24 (URA3) (3), YEUp3 (URA3) (A. Fujita, National Two-hybrid system. The transformant of strain L40 with plasmid pYH07 was Institute of Bioscience and Human Technology), pW1 (TRP1) (H. Hashimoto, introduced further with the pGAD bank. Trpϩ Leuϩ transformants were Nikka Whisky Co.), YEp13 (LEU2) (4), YEplac112 (TRP1) (12), and YEplac181 checked for the Hisϩ phenotype in the presence of 10 mM 3AT (3-amino-1,2,4- (LEU2) (12). Single-copy vectors YCUp4 (CEN4 ARS1 URA3) (A. Fujita), triazole) at 26ЊC. ␤-Galactosidase activity was also tested. YCp50 (CEN4 ARS1 URA3) (37), and pRS316 (CEN6 ARSH4 URA3) (50) were Assay for ␤-galactosidase. Cells were grown to mid-log phase in minimal used. The YEp24 library and YCp50 bank were kindly provided by D. Botstein medium and collected by centrifugation. ␤-Galactosidase activity was measured and R. Davis of Stanford University, respectively. The YEp13 library was gen- as described by Guarente (15). erously given by Y. Ohya of The University of Tokyo. The YCUp4 bank was Preparation of cell lysates. Transformants were grown in 5 ml of selective kindly provided by A. Fujita. DNA manipulations were performed as described media to an optical density at 600 nm (OD600) of 1.0 and collected by centrifu- elsewhere (43). pHY01 is YEp24 containing a defective BUL1 gene (Fig. 1A). gation. The pellets were suspended in 50 ␮l of lysis buffer (100 mM Tris-HCl [pH pHY02 is a plasmid with a deletion of the 3.5-kb SacI fragment from pHY01. 7.5], 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol) (54) pHY03 was constructed by deleting the 2␮m DNA from pHY02. pHY100 is containing 1 mM phenylmethylsulfonyl fluoride, 1 ␮g each of leupeptin, antipain, pRS316 carrying the 3-kb fragment from the junction of the insert (the SmaI site and pepstatin per ml and 2.1 ␮g of aprotinin per ml and broken by being vortexed of the vector) to the SacI site of pHY01. For pHY05, the 3-kb BamHI-SacI with glass beads, and the lysates were prepared by centrifugation at 12,000 ϫ g fragment of BUL1 was inserted into pTZ18R (Pharmacia) and the 1.1-kb XhoI- for 5 min at 5ЊC (56). KpnI fragment inside the BUL1 gene was replaced with the 1-kb BamHI-SalI Immunoblot analysis. The protein concentrations of cell lysates were deter- fragment of the TRP1 marker from pJJ280 (29) by ligation after treatment with mined by using the Bio-Rad protein assay. Samples (50 ␮g) were mixed with T4 DNA polymerase (Toyobo Co.). pHY06 is pRS316 containing the 4-kb equal amounts of sample buffer, incubated at 95ЊC for 5 min, and electropho- EcoRI-SacI fragment carrying the entire BUL1 gene. pHY66 is YEUp3 contain- resed through 7.5% polyacrylamide–sodium dodecyl sulfate (SDS) gels (31). The ing the same fragment as pHY06. For the two-hybrid system, we used pBTM116 gels were stained with Coomassie brilliant blue in 5% methanol–7.5% acetic (lexA DNA-binding domain, TRP1), kindly provided by R. Sternglanz of New acid. For immunoblotting, the proteins were transferred to Millipore membrane York State University, and GAD424 (Gal4-activating domain, LEU2) (Clontech) filters. The filters were incubated with 5% skim milk in TBST buffer (10 mM vectors. The gene libraries were constructed and generously given by R. Tris-HCl [pH 8.0], 0.15 M NaCl, 0.05% Tween 20) for blocking overnight at 4ЊC, Sternglanz; Sau3A partial digests of genomic DNA were ligated into the BamHI washed with 10 ml of TBST buffer three times for 5 min each, and incubated with site of either pGAD1, pGAD2, or pGAD3 (5). For pHY07, the 4.5-kb BamHI- 10 ml of 2-␮g/ml anti-HA (12CA5) (Boehringer Mannheim) or 1-␮g/ml anti-myc BglII fragment of BUL1 was inserted into the BamHI site of pBTM116, so that (9E10) monoclonal antibody in TBST buffer for2hatroom temperature. Then the BUL1 sequence from the BamHI site to the C terminus was fused in frame the membranes were washed three times with 10 ml of TBST buffer for 5 min to the lexA DNA-binding domain. pHY08 is YCUp4-RSP5. pYH09 is pGAD- each and incubated with 10 ml of TBST buffer containing 1.5 ␮l of secondary Rsp5, isolated from the pGAD bank (Fig. 1B). pHY14 is pRS316 containing the anti-mouse immunoglobulin G alkaline phosphatase (Tago Co.) or anti-mouse BUL1 fused gene with a double hemagglutinin (HA) epitope tag (amino acid immunoglobulin G horseradish peroxidase (Dupont) for1hatroom tempera- sequence MYPYDVPDYA) at the C terminus of Bul1. pHY15 is YEUp3-BUL1- ture. After three washes with 10 ml of TBST buffer for 5 min each, the detection HA. pTOP223 is YEp13-RSP5, isolated from the bank. For pTOP232, the 1.1-kb reagent was spread on the filters, as instructed by each manufacturer. HindIII fragment carrying URA3 was inserted into the PstI site inside RSP5 on Pulse-chase experiments. The protocol for the pulse-chase experiments was the Bluescript KSϩ vector containing LEU2 (Fig. 1B). pMS16 is YEp24-UBI1 essentially as described previously (35, 54). Cells of strain KA31 (RSP5)or VOL. 16, 1996 YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE 3257

