MOLECULAR AND CELLULAR BIOLOGY, July 1996, p. 3255–3263 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 16, No. 7
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 Spt2 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 which is highly conserved in eukaryotes (6, 11, 17, 19, 27). Not only are aberrant proteins recognized and conjugated with ubiquitin molecules, but also many important proteins in various processes of the mitotic cell cycle, signal transduction, or differentiation are known to be substrates of this system, such as cyclins and inhibitors of cyclin-dependent kinases (13, 25, 34, 48, 53). Subsequently, ATP-requiring proteolysis of those multiubiquitinated conjugates by the multicomponent proteasome occurs (40). Since the proteolysis is an irreversible process, the target proteins to be ubiquitinated should be recognized with a high degree of specificity and proper timing; thus, strict and complicated regulatory systems are needed. Recently, large complexes were reported to be required for the ubiquitination of cyclin B in various systems (34, 53). In Saccharomyces cerevisiae, many genes are known to be involved in the ubiquitination system. UBA1 encodes a ubiquitin-activating enzyme (E1) (39). At least 10 UBC genes (E2) encoding ubiquitin-conjugating enzymes have been isolated so far (27). These enzymes form thioester bonds with ubiquitin molecules and transfer them to substrate proteins. In some cases, additional (E3) factors (18) are known to be required; UBR1, which encodes an E3a as a substrate-recognizing protein involved in N-end rule degradation, was isolated (1, 7). Recently, another type of E3 enzyme, namely, a ubiquitin ligase containing a hect (homologous to E6-AP C terminus) domain, was reported (20). Human E6-AP was the first ubiquitin ligase described (45). When human cells are infected with the high-risk papillomavirus, the viral E6 protein binds to a host component, called
E6-AP (21). The E6 and E6-AP binary complex then binds to a tumor suppressor protein, p53, which is ubiquitinated by E6-AP, and the tagged p53 is degraded by the 26S proteasome, leading to tumorigenesis (47). With an in vitro system, Scheffner et al. established that ubiquitins, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), viral E6 protein, E6-AP (E3), and ATP are required for the ubiquitination of p53 (45). The cysteine residue near the C terminus of E6-AP is essential for the formation of a thioester bond with a ubiquitin molecule (46). Ubiquitin molecules are transferred through an E1-E2-E3 enzyme thioester cascade to their target proteins (46). In this system, viral E6 protein is a connector molecule between the enzyme and its substrate; it can recognize the target protein and activate the ubiquitin ligase. Many proteins have been found to contain a 30-kDa hect domain at their C termini (20). In S. cerevisiae, at least three proteins carry this hect domain. One of them, Rsp5, was suggested to be a ubiquitin ligase like E6-AP (20). rsp5 was isolated as a revertant of spt3 encoding a TFIID-binding protein (8, 16). UFD4 (identical to YKL162) was found to be involved in the UFD (Ub fusion degradation) pathway (28). We are also characterizing another hect gene, TOM1, which is required for the G2-M transition at a high temperature (56a). We have obtained temperature-sensitive (ts) mutants of S. cerevisiae, as isolated previously (33), and one of them was defective in the maintenance of minichromosomes at a high temperature. We cloned a new gene, BUL1, and RSP5, whose product interacted physically with this new gene product. In this report, we describe these new components involved in the ubiquitination pathway of S. cerevisiae.
* Corresponding author. Mailing address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3812-2111, ext. 4466. Fax: 81-3-5684-9420. Electronic mail address:
[email protected] -tokyo.ac.jp.
Strains and genetic manipulations. Escherichia coli K-12 strain DH5a [supE44 DlacU169 (f80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used for propagating plasmids. The following strains of S. cerevisiae were used: YPH499 (MATa ade2 his3 leu2 trp1 ura3 lys2) (50); YPH4992, a homozygous diploid strain of YPH499; KA31 (MATa his3 leu2 ura3 trp1) (24); RAY-3A (MATa leu2 ura3
MATERIALS AND METHODS
3255
3256
YASHIRODA ET AL.
FIG. 1. Restriction maps of BUL1 and RSP5 and structures of various plasmids. (A) Map of BUL1 and its neighboring regions, constructed from two plasmids, pHY01 and pHY06. Only inserts of various plasmids are given (lines). YEp, multicopy plasmid; YCp, single-copy plasmid; and Ylp, integration plasmid. The complementation activity of each plasmid is indicated on the right. Plasmid pHY05 was used to disrupt BUL1 (see text), and pHY07, which contains a gene encoding Bul1 fused to the LexA DNA binding domain, was used for screening plasmids by the two-hybrid system. (B) Map of RSP5 and structure of the plasmids. Plasmid pHY09 was isolated by the two-hybrid system; the Cterminal part of RSP5 was fused in frame to the Gal4 activation domain on plasmid pGAD. Plasmid pTOP232 was used for disrupting RSP5. Restriction sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; Pv, PvuII; RV, EcoRV; Sc, SacI; Sl, SalI; and X, XhoI.
