Gene UBP1 of Saccharomyces Cereuisiae”
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THEJOURNAL OF BIOLOGICALCHEMISTRY Vol. 266, No. 18, Issue of June 25, pp. 12021-1’2028,1991 (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cloning and Functional Analysisof the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cereuisiae” (Received for publication, February 5, 1991) John W. Tobias and Alexander Varshavsky From the Departmentof Biology, Massachusetts Instituteof Technology, Cambridge, Massachusetts02139 In eukaryotes, both natural and engineered fusions erone function,in that its transient(cotranslational) covalent of ubiquitin to itself or other proteins are cleaved by association with specific ribosomal proteins promotes the processing proteases after the last (G~Y‘~)residue of assembly of ribosomal subunits (Finley et al., 1989). In vivo ubiquitin. Using the method ofsib selection,and taking acceptors of ubiquitin include chromosomal histones (Bonner advantage of the fact that bacteria such as Escherichia et al., 1988), integral membrane proteins such as the lympho- coli lack ubiquitin-specific enzymes, we have cloned a cyte homing receptor and the growth hormone receptor (Sie- gene, named UBPl, of the yeast Saccharomyces cere- gelman et al., 1986; Leung et al., 1987), actin (Ball et al., visiae that encodes a ubiquitin-specific processing pro- 1987), the intracellular neurofibrillary tangles in a variety of tease. With the exception of polyubiquitin, the UBPl neurodegenerative diseases (Perry et al., 1987; Lowe et al., protease cleaves at the carboxyl terminus of the ubiqui- 1988), the plant regulatory protein phytochrome (Jabben et tin moiety in natural and engineered fusions irrespec- tive of their size or the presence of an amino-terminal al., 1989), the short-livedMATa2 repressor of the yeast ubiquitin extension. These properties of UBPl distin- Saccharomyces cereuisiae (Hochstrasser et al., 1991), and cy- guish it from the previously cloned yeast protease clins, a family of short-lived cell cycle regulators (Glotzer et YUH1, which deubiquitinates relatively short ubiqui- al., 1991). tin fusions but is virtually inactive withlonger fusions Unlike branched Ub-protein conjugates, which are formed such as ubiquitin-&galactosidase. The amino acid se- posttranslationally, linear Ub-protein adducts are formed as quence of the 809-residue UBPl lacks significantsim- the translational products of either natural or engineered ilarities to other known proteins, including the 236- ubiquitin gene fusions. Thus, in S. cereuisiae for example, residue YUHl protease. Null ubpl mutants are viable, ubiquitin is generated exclusively by proteolytic processing of and retain the ability to deubiquitinate ubiquitin-0- precursors in which ubiquitin is joined either to itself, as in galactosidase, indicating that the family of ubiquitin- the linear polyubiquitin protein UBI4, or to unrelated amino specific proteases in yeast is not limited to UBPl and acid sequences, as in the hybrid proteins UBI1-UBI3 (Ozkay- YUH1. nak et al., 1987). In growing yeast cells, ubiquitin is generated from the UBI1-UBI3 precursors whose “tails” are specific ribosomal proteins (Finley et al., 1989). The polyubiquitin (UBI4) gene is dispensable in growing cells but becomes Ubiquitin (Ub),’ a highly conserved 76-residue protein, is essential (as the main supplier of ubiquitin) during stress present in eukaryotic cells either free or covalently joined to (Finley et al., 1987, 1988). The lack of genes encoding mature a great variety of proteins. The posttranslational coupling of ubiquitin and the fusion structure of ubiquitin precursors in ubiquitin to other proteins is catalyzed by a family of Ub- yeast are characteristic of other eukaryotes as well (Schles- conjugating enzymes (also called E2 enzymes (Pickart and inger and Bond, 1987). Thus, there must exist Ub-specific Rose, 1985)) and involves formation of an isopeptide bond processing proteases that produce ubiquitin, a protein essen- between the carboxyl-terminal glycine residue of ubiquitin tial for cell viability (Finley et al., 1984,1987, 1988), by and the E-amino group of a lysine residue in an acceptor cleaving Gly-X peptide bonds at thejunctions between ubiqui- protein (Pickart, 1988; Jentsch et al., 1990). A major function tin and itscarboxyl-terminal extensions. A priori, these pro- of ubiquitin is to mark proteins destined for selective degra- dation (reviewed by Hershko, 1988; Finley et al., 1988; Cie- teases may also have an isopeptidase activity, i.e. the ability chanover and Schwartz, 1989; Olson and Dice, 1989; Monia to deubiquitinate posttranslationally formed, branched Ub- et al., 1990). Ubiquitin has also been shown to have a chap- protein conjugates. A multiubiquitin chain is one such con- jugate, in which ubiquitin itself serves as an acceptor, with * This work was supported by Grants AGO8991 and DK39520 (to several ubiquitin moieties attached