YAT2-1C (rsp5) containing pHY15 were grown overnight in minimal medium junction of the vector to the SacI site in the insert (pHY02). A (MV medium) in which all sulfate salts were replaced by chloride salts supple- 8 YIp plasmid (pHY03) carrying this region was digested with mented with 100 mM (NH4)2SO4 (41). Five OD600 units (3 ϫ 10 cells) of exponentially growing cells were harvested and resuspended in fresh MV me- restriction enzyme BamHI and integrated into the YHY001 dium to a density of 2 OD600 units. The cells were labeled for 20 min after genome by homologous recombination. The integrants were 35 addition of 15 ␮Ci of Tran[ S] (ICN) per OD600 unit of cells. Then a concen- able to grow at 37ЊC, and one of them (YHY002) was crossed trated chase solution [100 mM (NH4)2SO4, 0.3% cysteine, 0.4% methionine] was with the wild-type YPH499 and the resulting diploid was diluted 100-fold into the cultures for further incubation at 37ЊC. Aliquots of 1 sporulated. Almost all the spores (50 of 52) were able to OD600 unit of cells were removed at intervals, and the chase was terminated by the addition of an equal volume of ice-cold 20 mM sodium azide and rapid germinate and grow at 37ЊC, indicating that the integrated chilling on ice. The cells were washed once and resuspended in 50 ␮l of lysis gene was tightly linked to the ts mutation. However, when the buffer. Lysis was achieved by vortexing the suspension with glass beads, which DNA was cloned into a single-copy vector (pHY100), it could were washed with 100 ␮l of lysis buffer. For immunoprecipitation, aggregates were removed by centrifugation at 12,000 ϫ g for 15 min and 100 ␮lofthe not complement the ts mutation. The 5Ј upstream and N- supernatant was incubated with 1.5 ␮g of anti-HA antibody. After3hat0ЊC, 50 terminal regions of the gene were missing in this original iso- ␮l of a suspension of protein A-Sepharose beads (Pharmacia) (100 mg/ml in late. The neighboring region was cloned from the integrant radioimmunoprecipitation assay [RIPA] buffer, consisting of 50 mM Tris-HCl (YHY002) by the rolling method as described in Materials and [pH 7.5]–200 mM NaCl–1% Triton X-100–0.5% sodium deoxycholate–0.1% SDS, containing 1% bovine serum albumin) was added. The mixture was incu- Methods. From a recovered plasmid, we constructed a single- bated with gentle rocking for 2.5 h at 0ЊC. The beads were washed four times with copy plasmid (pHY06) containing the 4-kb EcoRI-SacI frag- 75% lysis buffer–25% RIPA buffer containing 1 mM phenylmethylsulfonyl flu- ment, which had the complementing activity, as shown below oride and then once with 50 mM Tris-HCl (pH 7.5)–50 mM NaCl. The beads (see Fig. 3A). As described below, we named the gene BUL1 were resuspended in 50 ␮lof2ϫsample buffer, and the mixture was heated for 5 min at 95ЊC. The supernatant was subjected to electrophoresis through 7.5% (for a protein that binds to the ubiquitin ligase). The BUL1 polyacrylamide–SDS gels. The gels were fixed in 7.5% acetic acid and 5% gene was physically mapped near SUP8 on chromosome XIII methanol, dried, and exposed to Imaging Plate (Fuji Film) for 2 days. The bands R, since the 3-kb BamHI-SacI fragment was hybridized to the were visualized by radioluminography with a BAS-Mac system (Fuji Film). lambda phage clones 70071 and 70218 on an American Type Sucrose gradient centrifugation. A 150-␮l volume of the cell lysates from 100 ml of culture was loaded on 10 to 30% sucrose gradients made on 0.5 ml of 60% Culture Collection membrane filter by Southern hybridization sucrose cushion in lysis buffer or phosphate-buffered saline (PBS) buffer (2.2 mM (data not shown). NaH2PO4 ⅐ 2H2O, 8.4 mM Na2HPO4 ⅐ 12H2O, 150 mM NaCl [pH 7.0]) and The nucleotide sequence and its deduced amino acid se- centrifuged in a Beckman SW41.Ti rotor at 32,000 rpm for 14 h at 5ЊC. Fractions quence are shown in Fig. 2A. The predicted gene product were collected from the top. Immunoprecipitation. A 300-␮l sample of the 40 or 22S sucrose gradient appears to be a basic (pI ϭ 8.8) and hydrophilic 110-kDa fractions in PBS buffer was mixed with 10 ␮g of anti-HA antibodies, and the protein, and its C terminus is very similar to the protein se- mixture was incubated at 4ЊC for 2 h. Then 50 ␮l of protein A-Sepharose beads quence predicted by a human cDNA which is expressed in T (Pharmacia) was added, and the mixture was incubated at 4ЊC for 2 h and washed cells (GenBank database accession no. Z16200) (Fig. 2B). Bul1 four times with 75% lysis buffer–25% RIPA buffer. After a further washing with 50 mM Tris-HCl (pH 7.5)–50 mM NaCl, sample buffer was added to the bead has many putative phosphorylation sites, such as those of mi- pellets and the samples were boiled for 5 min and subjected to gel electrophoresis. togen-activated protein (MAP) kinase (amino acid residues 59 Nucleotide sequence accession number. The nucleotide sequence of BUL1 has to 62), CDC28 kinase (residues 101 to 104), and A kinase been submitted to GenBank under accession no. D50083. (residues 104 to 107), C kinase, and casein kinase II. Two RESULTS serine-rich stretches are also