trp1 his3) (56); YHY001 (MATa bul1-1 ura3 trp1 lys2 his3) (33); YHY002, an integrant of YHY001 with the plasmid pHY03; YHY003 (bul1::TRP1 in the YPH499 background); YAT2-1C (MATa his3 leu2 trp1 ura3 rsp5-101) (this laboratory); and L40 (MATa his3 leu2 trp1 URA3::lexA-lacZ LYS2::lexA-HIS3) (57). The media and methods for mating, sporulation, and tetrad analysis were described previously (49). Plasmids and transformation. The following multicopy plasmid vectors were used in this study: YEp24 (URA3) (3), YEUp3 (URA3) (A. Fujita, National Institute of Bioscience and Human Technology), pW1 (TRP1) (H. Hashimoto, Nikka Whisky Co.), YEp13 (LEU2) (4), YEplac112 (TRP1) (12), and YEplac181 (LEU2) (12). Single-copy vectors YCUp4 (CEN4 ARS1 URA3) (A. Fujita), YCp50 (CEN4 ARS1 URA3) (37), and pRS316 (CEN6 ARSH4 URA3) (50) were used. The YEp24 library and YCp50 bank were kindly provided by D. Botstein and R. Davis of Stanford University, respectively. The YEp13 library was generously given by Y. Ohya of The University of Tokyo. The YCUp4 bank was kindly provided by A. Fujita. DNA manipulations were performed as described elsewhere (43). pHY01 is YEp24 containing a defective BUL1 gene (Fig. 1A). pHY02 is a plasmid with a deletion of the 3.5-kb SacI fragment from pHY01. pHY03 was constructed by deleting the 2mm DNA from pHY02. pHY100 is pRS316 carrying the 3-kb fragment from the junction of the insert (the SmaI site of the vector) to the SacI site of pHY01. For pHY05, the 3-kb BamHI-SacI fragment of BUL1 was inserted into pTZ18R (Pharmacia) and the 1.1-kb XhoIKpnI fragment inside the BUL1 gene was replaced with the 1-kb BamHI-SalI fragment of the TRP1 marker from pJJ280 (29) by ligation after treatment with T4 DNA polymerase (Toyobo Co.). pHY06 is pRS316 containing the 4-kb EcoRI-SacI fragment carrying the entire BUL1 gene. pHY66 is YEUp3 containing the same fragment as pHY06. For the two-hybrid system, we used pBTM116 (lexA DNA-binding domain, TRP1), kindly provided by R. Sternglanz of New York State University, and GAD424 (Gal4-activating domain, LEU2) (Clontech) vectors. The gene libraries were constructed and generously given by R. Sternglanz; Sau3A partial digests of genomic DNA were ligated into the BamHI site of either pGAD1, pGAD2, or pGAD3 (5). For pHY07, the 4.5-kb BamHIBglII fragment of BUL1 was inserted into the BamHI site of pBTM116, so that the BUL1 sequence from the BamHI site to the C terminus was fused in frame to the lexA DNA-binding domain. pHY08 is YCUp4-RSP5. pYH09 is pGADRsp5, isolated from the pGAD bank (Fig. 1B). pHY14 is pRS316 containing the BUL1 fused gene with a double hemagglutinin (HA) epitope tag (amino acid sequence MYPYDVPDYA) at the C terminus of Bul1. pHY15 is YEUp3-BUL1HA. pTOP223 is YEp13-RSP5, isolated from the bank. For pTOP232, the 1.1-kb HindIII fragment carrying URA3 was inserted into the PstI site inside RSP5 on the Bluescript KS1 vector containing LEU2 (Fig. 1B). pMS16 is YEp24-UBI1
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 replacement 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). DNA sequencing. Nucleotide sequences were determined by the dideoxy chain-termination method (44), using double-stranded DNAs as templates and a Dye Deoxy Cycle sequencing kit (Applied Biosystems). After PCRs, the samples were analyzed by using 6% polyacrylamide gels and an ABI model 370 sequencer. DNA isolation and Southern hybridization. Yeast DNA was isolated (43), digested with appropriate restriction enzymes, and run on 1% agarose gels. During the treatment of the gel with 0.4 N NaOH for 4 h, the denatured DNA was blotted onto Hybond N1 membrane filters (Amersham). Southern hybridization was performed by using the enhanced chemiluminescence direct nucleic acid labeling system, according to the procedure recommended by the manufacturer (Amersham). For the evaluation of the BUL1 gene disruption, the 3-kb 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 Collection, 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 fragment of pTOP223 was used as a probe. Cell lysis assay. Yeast cells were streaked as patches on yeast-peptone-dextrose (YPD) plates, which were incubated at 378C overnight. Then the patches were covered with a mixture of 2 ml of 1% melted agar, 2.5 ml of 100 mM glycine-HCl (pH 9.5), and 434 ml of BCIP (5-bromo-4-chloro-3-indolylphosphate toluidinium) (Promega). After incubation at 378C for 1 h, the patches of lysed cells should turn blue. Two-hybrid system. The transformant of strain L40 with plasmid pYH07 was introduced further with the pGAD bank. Trp1 Leu1 transformants were checked for the His1 phenotype in the presence of 10 mM 3AT (3-amino-1,2,4triazole) at 268C. b-Galactosidase activity was also tested. Assay for b-galactosidase. Cells were grown to mid-log phase in minimal medium and collected by centrifugation. b-Galactosidase activity