sequentially to the initial A. V.) from the National Institutes of Health. The costs of publication acceptor protein to form a chain of branched Ub-Ub adducts of this article were defrayed in part by the payment of page charges. (Chau et al., 1989). Formation of a multiubiquitin chain on a This article must therefore be hereby marked “aduertisement” in targeted protein is required for the protein’s subsequent deg- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. radation (Chau et al., 1989; Gregori et al., 1990). The nucleotide sequence(s) reportedin thispaper has been submitted tothe GenBankTM/EMBL Data Bunk with accessionnumberls) Ub-specific, ATP-independent proteases capable of cleav- M63484. ing ubiquitin from its linearor branched conjugates have been ’ The abbreviations used are: Ub, ubiquitin; DHFR, dihydrofolate detected in all eukaryotes examined (Matsui et al., 1982; Rose, reductase; UB-X-DHFR, ubiquitin-X-mouse dihydrofolate reductase, 1988; Mayer and Wilkinson, 1989; Jonnalagadda et al., 1989) where X is an amino acid residue at theUb-DHFR junction; Ub-X- but not in bacteria such asEscherichia coli, which lack eukar- Pgal, ubiquitin-X-E. coli (%galactosidase;MTX, methothrexate; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; yotic ubiquitin and Ub-specific enzymes (Finley et al., 1988). DTT, dithiothreitol; LB, Luria broth; HEPES, 4-(2-hydroxyethyl)-l- Recently, Miller et al. (1989) have cloned a S. cereuisiae gene, piperazineethanesulfonic acid; kb, kilobase(s); bp, base pair(s); ORF, named YUHl, for a Ub-specific protease that cleaves ubiqui- open reading frame. tin from its carboxyl-terminal fusions to relatively short 12021 12022 Ubiquitin-specific Protease amino acid sequences but is virtually inactive with larger centrifuge tube containing0.975 mlof 65 mM Na-EDTA, 50 mM Tris- fusions such as Ub-@-galactosidase(Ub-@gal). Wilkinson et HCl (pH 8.0), and protease inhibitors antipain, chymostatin, leupep- al. (1989) havealso cloned a cDNA for a mammalian homolog tin, pepstatin, and aprotinin, each at 25 pg/ml. 10 p1 of 10% (v/v) of the yeast YUHl protease. Triton X-100 was then added and dispersed by pipetting. The lysate was centrifuged at 40,000 X g for 30 min. The supernatant, containing We now report the cloning and functional analysis of an S. the 35S-labeledUb-M-DHFR, was frozen in liquid nitrogen and stored cereuisiue gene, named UBPl, which encodes a Ub-specific at -85 "C. To purify Ub-M-DHFR, a methothrexate (MTX) affinity processing protease whose amino acid sequence is dissimilar matrix was prepared as described by Kaufman (1974), using amino- to those of the YUHl protease and other known proteins. hexyl-Sepharose (Sigma). The MTX-Sepharose column (0.5-ml bed The UBPl protease cleaves at the carboxyl terminus of the volume) was washed with 10 ml of MTX buffer (0.2 M NaCl, 1 mM Na-EDTA, 0.2 mM dithiothreitol (DTT), 20mM Na-HEPES, pH ubiquitin moiety in linear fusions irrespective of their size or 7.5). The Ub-M-DHFR-containing 35Ssupernatant was thawed, clar- the presence of an amino-terminal ubiquitin extension. We ified by centrifugation at 12,000 X g for 1 min, and applied to the also show that null ubpl mutants are viable and have prop- column, which was then washed with 50 ml of MTX buffer, 50 ml of erties indicating that the family of Ub-specific proteases in MTX buffer containing 2M urea, and thenagain with 50 ml of MTX yeast is not limited to UBPl andYUH1. buffer. Ub-M-DHFR was then eluted from the column with folic acid Since Ub-specific proteases cleave ubiquitin from its car- buffer (10 mM folic acid, 1 M KCI, 1 mM DTT, 1 mM Na-EDTA, 0.2 M potassium borate, pH 9.0). The folic acid buffer was applied in 1- boxyl-terminal extensions irrespective of the identity of the ml aliquots, and 1-ml fractions were collected. The fractions were extension's residue abutting the cleavage site (Bachmair et assayed for 35S;pooled fractions containing the peak of 35Swere al., 1986; Gonda et al., 1989), UBPl and its functional coun- dialyzed for 20 h at 4 "C against two changes of storage buffer (50% terparts make possible the in vivo or in vitro generation of glycerol, 1 mM MgCI2, 0.1 mM Na-EDTA, 40 mM Na-HEPES, pH proteins (including short polypeptides) bearing predetermined 7.5), and stored at -20 "C. The purified [35S]Ub-M-DHFRwas as- residues at their amino termini, a method with applications sayed by SDS-PAGE and fluorography and found to be greater than 95% pure. Other test proteins such as DHFR-Ub-M-pgal (Fig. L4, in both basic research (Baker and Varshavsky, 1991; Bartel VI), and the natural yeast ubiquitin fusions UBI2 (Fig. L4, I), UBI3 et al., 1990; Johnson et al., 1990; Townsend et al., 1988) and (Fig. lA, II), and UBI4 (polyubiquitin; Fig. lA, III), were expressed biotechnology (Lu et al., 1990; Butt et al., 1989; Sabin et al., in E. coli and assayed for deubiquitination with UBPl in mixed E. 1989; Ecker et al., 1989; Yo0 et al., 1989; Mak et al., 1989).