found near the C-terminal region (amino acids 619 to 640 and 868 to 898). A ts mutant defective in the maintenance of minichromo- The bul1::TRP1 disruptant (YHY003) in the YPH499 strain somes. From our ts mutants isolated previously (33), we se- background showed ts growth like the original ts mutant, and lected one (YHY001) in which a YCp50 minichromosome (37) its temperature sensitivity was complemented by a single copy containing a replication origin (ARS1) (52) and a centromere of BUL1 (pHY06), as shown in Fig. 3A. The disruptant was (CEN4) (51) was maintained less stably at 35ЊC (a semiper- also moderately defective in the stability of the minichromo- missive temperature) than at 26ЊC (a permissive temperature). some at a semipermissive temperature (33% at 35ЊC compared The mutant carrying YCp50 was grown in a selective medium with 62% at 26ЊC). Furthermore, the disruptant cells were at 26ЊC, diluted in a nonselective medium (YPD), and grown at partially lysed on YPD plates at 37ЊC, since the cell patches either 35 or 26ЊC for seven generations. Then cells were spread turned blue when assayed with BCIP (data not shown). The on plates for single colonies, and each colony was checked for temperature sensitivity of the bul1 disruptant as well as that of uracil prototrophy, as described previously (30). Only 23% of the original ts mutant was suppressed by adding 1 M sorbitol as the cells maintained YCp50 at 35ЊC, whereas 56% of the cells an osmotic stabilizer in the medium (Fig. 3B). contained it at 26ЊC. In contrast, the wild-type strain YPH499 SSD1/MCS1 is a single-copy suppressor of bul1. We isolated (50) could maintain YCp50 equally well at either temperature SSD1/MCS1 (54, 56) on a single-copy vector, which was able to (63% at 26ЊC and 57% at 35ЊC). Thus, the mutant appeared to suppress the temperature sensitivity of the bul1 disruptant be moderately defective in the stable maintenance of the even at 37ЊC (Fig. 3A). Although the function of SSD1/MCS1 minichromosome at the higher temperature. When this ts mu- is still obscure at present, it may encode an exoribonuclease II, tant was crossed with the wild-type YPH499, the ts phenotype since the amino acid sequence is similar to that of the Shigella was segregated at 2ϩ:2Ϫ, indicating that a single mutation flexneri VacB protein (Swiss-Prot database accession no. should cause the ts growth. The morphology of the arrested P30851). Previous work indicated that the intact Ssd1/Mcs1 cells at 37ЊC was not cell cycle specific, but large cells with an protein was missing in strain YPH499 (56), and some ts mu- elongated bud were frequently observed at 6 h after the tem- tations, for example, htr1, are suppressed by introducing a perature shift (data not shown). Fluorescence-activated cell single copy of SSD1/MCS1 (32). The bul1 disruptant in other sorter analysis did not show any cell cycle-specific arrest (data strain backgrounds, such as strain RAY-3A carrying intact not shown). SSD1/MCS1 (56), was not ts (data not shown). Thus, BUL1 and Isolation of a new gene, BUL1. By introducing the YEp24 SSD1/MCS1 might function redundantly in some step for mi- library (3) into strain YHY001 (bul1-1), we isolated a plasmid totic growth, at least at a high temperature. (pHY01) by the ability to complement the ts phenotype. As Bul1 physically interacts with Rsp5. In order to search for shown in Fig. 1A, subcloning experiments could locate the proteins which interact with Bul1, we took advantage of the responsible region within the 3.0-kb DNA fragment from one two-hybrid system (9, 10). First we constructed plasmid 3258 YASHIRODA ET AL. MOL.CELL.BIOL.

FIG. 2. Nucleotide sequence of BUL1 and corresponding amino acid sequence. (A) Nucleotide sequence of BUL1 encoding 976 amino acids. Putative phosphorylation sites of MAP kinase (shaded), Cdc28 kinase (boxed), and A kinase (double underlined) and a putative polyadenylation signal (underlined) are indicated. (B) Identical amino acids between the C terminus of the yeast Bul1 and the predicted sequence of a human cDNA (GenBank accession no. Z16200) are shown (black boxes). VOL. 16, 1996 YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE 3259

(2). The overall amino acid sequence homology with yeast RSP5 is found in mammalian Nedd-4; mouse Nedd-4 was isolated as a gene with developmentally regulated expression in the mouse brain (36). RSP5 was essential for mitotic growth. We disrupted one of the RSP5 genes of the wild-type diploid strain YPH4992. Two spores from one ascus were able to make colonies (Fig. 4A), and all the viable clones were UraϪ, indicating that RSP5 is essential for mitotic growth, which is consistent with recent results of others (58). We had a ts rsp5-101 mutant. The mutation was recessive to the wild type. The arrested morphology of the mutant was not cell cycle speciﬁc, but the cells were partially lysed on YPD FIG. 3. Complementation and suppression of temperature sensitivity of the plates at 37ЊC, when assayed by using BCIP (data not shown). bul1 mutants. (A) Strain YHY003 (bul1::TRP1) was transformed with YCUp4 As shown in Fig. 4B, this temperature sensitivity was partially (a), YCp-BUL1 (b), and YCp-SSD1/MCS1 (c) at 26ЊC. Strain YHY001 (bul1-1) suppressed by adding 10% sorbitol to the medium, although was transformed with YCp-SSD1/MCS1 (d), YCp-BUL1 (e), and YCUp4 (f) at the rsp5 disruptant spore was not able to recover on 10% 26ЊC. Each Uraϩ transformant on a YPD plate was incubated at 37ЊC for 2 days. (B) The same transformants were streaked on a YPD plate containing 1 M sorbitol medium (data not shown). Other than the ts pheno- sorbitol and incubated at 37ЊC for 2 days. type of this rsp5 mutant, it was sensitive to 1 ␮g of canavanine per ml in minimal medium at 26ЊC (Fig. 4C), suggesting that the mutant is defective in ubiquitination and/or degradation of pHY07, encoding a LexA-Bul1 fusion protein (Fig. 1A), aberrant proteins. screened the pGAD library, and isolated pHY09. Cotransfor- Genetic interactions between RSP5 and BUL1. Multiple cop- mation of pHY07 with pHY09 caused a Hisϩ phenotype and a ies of BUL1 (pHY66) were not able to suppress the rsp5-101 high ␤-galactosidase activity (164.3 U), whereas the ␤-galacto- mutation, and a high dose of RSP5 (pTOP223) could not sidase activity of the transformant of pHY07 plus GAD424 suppress the ts phenotype of the bul1 disruptant. The rsp5 vector or pHY09 plus lexA vector was 2.1 or 1.3 U, respectively. bul1::TRP1 double mutant in the KA31 background did not DNA sequencing revealed that Gal4 was fused in frame to the show any additive phenotype, possibly because this strain con- C-terminal region (517 amino acids) of Rsp5 (Fig. 1B). RSP5 tains intact SSD1/MCS1. On the other hand, a high dose of stands for “reverses SptϪ phenotype” (16). The C-terminal half UBI1 encoding a ubiquitin molecule (pMS16) partially sup- of the gene product (380 amino acids) is homologous (34.5% pressed the rsp5 mutation (Fig. 5B) and also the temperature identical) to the C-terminal region of the human E6-AP ubiq- sensitivity of the bul1 disruptant (Fig. 5A). Furthermore, si- uitin ligase (22). The N-terminal half of Rsp5 contains the C2 multaneous overexpression of both genes on a multicopy plas- region, found in some protein kinases C (38), and three WW mid (pHY19) was toxic to the wild-type cell growth, while the domains which may be involved in protein-protein interactions overexpression of each plasmid individually (pHY17 or

FIG. 4. RSP5 is essential for mitotic growth. (A) A diploid strain, YPH4992, carrying a deletion in one of the RSP5 genes, was sporulated for tetrad dissection on a YPD plate and incubated at 25ЊC for 2 days. (B) Strain YAT2-1C (rsp5-101) and its cognate wild-type (WT) strain KA31, were streaked on YPD plates with or without 10% sorbitol, which were incubated at 36.5ЊC for 2 days. (C) Strain YAT2-1C was transformed with pHY08 (YCp-RSP5) or YCp50, and each transformant was streaked on a minimal medium (SD-Ura) plate containing 1 ␮g of canavanine per ml, which was incubated at 25ЊC for 2 days. 3260 YASHIRODA ET AL. MOL.CELL.BIOL.

was prepared for immunoblotting analysis, a very faint 140- kDa band could be detected (Fig. 7A, lane 2), while no bands were detected at the corresponding position in the negative control (lane 3). When the same fusion gene was introduced on a multicopy plasmid (pHY15), a much larger amount of the protein was detected (Fig. 7A, lane 1). Thus, the 140-kDa band was identified as the gene product of BUL1 tagged with HA, and the protein was a bit larger than expected from the sequence data (110 kDa). Frequently the Bul1 bands appeared as a doublet, suggesting that there may be modified forms. Next, we performed pulse-chase experiments. Wild-type cells or rsp5 mutant cells carrying pHY15 were labeled with 35S for 20 min at 26ЊC and chased for various times at 37ЊC. Cell lysates were prepared, Bul1 was immunoprecipitated with anti-HA antibody, and the precipitates were subjected to gel electrophoresis, as shown in Fig. 7B. The amount of labeled FIG. 5. The ubiquitin-encoding gene (UBI1) is a multicopy suppressor of the Bul1 in the wild-type cells after 2 h was almost unchanged, temperature sensitivity of the bul1 and rsp5 mutants. (A) Strain YHY003 (bul1::TRP1) was transformed with plasmid YEUp3 (a), pMS16 (YEp24-UBI1) indicating that Bul1 is a stable protein. In contrast, the bands (b), or pYH06 (YCp-BUL1) (c), and each transformant was streaked on a YPD of Bul1 in the rsp5 mutant were faint, even from the sample of plate which was incubated at 36.5ЊC for 3 days. (B) Strain YAT2-1C (rsp5-101) 0 min. When Bul1 was detected directly from crude lysates by was transformed with plasmid YEUp3 (a), pMS16 (YEp24-UBI1) (b), or pHY08 immunoblotting, the amount of Bul1 in the mutant was similar (YCUp4-RSP5) (c). Each transformant was streaked on a YPD plate which was incubated at 35.7ЊC for 2 days. to that in the wild type at either the normal or the high temperature (Fig. 7C). Probably, Bul1 was degraded during the immunoprecipitation in the absence of intact Rsp5, suggesting pHY18) did