was measured as described by Guarente (15). Preparation of cell lysates. Transformants were grown in 5 ml of selective media to an optical density at 600 nm (OD600) of 1.0 and collected by centrifugation. The pellets were suspended in 50 ml of lysis buffer (100 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol) (54) containing 1 mM phenylmethylsulfonyl fluoride, 1 mg each of leupeptin, antipain, and pepstatin per ml and 2.1 mg of aprotinin per ml and broken by being vortexed with glass beads, and the lysates were prepared by centrifugation at 12,000 3 g for 5 min at 58C (56). Immunoblot analysis. The protein concentrations of cell lysates were determined by using the Bio-Rad protein assay. Samples (50 mg) were mixed with equal amounts of sample buffer, incubated at 958C for 5 min, and electrophoresed through 7.5% polyacrylamide–sodium dodecyl sulfate (SDS) gels (31). The gels were stained with Coomassie brilliant blue in 5% methanol–7.5% acetic acid. For immunoblotting, the proteins were transferred to Millipore membrane filters. The filters were incubated with 5% skim milk in TBST buffer (10 mM Tris-HCl [pH 8.0], 0.15 M NaCl, 0.05% Tween 20) for blocking overnight at 48C, washed with 10 ml of TBST buffer three times for 5 min each, and incubated with 10 ml of 2-mg/ml anti-HA (12CA5) (Boehringer Mannheim) or 1-mg/ml anti-myc (9E10) monoclonal antibody in TBST buffer for 2 h at room temperature. Then the membranes were washed three times with 10 ml of TBST buffer for 5 min each and incubated with 10 ml of TBST buffer containing 1.5 ml of secondary anti-mouse immunoglobulin G alkaline phosphatase (Tago Co.) or anti-mouse immunoglobulin G horseradish peroxidase (Dupont) for 1 h at room temperature. After three washes with 10 ml of TBST buffer for 5 min each, the detection reagent was spread on the filters, as instructed by each manufacturer. Pulse-chase experiments. The protocol for the pulse-chase experiments was essentially as described previously (35, 54). Cells of strain KA31 (RSP5) or
VOL. 16, 1996
YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE
YAT2-1C (rsp5) containing pHY15 were grown overnight in minimal medium (MV medium) in which all sulfate salts were replaced by chloride salts supplemented with 100 mM (NH4)2SO4 (41). Five OD600 units (3 3 108 cells) of exponentially growing cells were harvested and resuspended in fresh MV medium to a density of 2 OD600 units. The cells were labeled for 20 min after addition of 15 mCi of Tran[35S] (ICN) per OD600 unit of cells. Then a concentrated chase solution [100 mM (NH4)2SO4, 0.3% cysteine, 0.4% methionine] was diluted 100-fold into the cultures for further incubation at 378C. Aliquots of 1 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 chilling on ice. The cells were washed once and resuspended in 50 ml of lysis buffer. Lysis was achieved by vortexing the suspension with glass beads, which were washed with 100 ml of lysis buffer. For immunoprecipitation, aggregates were removed by centrifugation at 12,000 3 g for 15 min and 100 ml of the supernatant was incubated with 1.5 mg of anti-HA antibody. After 3 h at 08C, 50 ml of a suspension of protein A-Sepharose beads (Pharmacia) (100 mg/ml in radioimmunoprecipitation assay [RIPA] buffer, consisting of 50 mM Tris-HCl [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 incubated with gentle rocking for 2.5 h at 08C. The beads were washed four times with 75% lysis buffer–25% RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride and then once with 50 mM Tris-HCl (pH 7.5)–50 mM NaCl. The beads were resuspended in 50 ml of 23 sample buffer, and the mixture was heated for 5 min at 958C. The supernatant was subjected to electrophoresis through 7.5% polyacrylamide–SDS gels. The gels were fixed in 7.5% acetic acid and 5% methanol, dried, and exposed to Imaging Plate (Fuji Film) for 2 days. The bands were visualized by radioluminography with a BAS-Mac system (Fuji Film). Sucrose gradient centrifugation. A 150-ml 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% sucrose cushion in lysis buffer or phosphate-buffered saline (PBS) buffer (2.2 mM NaH2PO4 z 2H2O, 8.4 mM Na2HPO4 z 12H2O, 150 mM NaCl [pH 7.0]) and centrifuged in a Beckman SW41.Ti rotor at 32,000 rpm for 14 h at 58C. Fractions were collected from the top. Immunoprecipitation. A 300-ml sample of the 40 or 22S sucrose gradient fractions in PBS buffer was mixed with 10 mg of anti-HA antibodies, and the mixture was incubated at 48C for 2 h. Then 50 ml of protein A-Sepharose beads (Pharmacia) was added, and the mixture was incubated at 48C for 2 h and washed 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 pellets and the samples were boiled for 5 min and subjected to gel electrophoresis. Nucleotide sequence accession number. The nucleotide sequence of BUL1 has been submitted to GenBank under accession no. D50083.
