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

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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/$04.0010 Copyright q 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 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 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 E3a 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 DH5a [supE44 Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, DlacU169 (f80lacZDM15) 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 down- stream 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 N1 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.
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