not interfere with wild-type cell growth (Fig. 6). that Bul1 physically interacts with Rsp5. Thus, it seems unlikely These results support the idea that BUL1 is involved in the that Bul1 is a target protein which is ubiquitinated by the Rsp5 ubiquitination pathway by collaborating with RSP5 by an un- ubiquitin ligase for its degradation. known mechanism. Bul1 and Rsp5 appeared to make large complexes. In order Since we do not know at present what proteins are directly to prove that Bul1 and Rsp5 physically interact in vivo, we ubiquitinated in vivo by this Rsp5-Bul1 system, we tested the attempted to detect a complex of Bul1 and Rsp5 by centrifuging protein stability of artificially designed substrates for the N-end the cell lysates through a sucrose density gradient. Bul1-HA rule pathway (1, 7). We tested five types of Ub-X-LacZ con- was expressed from its own promoter on a multicopy vector structs (with Leu, Arg, Ala, Met, and Pro as amino acid X) and (pHY20), and Rsp5 was tagged with myc at the N terminus and found that the ␤-galactosidase activity in the bul1 disruptant expressed under the control of the GAPDH promoter (YHY003) in each case was similar to that in the parental (pHY16). The cell lysate of the bul1 disruptant carrying both wild-type strain (YPH499), although the activity of Leu-LacZ plasmids was prepared and centrifuged through a sucrose den- in the bul1 disruptant was a few times higher than that in the sity gradient. Proteins of each fraction were separated through wild-type strain. Thus, BUL1 is not directly involved in the a gel electrophoresis and subjected to immunoblotting, as N-end rule pathway (1, 7). shown in Fig. 8. Bul1 sedimented with three different sizes Bul1 is a stable protein. If Bul1 is a substrate of Rsp5 (around 40, 22, and 7S), and Rsp5 sedimented similarly but ubiquitin ligase, it might be an unstable protein, since ubiq- more broadly, possibly because of its overexpression. A bulk of uitinated proteins are most likely to be degraded by the pro- proteins in the same gradient sedimented in narrower ranges, teasome. In order to determine the stability of Bul1, we con- judging from the pattern of the Coomassie-stained gel. A structed a functional BUL1 gene tagged with HA. When higher salt concentration (150 mM NaCl) in the gradient de- BUL1-HA was introduced into the bul1 disruptant on a single- creased the fast-sedimenting fractions of Bul1 or Rsp5 (data copy plasmid (pHY14) and the cell lysate of the transformant not shown). Next, we tested whether the sedimentation pattern of Rsp5 was influenced by the absence of Bul1. The cell lysate of the bul1 disruptant carrying both pHY16 (myc-RSP5) and YEplac181 (vector) was prepared and centrifuged through a gradient in parallel, and the fractions were subjected to immunoblotting in the same way. The sedimentation pattern of Rsp5 was not dramatically different from the previous pattern (data not shown). These experiments indicated that some fraction of the Rsp5 or Bul1 protein population cosedimented in the centrifugation, but they did not prove that the two proteins actually formed a complex. We immunoprecipitated Bul1-HA in the 40S fractions with anti-HA antibody, and the precipitates were subjected to immunoblotting. myc-Rsp5 was indeed detected in the precipitates (Fig. 9, lane 4), as well as Bul1-HA (lane 2). In contrast, no myc-Rsp5 was detected in the negative control (Fig. 9, lane FIG. 6. Simultaneous overexpression of RSP5 and BUL1 inhibits wild-type 3) when the corresponding gradient fractions from the cell cell growth. Multicopy plasmid pHY18 (pW1-BUL1), pHY17 (YEplac112- lysates without Bul1-HA were used, as mentioned above. A RSP5), or pHY19 (YEplac112-RSP5,-BUL1) was introduced into wild-type strain KA31 at 25ЊC, and each transformant was streaked on a minimal medium plate small amount of myc-Rsp5 was also immunoprecipitated with which was incubated at 34ЊC for 2 days. Bul1-HA in the 22S fraction (data not shown). Thus, at least VOL. 16, 1996 YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE 3261

FIG. 7. Bul1 is a stable protein. (A) Identification of Bul1-HA. Cell lysates of YHY003 (bul1::TRP1) carrying the indicated plasmids were prepared and subjected to immunoblotting as described in Materials and Methods. Lane 1, pHY15 (YEp-BUL1-HA); lane 2, pHY14 (YCp-BUL1-HA); lane 3, pRS316. (B) Pulse-chase experiment. Cells of YAT2-1C (rsp5) or KA31 (RSP5) carrying pHY15 were pulse-labeled with 35S for 20 min at 26ЊC and chased for various times at 37ЊC. Cell extracts were prepared and mixed with anti-HA antibody for 2 h. Protein A-Sepharose beads were added and the mixture was incubated for another 2 h, and the precipitates were subjected to gel electrophoresis. The bands were visualized by radioluminography. Lane 1, KA31; lanes 2 to 6, YAT2-1C(pHY15); lanes 7 to 11, KA31(pHY15). Lanes are shown 0 (lanes 1, 2, and 7) 30 (lanes 3 and 8), 60 (lanes 4 and 9), 90 (lanes 5 and 10), and 120 (lanes 6 and 11) min after the chase. (C) Cells of the same transformants as in panel B were cultivated