RESULTS A ts mutant defective in the maintenance of minichromosomes. From our ts mutants isolated previously (33), we selected one (YHY001) in which a YCp50 minichromosome (37) containing a replication origin (ARS1) (52) and a centromere (CEN4) (51) was maintained less stably at 358C (a semipermissive temperature) than at 268C (a permissive temperature). The mutant carrying YCp50 was grown in a selective medium at 268C, diluted in a nonselective medium (YPD), and grown at either 35 or 268C for seven generations. Then cells were spread on plates for single colonies, and each colony was checked for uracil prototrophy, as described previously (30). Only 23% of the cells maintained YCp50 at 358C, whereas 56% of the cells contained it at 268C. In contrast, the wild-type strain YPH499 (50) could maintain YCp50 equally well at either temperature (63% at 268C and 57% at 358C). Thus, the mutant appeared to be moderately defective in the stable maintenance of the minichromosome at the higher temperature. When this ts mutant was crossed with the wild-type YPH499, the ts phenotype was segregated at 21:22, indicating that a single mutation should cause the ts growth. The morphology of the arrested cells at 378C was not cell cycle specific, but large cells with an elongated bud were frequently observed at 6 h after the temperature shift (data not shown). Fluorescence-activated cell sorter analysis did not show any cell cycle-specific arrest (data not shown). Isolation of a new gene, BUL1. By introducing the YEp24 library (3) into strain YHY001 (bul1-1), we isolated a plasmid (pHY01) by the ability to complement the ts phenotype. As shown in Fig. 1A, subcloning experiments could locate the responsible region within the 3.0-kb DNA fragment from one
3257
junction of the vector to the SacI site in the insert (pHY02). A YIp plasmid (pHY03) carrying this region was digested with restriction enzyme BamHI and integrated into the YHY001 genome by homologous recombination. The integrants were able to grow at 378C, and one of them (YHY002) was crossed with the wild-type YPH499 and the resulting diploid was sporulated. Almost all the spores (50 of 52) were able to germinate and grow at 378C, indicating that the integrated gene was tightly linked to the ts mutation. However, when the DNA was cloned into a single-copy vector (pHY100), it could not complement the ts mutation. The 59 upstream and Nterminal regions of the gene were missing in this original isolate. The neighboring region was cloned from the integrant (YHY002) by the rolling method as described in Materials and Methods. From a recovered plasmid, we constructed a singlecopy plasmid (pHY06) containing the 4-kb EcoRI-SacI fragment, which had the complementing activity, as shown below (see Fig. 3A). As described below, we named the gene BUL1 (for a protein that binds to the ubiquitin ligase). The BUL1 gene was physically mapped near SUP8 on chromosome XIII R, since the 3-kb BamHI-SacI fragment was hybridized to the lambda phage clones 70071 and 70218 on an American Type Culture Collection membrane filter by Southern hybridization (data not shown). The nucleotide sequence and its deduced amino acid sequence are shown in Fig. 2A. The predicted gene product appears to be a basic (pI 5 8.8) and hydrophilic 110-kDa protein, and its C terminus is very similar to the protein sequence predicted by a human cDNA which is expressed in T cells (GenBank database accession no. Z16200) (Fig. 2B). Bul1 has many putative phosphorylation sites, such as those of mitogen-activated protein (MAP) kinase (amino acid residues 59 to 62), CDC28 kinase (residues 101 to 104), and A kinase (residues 104 to 107), C kinase, and casein kinase II. Two serine-rich stretches are also found near the C-terminal region (amino acids 619 to 640 and 868 to 898). The bul1::TRP1 disruptant (YHY003) in the YPH499 strain background showed ts growth like the original ts mutant, and its temperature sensitivity was complemented by a single copy of BUL1 (pHY06), as shown in Fig. 3A. The disruptant was also moderately defective in the stability of the minichromosome at a semipermissive temperature (33% at 358C compared with 62% at 268C). Furthermore, the disruptant cells were partially lysed on YPD plates at 378C, since the cell patches turned blue when assayed with BCIP (data not shown). The temperature sensitivity of the bul1 disruptant as well as that of the original ts mutant was suppressed by adding 1 M sorbitol as an osmotic stabilizer in the medium (Fig. 3B). SSD1/MCS1 is a single-copy suppressor of bul1. We isolated SSD1/MCS1 (54, 56) on a single-copy vector, which was able to suppress the temperature sensitivity of the bul1 disruptant even at 378C (Fig. 3A). Although the function of SSD1/MCS1 is still obscure at present, it may encode an exoribonuclease II, since the amino acid sequence is similar to that of the Shigella flexneri VacB protein (Swiss-Prot database accession no. P30851). Previous work indicated that the intact Ssd1/Mcs1 protein was missing in strain YPH499 (56), and some ts mutations, for example, htr1, are suppressed by introducing a single copy of SSD1/MCS1 (32). The bul1 disruptant in other strain backgrounds, such as strain RAY-3A carrying intact SSD1/MCS1 (56), was not ts (data not shown). Thus, BUL1 and SSD1/MCS1 might function redundantly in some step for mitotic growth, at least at a high temperature. Bul1 physically interacts with Rsp5. In order to search for proteins which interact with Bul1, we took advantage of the 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
FIG. 3. Complementation and suppression of temperature sensitivity of the bul1 mutants. (A) Strain YHY003 (bul1::TRP1) was transformed with YCUp4 (a), YCp-BUL1 (b), and YCp-SSD1/MCS1 (c) at 268C. Strain YHY001 (bul1-1) was transformed with YCp-SSD1/MCS1 (d), YCp-BUL1 (e), and YCUp4 (f) at 268C. Each Ura1 transformant on a YPD plate was incubated at 378C for 2 days. (B) The same transformants were streaked on a YPD plate containing 1 M sorbitol and incubated at 378C for 2 days.