in a selective medium at 26ЊC and divided into two samples for another 2-h incubation at 26 or 37ЊC. Cell lysates were prepared and subjected to immunoblotting. The band positions of Bul1 (arrows on the left of each gel) and standard molecular mass markers (arrows on the right of the gel) are indicated. WT, wild type. some fraction in the Rsp5 protein population actually forms found in the amino acid sequence of Bul1 (Fig. 2). In the case large complexes with Bul1. of E6-AP (45), the E6-AP ubiquitin ligase alone cannot act on p53, but E6-AP forms a binary complex with E6 which can bind DISCUSSION its substrate, so that the substrate recognition and activity of E6-AP are regulated by the presence of another factor(s). Bul1 and Rsp5 ubiquitin ligase are associated with large Thus, one attractive possibility is that the Rsp5 ubiquitin complexes. By the two-hybrid system and cosedimentation and ligase and Bul1 make large complexes which may be acting as coimmunoprecipitation experiments, we have shown that Bul1 an E3 protein in the ubiquitination pathway. In fact, recent physically interacts with the Rsp5 ubiquitin ligase. As de- findings in various systems for cyclin ubiquitination and deg- scribed above, the C terminus of the Rsp5 is homologous to radation indicated that the E3 components in the ubiquitina- human E6-AP ubiquitin ligase (22), and the N-terminal half tion system form large complexes. For example, a cyclosome contains three repeats of the WW domain (2). The WW do- from a clam extract and the 20S anaphase-promoting complex main, containing characteristic tryptophan residues, is thought containing Cdc27 and Cdc16 from Xenopus oocytes are re- to be involved in protein-protein interaction, and many pro- quired as E3 components for cyclin ubiquitination (34, 53). In teins are found to carry this domain, such as human dystrophin S. cerevisiae, Cdc23, Cdc16, and Cdc27, which contain TPR and YAP (2). Experiments are under way to clarify whether motifs (14), form a complex (37a). Also, Cdc23, Cdc16, and the WW domains of Rsp5 are required for the physical inter- Cse1 are involved in the degradation of the G2 cyclin at both action with Bul1. Since the C-terminal half from the BamHI the metaphase-to-anaphase transition and the telophase-to-G1 site of Rsp5 (Fig. 1) is sufficient for the interaction with Bul1 in transition (25). It is not known, however, whether those com- the two-hybrid system (unpublished result), one WW domain plexes contain an enzymatic ubiquitin ligase, such as hect pro- could be sufficient for the Bul1 interaction. Otherwise, the hect teins. domain or the other regions of Rsp5 are necessary for its The protein degradation system must be strictly regulated, interaction. since it is an irreversible process. Thus, large complexes of E3 Since Bul1 interacts with a ubiquitin ligase, we considered containing Rsp5, which may usually be silent, must communi- two simple possibilities. (i) Bul1 is a substrate for the Rsp5 cate with various signaling pathways by interacting with various ubiquitin ligase. (ii) Bul1 is a modulator for the Rsp5 ubiquitin associated components and be activated at the proper time to ligase. If it is a substrate, the ubiquitinated Bul1 is likely to be tag the right target proteins, leading to the destruction of those degraded by the proteasome. In this case, Bul1 should be an targets. unstable protein. On the other hand, if it is a regulator of the Minichromosome stability. The bul1 mutant is moderately enzyme, it is not necessarily unstable. The results shown in Fig. defective in maintaining a minichromosome at a high temper- 7 suggested the latter possibility. In this regard, it is interesting ature; thus, the Rsp5-Bul1 complex might be involved in mini- that Bul1 appeared to be modified; the protein band identified chromosome stability, either directly or indirectly. However, as Bul1 was frequently seen as a doublet. Preliminary experi- the rsp5-101 mutant in our laboratory was not defective in ments indicated that the protein was phosphorylated (unpub- minichromosome stability. We might clarify this discrepancy by lished results). As described above, many putative phosphory- isolating other types of rsp5 mutations, if the phenotype of the lation sites which could be involved in receiving putative mutants is allele specific. Our rsp5-101 mutant causes cell lysis signals and/or modulating protein-protein interaction can be at a high temperature, which is a common phenotype of the 3262 YASHIRODA ET AL. MOL.CELL.BIOL.

FIG. 8. Bul1 and Rsp5 cosediment as large complexes in sucrose gradient centrifugation. The cell lysate of the transformant carrying pHY20 (YEp-BUL1-HA) and pHY16 (YEp-myc-RSP5) was prepared, loaded on a 10 to 30% sucrose density gradient, and centrifuged in a Beckman SW41.Ti rotor at 32,000 rpm for 14 h at 5ЊC. Proteins of each fraction were analyzed for immunoblotting. The Coomassie brilliant blue-stained gel (bottom panel) assures the successful density gradient. The position of bacteriophage M13mp19 is indicated (top).

bul1 disruptant, and this phenotype can be suppressed by adding 1 M sorbitol as an osmotic stabilizer in the medium. This phenotype is seen in the mutants of the C-kinase–MAP-kinase cascade in S. cerevisiae, although the rsp5 disruptant cannot survive even in the presence of the osmotic stabilizer, unlike the pkc1 disruptant (38). It is still possible that the defect in the minichromosome stability of the bul1 mutant is independent of RSP5. For example, Bul1 is involved in multiple pathways, one for making a complex with Rsp5 to function in unidentiﬁed processes and another involved in minichromosome stability. Genetic interaction of BUL1 and RSP5 with another gene. We have additional genetic evidence supporting the functional relationship between Bul1 and Rsp5. The temperature sensitivity of another ts (ytg1 [yeast ts growth]) mutant in our laboratory could be suppressed by a single copy of either RSP5 or BUL1 (unpublished results). UBI1 was also a suppressor of ytg1 when the gene dosage was high. Furthermore, we have noticed FIG. 9. Rsp5 coimmunoprecipitates with Bul1. The 40S fraction from the cell very recently the existence of a Bul1 homolog (GenBank ac- lysate containing both Bul1-HA and myc-Rsp5 (lanes 2 and 4) or myc-Rsp5 alone cession no. Z49210) with a 51% identical amino acid sequence. (lanes 1 and 3) was mixed with anti-HA antibody and incubated for 2 h, after Characterization of those genes will deﬁnitely help to solve the which protein A-Sepharose beads were added and the mixture was incubated for another 2 h. The precipitates were then subjected to immunoblotting. The questions of what Bul1 does and what the target proteins of the blotted membrane was treated with anti-HA (lanes 1 and 2) or anti-myc (lanes Rsp5 ubiquitin ligase are. These experiments are under way. 3 and 4) antibody. VOL. 16, 1996 YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE 3263

ACKNOWLEDGMENTS 31. Kikuchi, Y., and J. King. 1975. Genetic control of bacteriophage T4 base- plate morphogenesis. I. Sequential assembly of the major precursor, in vivo We thank D. Botstein, R. Davis, R. Sternglanz, A. Fujita, Y. Ohya, and in vitro. J. Mol. Biol. 99:645–672. A. Varshavsky, K. Tanaka, and H. Hashimoto for providing plasmids 32. Kikuchi, Y., Y. Oka, M. Kobayashi, Y. Uesono, A. Toh-e, and A. Kikuchi. or yeast strains. We are grateful to A. Kikuchi for his critical reading 1994. A new yeast gene, HTR1, required for growth at high temperature, is of the manuscript. needed for recovery from mating pheromone-induced G1 arrest. Mol. Gen. H. Yashiroda is a research fellow of the Japan Society for the Genet. 245:107–116. Promotion of Science. 33. Kikuchi, Y., H. Shimatake, and A. Kikuchi. 1988. A yeast gene required for the G1-to-S transition encodes a protein containing an A-kinase target site REFERENCES and GTPase domain. EMBO J. 7:1175–1182. 1. Bartel, B., I. Wu¨nning, and A. Varshavsky. 1990. The recognition component 34. King, R. W., J.-M. Peters, S. Tugendreich, M. Rolfe, P. Hieter, and M. W. of the N-end rule pathway. EMBO J. 9:3179–3189. Kirschner. 1995. A 20S complex containing CDC27 and CDC16 catalyzes 2. Bork, P., and M. Sudol. 1994. The WW domain: a signaling site in dystro- the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 81:279–288. phin? Trends Biochem. Sci. 19:531–533. 35. Ko¨lling, R., and C. P. Hollenberg. 1994. The ABC-transporter Ste6 accumu- 3. Botstein, D., S. C. Fallo, S. E. Stewart, M. Brennan, S. Scherer, D. T. lates in the plasma membrane in a ubiquitinated form in endocytosis mu- Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeast (SHY); a tants. EMBO J. 13:3261–3271. eukaryotic system of biological containment for recombinant DNA experi- 36. Kumar, S., Y. Tomooka, and M. Noda. 1992. Identification of a set of genes ments. Gene 8:17–24. with developmentally down-regulated expression in the mouse brain. Bio- 4. Broach, J. R., J. N. Strathern, and J. B. Hicks. 1979. Transformation in chem. Biophys. Res. Commun. 185:1155–1161. yeast: development of a hybrid cloning vector and isolation of the CAN1 37. Kuo, C.-L., and J. Campbell. 1983. Cloning of Saccharomyces cerevisiae DNA gene. Gene 8:121–133. replication genes: isolation of the CDC8 gene and two genes that compen- 5. Chien, C.-T., P. L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid sate for the cdc8-1 mutation. Mol. Cell. Biol. 3:1730–1737. system: a method to identify and clone genes for proteins that interact with 37a.Lamb, J. R., W. A. Michaud, R. S. Sikorski, and P. A. Hieter. 1994. Cdc16p, Cdc23p a protein of interest. Proc. Natl. Acad. Sci. USA 88:9578–9582. and Cdc27p form a complex essential for mitosis. EMBO J. 13:4321–4328. 6. Ciechanover, A. 1994. The ubiquitin-proteasome proteolytic pathway. Cell 38. Levin, D. E., and E. B. Heubusch. 1992. Mutants in S. cerevisiae PKC1 gene 79:13–21. display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221–1229. 7. Dohmen, J., K. Madura, B. Bartel, and A. Varshavsky. 1991. The N-end rule 39. McGrath, J. P., S. Jentsch, and A. Varshavsky. 1991. UBA1: an essential is mediated by the UBC2 (RAD6) ubiquitin-conjugating enzyme. Proc. Natl. yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10:227–236. Acad. Sci. USA 88:7351–7355. 40. Peters, J.-M. 1994. Proteasomes: protein degradation machines of the cell. 8. Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rooney, and F. Trends Biochem. Sci. 19:377–382. Winston. 1992. SPT3 interacts with TFIID to allow normal transcription in 41. Rothblatt, J., and R. Schekman. 1989. A hitchhiker’s guide to analysis of the Saccharomyces cerevisiae. Genes Dev. 6:1319–1331. secretory pathway in yeast. Methods Cell Biol. 32:3–36. 9. Fields, S., and O. Song. 1989. A novel genetic system to detect protein- 42. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. protein interactions. Nature (London) 340:245–246. 101:202–211. 10. Fields, S., and R. Sternglanz. 