pHY07, encoding a LexA-Bul1 fusion protein (Fig. 1A), screened the pGAD library, and isolated pHY09. Cotransformation of pHY07 with pHY09 caused a His1 phenotype and a high b-galactosidase activity (164.3 U), whereas the b-galactosidase activity of the transformant of pHY07 plus GAD424 vector or pHY09 plus lexA vector was 2.1 or 1.3 U, respectively. DNA sequencing revealed that Gal4 was fused in frame to the C-terminal region (517 amino acids) of Rsp5 (Fig. 1B). RSP5 stands for “reverses Spt2 phenotype” (16). The C-terminal half of the gene product (380 amino acids) is homologous (34.5% identical) to the C-terminal region of the human E6-AP ubiquitin ligase (22). The N-terminal half of Rsp5 contains the C2 region, found in some protein kinases C (38), and three WW domains which may be involved in protein-protein interactions
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 Ura2, 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 plates at 378C, when assayed by using BCIP (data not shown). As shown in Fig. 4B, this temperature sensitivity was partially suppressed by adding 10% sorbitol to the medium, although the rsp5 disruptant spore was not able to recover on 10% sorbitol medium (data not shown). Other than the ts phenotype of this rsp5 mutant, it was sensitive to 1 mg of canavanine per ml in minimal medium at 268C (Fig. 4C), suggesting that the mutant is defective in ubiquitination and/or degradation of aberrant proteins. Genetic interactions between RSP5 and BUL1. Multiple copies of BUL1 (pHY66) were not able to suppress the rsp5-101 mutation, and a high dose of RSP5 (pTOP223) could not suppress the ts phenotype of the bul1 disruptant. The rsp5 bul1::TRP1 double mutant in the KA31 background did not show any additive phenotype, possibly because this strain contains intact SSD1/MCS1. On the other hand, a high dose of UBI1 encoding a ubiquitin molecule (pMS16) partially suppressed the rsp5 mutation (Fig. 5B) and also the temperature sensitivity of the bul1 disruptant (Fig. 5A). Furthermore, simultaneous overexpression of both genes on a multicopy plasmid (pHY19) was toxic to the wild-type cell growth, while the 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 258C 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 36.58C 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 mg of canavanine per ml, which was incubated at 258C for 2 days.
3260
YASHIRODA ET AL.
FIG. 5. The ubiquitin-encoding gene (UBI1) is a multicopy suppressor of the temperature sensitivity of the bul1 and rsp5 mutants. (A) Strain YHY003 (bul1::TRP1) was transformed with plasmid YEUp3 (a), pMS16 (YEp24-UBI1) (b), or pYH06 (YCp-BUL1) (c), and each transformant was streaked on a YPD plate which was incubated at 36.58C for 3 days. (B) Strain YAT2-1C (rsp5-101) was transformed with plasmid YEUp3 (a), pMS16 (YEp24-UBI1) (b), or pHY08 (YCUp4-RSP5) (c). Each transformant was streaked on a YPD plate which was incubated at 35.78C for 2 days.
pHY18) did not interfere with wild-type cell growth (Fig. 6). These results support the idea that BUL1 is involved in the ubiquitination pathway by collaborating with RSP5 by an unknown mechanism. Since we do not know at present what proteins are directly ubiquitinated in vivo by this Rsp5-Bul1 system, we tested the protein stability of artificially designed substrates for the N-end rule pathway (1, 7). We tested five types of Ub-X-LacZ constructs (with Leu, Arg, Ala, Met, and Pro as amino acid X) and found that the b-galactosidase activity in the bul1 disruptant (YHY003) in each case was similar to that in the parental wild-type strain (YPH499), although the activity of Leu-LacZ in the bul1 disruptant was a few times higher than that in the wild-type strain. Thus, BUL1 is not directly involved in the N-end rule pathway (1, 7). Bul1 is a stable protein. If Bul1 is a substrate of Rsp5 ubiquitin ligase, it might be an unstable protein, since ubiquitinated proteins are most likely to be degraded by the proteasome. In order to determine the stability of Bul1, we constructed a functional BUL1 gene tagged with HA. When BUL1-HA was introduced into the bul1 disruptant on a singlecopy plasmid (pHY14) and the cell lysate of the transformant
FIG. 6. Simultaneous overexpression of RSP5 and BUL1 inhibits wild-type cell growth. Multicopy plasmid pHY18 (pW1-BUL1), pHY17 (YEplac112RSP5), or pHY19 (YEplac112-RSP5,-BUL1) was introduced into wild-type strain KA31 at 258C, and each transformant was streaked on a minimal medium plate which was incubated at 348C for 2 days.
MOL. CELL. BIOL.
was prepared for immunoblotting analysis, a very faint 140kDa 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 268C and chased for various times at 378C. 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 Bul1 in the wild-type cells after 2 h was almost unchanged, indicating that Bul1 is a stable protein. In contrast, the bands of Bul1 in the rsp5 mutant were faint, even from the sample of 0 min. When Bul1 was detected directly from crude lysates by immunoblotting, the amount of Bul1 in the mutant was similar 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 that Bul1 physically interacts with Rsp5. Thus, it seems unlikely that Bul1 is a target protein which is ubiquitinated by the Rsp5 ubiquitin ligase for its degradation. Bul1 and Rsp5 appeared to make large complexes. In order to prove that Bul1 and Rsp5 physically interact in vivo, we attempted to detect a complex of Bul1 and Rsp5 by centrifuging the cell lysates through a sucrose density gradient. Bul1-HA was expressed from its own promoter on a multicopy vector (pHY20), and Rsp5 was tagged with myc at the N terminus and expressed under the control of the GAPDH promoter (pHY16). The cell lysate of the bul1 disruptant carrying both plasmids was prepared and centrifuged through a sucrose density gradient. Proteins of each fraction were separated through a gel electrophoresis and subjected to immunoblotting, as shown in Fig. 8. Bul1 sedimented with three different sizes (around 40, 22, and 7S), and Rsp5 sedimented similarly but more broadly, possibly because of its overexpression. A bulk of proteins in the same gradient sedimented in narrower ranges, judging from the pattern of the Coomassie-stained gel. A higher salt concentration (150 mM NaCl) in the gradient decreased the fast-sedimenting fractions of Bul1 or Rsp5 (data 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 3) when the corresponding gradient fractions from the cell lysates without Bul1-HA were used, as mentioned above. A small amount of myc-Rsp5 was also immunoprecipitated with 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 268C and chased for various times at 378C. 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 268C and divided into two samples for another 2-h incubation at 26 or 378C. 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 large complexes with Bul1. DISCUSSION Bul1 and Rsp5 ubiquitin ligase are associated with large complexes. By the two-hybrid system and cosedimentation and coimmunoprecipitation experiments, we have shown that Bul1 physically interacts with the Rsp5 ubiquitin ligase. As described above, the C terminus of the Rsp5 is homologous to human E6-AP ubiquitin ligase (22), and the N-terminal half contains three repeats of the WW domain (2). The WW domain, containing characteristic tryptophan residues, is thought to be involved in protein-protein interaction, and many proteins are found to carry this domain, such as human dystrophin and YAP (2). Experiments are under way to clarify whether the WW domains of Rsp5 are required for the physical interaction with Bul1. Since the C-terminal half from the BamHI site of Rsp5 (Fig. 1) is sufficient for the interaction with Bul1 in the two-hybrid system (unpublished result), one WW domain could be sufficient for the Bul1 interaction. Otherwise, the hect domain or the other regions of Rsp5 are necessary for its interaction. Since Bul1 interacts with a ubiquitin ligase, we considered two simple possibilities. (i) Bul1 is a substrate for the Rsp5 ubiquitin ligase. (ii) Bul1 is a modulator for the Rsp5 ubiquitin ligase. If it is a substrate, the ubiquitinated Bul1 is likely to be degraded by the proteasome. In this case, Bul1 should be an unstable protein. On the other hand, if it is a regulator of the enzyme, it is not necessarily unstable. The results shown in Fig. 7 suggested the latter possibility. In this regard, it is interesting that Bul1 appeared to be modified; the protein band identified as Bul1 was frequently seen as a doublet. Preliminary experiments indicated that the protein was phosphorylated (unpublished results). As described above, many putative phosphorylation sites which could be involved in receiving putative signals and/or modulating protein-protein interaction can be