1994. The two-hybrid system: an assay for 43. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a protein-protein interactions. Trends Genet. 10:286–292. laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold 11. Finley, D., and V. Chaw. 1991. Ubiquitination. Annu. Rev. Cell Biol. 7:25–69. Spring Harbor, N.Y. 12. Gietz, R. D., and A. Sugino. 1988. New yeast-Escherichia coli shuttle vectors 44. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with constructed with in vitro mutagenized yeast genes lacking six-base pair re- chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467. striction sites. Gene 74:527–534. 45. Scheffner, M., J. M. Huibregtse, R. D. Vierstra, and P. M. Howley. 1993. The 13. Glotzer, M., A. W. Murray, and M. W. Kirschner. 1991. Cyclin is degraded HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the by the ubiquitin pathway. Nature (London) 349:132–138. ubiquitination of p53. Cell 75:495–505. 14. Goebl, M., and M. Yanagida. 1991. The TPR snap helix: a novel protein 46. Scheffner, M., U. Nuber, and J. M. Huibregtse. 1995. Protein ubiquitination involv- repeat motif from mitosis to transcription. Trends Biochem Sci. 16:173–177. ing an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature (London) 373:81–83. 15. Guarente, L. 1983. Yeast promoters and lacZ fusions designed to study 47. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. expression of cloned genes in yeast. Methods Enzymol. 101:181–191. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus types 16. Happel, A. M., and F. Winston. 1992. A mutant tRNA affects ␦-mediated 16 and 18 promotes the degradation of p53. Cell 63:1129–1136. transcription in Saccharomyces cerevisiae. Genetics 132:361–374. 48. Schwob, E., T. Bo¨hm, M. D. Mendenhall, and K. Nasmyth. 1994. The B-type 17. Hershko, A., and A. Ciechanover. 1992. The ubiquitin system for protein cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevi- degradation. Annu. Rev. Biochem. 61:761–807. siae. Cell 79:233–244. 18. Hershko, A., H. Heller, S. Elias, and A. Ciechanover. 1983. Components of 49. Sherman, F., G. R. Fink, and J. M. Hicks. 1986. Laboratory course manual the ubiquitin-protein ligase system. J. Biol. Chem. 258:8206–8214. for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring 19. Hochstrasser, M. 1995. Ubiquitin, proteasome, and the regulation of intra- Harbor, N.Y. cellular protein degradation. Curr. Opin. Cell Biol. 7:215–223. 50. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast 20. Huibregtse, J. M., M. Scheffner, S. Beaudenon, and P. M. Howley. 1995. A host strains designed for efficient manipulation of DNA in Saccharomyces family of proteins structurally and functionally related to the E6-AP ubiq- cerevisiae. Genetics 122:19–27. uitin-protein ligase. Proc. Natl. Acad. Sci. USA 92:2563–2567. 51. Stinchcomb, D. T., C. Mann, and R. W. Davis. 1982. Centromeric DNA from 21. Huibregtse, J. M., Scheffner, and P. M. Howley. 1991. A cellular protein Saccharomyces cerevisiae. J. Mol. Biol. 158:157–179. mediates association of p53 with the E6 oncoprotein of human papilloma- 52. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979. High- virus types 16 or 18. EMBO J. 10:4129–4135. frequency transformation of yeast: autonomous replication of hybrid DNA 22. Huibregtse, J. M., Scheffner, and P. M. Howley. 1993. Cloning and expres- molecules. Proc. Natl. Acad. Sci. USA 76:1035–1039. sion of the cDNA for E6-AP, a protein that mediates the interaction of the 53. Sudakin, V., D. Ganoth, A. Dahan, H. Heller, J. Hershko, F. C. Luca, J. V. human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13:775–784. Ruderman, and A. Hershko. 1995. The cyclosome, a large complex contain- 23. Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation ing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at of Escherichia coli with plasmids. Gene 96:23–28. the end of mitosis. Mol. Biol. Cell 6:185–198. 24. Irie, K., S. Nomoto, I. Miyajima, and K. Motsumoto. 1991. SGV1 encodes a 54. Sutton, A., D. Immanuel, and K. T. Arndt. 1991. The SIT4 protein phos- CDC28/cdc2-related kinase required for a G␣ subunit-mediated adaptive phatase function in late G1 for progression into S phase. Mol. Cell. Biol. response to pheromone in S. cerevisiae. Cell 65:785–795. 11:2133–2148. 25. Irniger, S., S. Piatti, C. Michaelis, and K. Nasmyth. 1995. Genes involved in 55. Tanaka, K., M. Nakafuku, F. Tamanoi, Y. Kajiro, K. Matsumoto, and A. sister chromatid separation are needed for B-type cyclin proteolysis in bud- Toh-e. 1990. IRA2, a second gene of Saccharomyces cerevisiae that encodes a ding yeast. Cell 81:269–277. protein with a domain homologous to mammalian ras GTPase-activating 26. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of protein. Mol. Cell. Biol. 10:4304–4313. intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168. 56. Uesono, Y., A. Fujita, A. Toh-e, and Y. Kikuchi. 1994. The MCS1/SSD1/ 27. Jentsch, S. 1992. The ubiquitin-conjugation system. Annu. Rev. Genet. 26: SRK1/SSL1 gene is involved in stable maintenance of the chromosome in 179–207. yeast. Gene 143:135–138. 28. Johnson, E. S., P. C. M. Ma, I. M. Ota, and A. Varshavsky. 1995. A proteo- 56a.Utsugi, T., et al. Unpublished data. lytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 57. Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper. 1993. Mammalian ras 270:17442–17456. interacts directly with the serine/threonine kinase Raf. Cell 74:205–214. 29. Jones, J. R., and L. Parkash. 1990. Yeast Saccharomyces cerevisiae selectable 58. Watanabe, Y., K. Irie, and K. Matsumoto. 1995. Yeast RLM1 encodes a markers in pUC18 poly linkers. Yeast 6:363–366. serum response factor-like protein that may function downstream of the 30. Kikuchi, Y. 1983. Yeast plasmid requires a cis-acting locus and two plasmid Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 15: proteins for its stable maintenance. Cell 35:487–493. 5740–5749.