found in the amino acid sequence of Bul1 (Fig. 2). In the case 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 its substrate, so that the substrate recognition and activity of E6-AP are regulated by the presence of another factor(s). Thus, one attractive possibility is that the Rsp5 ubiquitin ligase and Bul1 make large complexes which may be acting as an E3 protein in the ubiquitination pathway. In fact, recent findings in various systems for cyclin ubiquitination and degradation indicated that the E3 components in the ubiquitination system form large complexes. For example, a cyclosome from a clam extract and the 20S anaphase-promoting complex containing Cdc27 and Cdc16 from Xenopus oocytes are required as E3 components for cyclin ubiquitination (34, 53). In S. cerevisiae, Cdc23, Cdc16, and Cdc27, which contain TPR motifs (14), form a complex (37a). Also, Cdc23, Cdc16, and Cse1 are involved in the degradation of the G2 cyclin at both the metaphase-to-anaphase transition and the telophase-to-G1 transition (25). It is not known, however, whether those complexes contain an enzymatic ubiquitin ligase, such as hect proteins. The protein degradation system must be strictly regulated, since it is an irreversible process. Thus, large complexes of E3 containing Rsp5, which may usually be silent, must communicate with various signaling pathways by interacting with various associated components and be activated at the proper time to tag the right target proteins, leading to the destruction of those targets. Minichromosome stability. The bul1 mutant is moderately defective in maintaining a minichromosome at a high temperature; thus, the Rsp5-Bul1 complex might be involved in minichromosome stability, either directly or indirectly. However, the rsp5-101 mutant in our laboratory was not defective in minichromosome stability. We might clarify this discrepancy by isolating other types of rsp5 mutations, if the phenotype of the mutants is allele specific. Our rsp5-101 mutant causes cell lysis 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 58C. 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 very recently the existence of a Bul1 homolog (GenBank accession no. Z49210) with a 51% identical amino acid sequence. Characterization of those genes will definitely help to solve the questions of what Bul1 does and what the target proteins of the Rsp5 ubiquitin ligase are. These experiments are under way.
FIG. 9. Rsp5 coimmunoprecipitates with Bul1. The 40S fraction from the cell lysate containing both Bul1-HA and myc-Rsp5 (lanes 2 and 4) or myc-Rsp5 alone (lanes 1 and 3) was mixed with anti-HA antibody and incubated for 2 h, after which protein A-Sepharose beads were added and the mixture was incubated for another 2 h. The precipitates were then subjected to immunoblotting. The blotted membrane was treated with anti-HA (lanes 1 and 2) or anti-myc (lanes 3 and 4) antibody.
VOL. 16, 1996
YEAST Bul1, A PROTEIN BINDING TO UBIQUITIN LIGASE ACKNOWLEDGMENTS
We thank D. Botstein, R. Davis, R. Sternglanz, A. Fujita, Y. Ohya, A. Varshavsky, K. Tanaka, and H. Hashimoto for providing plasmids or yeast strains. We are grateful to A. Kikuchi for his critical reading of the manuscript. H. Yashiroda is a research fellow of the Japan Society for the Promotion of Science. REFERENCES 1. Bartel, B., I. Wu ¨nning, and A. Varshavsky. 1990. The recognition component of the N-end rule pathway. EMBO J. 9:3179–3189. 2. Bork, P., and M. Sudol. 1994. The WW domain: a signaling site in dystrophin? Trends Biochem. Sci. 19:531–533. 3. Botstein, D., S. C. Fallo, S. E. Stewart, M. Brennan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeast (SHY); a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24. 4. Broach, J. R., J. N. Strathern, and J. B. Hicks. 1979. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133. 5. Chien, C.-T., P. L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88:9578–9582. 6. Ciechanover, A. 1994. The ubiquitin-proteasome proteolytic pathway. Cell 79:13–21. 7. Dohmen, J., K. Madura, B. Bartel, and A. Varshavsky. 1991. The N-end rule is mediated by the UBC2 (RAD6) ubiquitin-conjugating enzyme. Proc. Natl. Acad. Sci. USA 88:7351–7355. 8. Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rooney, and F. Winston. 1992. SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6:1319–1331. 9. Fields, S., and O. Song. 1989. A novel genetic system to detect proteinprotein interactions. Nature (London) 340:245–246. 10. Fields, S., and R. Sternglanz. 1994. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10:286–292. 11. Finley, D., and V. Chaw. 1991. Ubiquitination. Annu. Rev. Cell Biol. 7:25–69. 12. Gietz, R. D., and A. Sugino. 1988. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534. 13. Glotzer, M., A. W. Murray, and M. W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature (London) 349:132–138. 14. Goebl, M., and M. Yanagida. 1991. The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci. 16:173–177. 15. Guarente, L. 1983. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 101:181–191. 16. Happel, A. M., and F. Winston. 1992. A mutant tRNA affects d-mediated transcription in Saccharomyces cerevisiae. Genetics 132:361–374. 17. Hershko, A., and A. Ciechanover. 1992. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61:761–807. 18. Hershko, A., H. Heller, S. Elias, and A. Ciechanover. 1983. Components of the ubiquitin-protein ligase system. J. Biol. Chem. 258:8206–8214. 19. Hochstrasser, M. 1995. Ubiquitin, proteasome, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol. 7:215–223. 20. Huibregtse, J. M., M. Scheffner, S. Beaudenon, and P. M. Howley. 1995. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92:2563–2567. 21. Huibregtse, J. M., Scheffner, and P. M. Howley. 1991. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 10:4129–4135. 22. Huibregtse, J. M., Scheffner, and P. M. Howley. 1993. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13:775–784. 23. Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28. 24. Irie, K., S. Nomoto, I. Miyajima, and K. Motsumoto. 1991. SGV1 encodes a CDC28/cdc2-related kinase required for a Ga subunit-mediated adaptive response to pheromone in S. cerevisiae. Cell 65:785–795. 25. Irniger, S., S. Piatti, C. Michaelis, and K. Nasmyth. 1995. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell 81:269–277. 26. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168. 27. Jentsch, S. 1992. The ubiquitin-conjugation system. Annu. Rev. Genet. 26: 179–207. 28. Johnson, E. S., P. C. M. Ma, I. M. Ota, and A. Varshavsky. 1995. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270:17442–17456. 29. Jones, J. R., and L. Parkash. 1990. Yeast Saccharomyces cerevisiae selectable markers in pUC18 poly linkers. Yeast 6:363–366. 30. Kikuchi, Y. 1983. Yeast plasmid requires a cis-acting locus and two plasmid proteins for its stable maintenance. Cell 35:487–493.
3263
31. Kikuchi, Y., and J. King. 1975. Genetic control of bacteriophage T4 baseplate morphogenesis. I. Sequential assembly of the major precursor, in vivo and in vitro. J. Mol. Biol. 99:645–672. 32. Kikuchi, Y., Y. Oka, M. Kobayashi, Y. Uesono, A. Toh-e, and A. Kikuchi. 1994. A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest. Mol. Gen. Genet. 245:107–116. 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 and GTPase domain. EMBO J. 7:1175–1182. 34. King, R. W., J.-M. Peters, S. Tugendreich, M. Rolfe, P. Hieter, and M. W. Kirschner. 1995. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 81:279–288. 35. Ko ¨lling, R., and C. P. Hollenberg. 1994. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 13:3261–3271. 36. Kumar, S., Y. Tomooka, and M. Noda. 1992. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185:1155–1161. 37. Kuo, C.-L., and J. Campbell. 1983. Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol. Cell. Biol. 3:1730–1737. 37a.Lamb, J. R., W. A. Michaud, R. S. Sikorski, and P. A. Hieter. 1994. Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J. 13:4321–4328. 38. Levin, D. E., and E. B. Heubusch. 1992. Mutants in S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221–1229. 39. McGrath, J. P., S. Jentsch, and A. Varshavsky. 1991. UBA1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10:227–236. 40. Peters, J.-M. 1994. Proteasomes: protein degradation machines of the cell. Trends Biochem. Sci. 19:377–382. 41. Rothblatt, J., and R. Schekman. 1989. A hitchhiker’s guide to analysis of the secretory pathway in yeast. Methods Cell Biol. 32:3–36. 42. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202–211. 43. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 44. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467. 45. Scheffner, M., J. M. Huibregtse, R. D. Vierstra, and P. M. Howley. 1993. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:495–505. 46. Scheffner, M., U. Nuber, and J. M. Huibregtse. 1995. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature (London) 373:81–83. 47. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136. 48. Schwob, E., T. Bo ¨hm, M. D. Mendenhall, and K. Nasmyth. 1994. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell 79:233–244. 49. Sherman, F., G. R. Fink, and J. M. Hicks. 1986. Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 50. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27. 51. Stinchcomb, D. T., C. Mann, and R. W. Davis. 1982. Centromeric DNA from Saccharomyces cerevisiae. J. Mol. Biol. 158:157–179. 52. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979. Highfrequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. USA 76:1035–1039. 53. Sudakin, V., D. Ganoth, A. Dahan, H. Heller, J. Hershko, F. C. Luca, J. V. Ruderman, and A. Hershko. 1995. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol. Biol. Cell 6:185–198. 54. Sutton, A., D. Immanuel, and K. T. Arndt. 1991. The SIT4 protein phosphatase function in late G1 for progression into S phase. Mol. Cell. Biol. 11:2133–2148. 55. Tanaka, K., M. Nakafuku, F. Tamanoi, Y. Kajiro, K. Matsumoto, and A. Toh-e. 1990. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol. Cell. Biol. 10:4304–4313. 56. Uesono, Y., A. Fujita, A. Toh-e, and Y. Kikuchi. 1994. The MCS1/SSD1/ SRK1/SSL1 gene is involved in stable maintenance of the chromosome in yeast. Gene 143:135–138. 56a.Utsugi, T., et al. Unpublished data. 57. Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper. 1993. Mammalian ras interacts directly with the serine/threonine kinase Raf. Cell 74:205–214. 58. Watanabe, Y., K. Irie, and K. Matsumoto. 1995. Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 15: 5740–5749.
