The N-end rule is mediated by the UBC 2 ( RAD 6 ) ubiquitin-conjugating enzyme ( protein degradation / yeast / RAD 6 / UBC 2 )

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway. Selective protein degradation underlies the elimination of damaged or otherwise abnormal proteins and the temporal control of many cellular processes that involve short-lived regulators. At least some proteins are short-lived in vivo because they contain sequences (degradation signals) that make these proteins substrates of specific proteolytic pathways. An essential component of one degradation signal is the protein's amino-terminal residue (1). The presence of this signal, named the N-degron (2), is manifested as the N-end rule, which relates the metabolic stability of a protein to the identity of its amino-terminal residue (1). Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria (refs. 1-12; J. Tobias, T. Shrader, G. Rocap, and A.V., unpublished data). The eukaryotic N-degron is a bipartite signal, comprising a destabilizing aminoterminal residue (1) and a specific internal Lys residue (6, 8, 9). The N-end rule is organized hierarchically (Fig. 1). Specifically, amino-terminal Asp and Glu (and Cys in mammalian reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to Arg, one of the primary destabilizing residues (1, 7, 10, 12). Amino-terminal Asn and Gln are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into the secondary destabilizing residues Asp and Glu (Fig. 1) (7). In the yeast Saccharomyces cerevisiae, the recognition component of the N-end rule pathway is encoded by the UBRI gene (11). The 225-kDa UBR1 protein, named N-recognin [also known as the type 1, 2 E3 protein (5, 7, 13)], selects potential proteolytic substrates by binding to their primary destabilizing amino-terminal residues (Fig. 1) (6, 11). The yeast N-recognin (11, 14) and its mammalian counterparts (5, 7, 13, 16, 18) each possess distinct binding sites for the two classes of primary destabilizing residues. The type 1 binding site is specific for the positively charged aminoterminal residues Arg, Lys, and His. The type 2 binding site is specific for the bulky hydrophobic amino-terminal residues Phe, Trp, Tyr, and Leu (and Ile in yeast) (5, 7, 11, 13). Ifa substrate bears both determinants of the N-degron, the binding of N-recognin is followed by formation of a multiubiquitin chain linked to the N-degron's second determinant, a specific internal Lys residue (6-9). The coupling of ubiquitin (Ub) to proteins is catalyzed by a family of Ubconjugating (UBC) enzymes [also called E2 enzymes (13)] and involves formation of an isopeptide bond between the carboxyl-terminal Gly residue of Ub and the E-amino group of a Lys residue in an acceptor protein (13, 19, 20). In a multiubiquitin chain, Ub itself serves as an acceptor, with several Ub moieties attached sequentially to the initial acceptor protein to form a chain ofbranched Ub-Ub conjugates (8, 21). There are seven or more distinct UBC enzymes in S. cerevisiae; other eukaryotes appear to have at least as many (20). Two of the yeast UBC enzymes have been identified as the products of the previously known genes RAD6 (renamed UBC2) (22), which participates in DNA repair, induced mutagenesis, sporulation (23-33), and suppression of retrotransposition (34), and CDC34 (renamed UBC3), which is required for the transition from G1 to S phase ofthe cell cycle (35). Two of the other yeast UBC enzymes, UBC4 and UBC5, have been shown to be required for most of the Ub-dependent protein degradation in this organism (18, 36). We now report that UBC2(RAD6) is essential for the degradation of N-end rule substrates and that UBC2 is physically associated with the UBRI-encoded N-recognin. Thus, at least one of the multiple functions of UBC2 is likely to be mediated by protein degradation via the N-end rule pathway. MATERIALS AND METHODS Plasmids. The plasmid pSOB44 is a derivative of pSOB37 (11), in which the UBRI promoter was replaced with the 1.5-kilobase (kb) BamHI fragment from the ADHI promoter-containing plasmid pJDcAG1 (37). The plasmid pADHUBC2 was constructed by inserting the -1.5-kb BamHIEcoRI fragment from pJDcAG1 into BamHI/EcoRI-cut YEplacl95 (38). The resultant plasmid was cut with EcoRI, and the ""0.6-kb EcoRI fragment containing the UBC2 coding sequence (22) was inserted. pADHUBC2 fully complemented the radiation sensitivity and slow growth phenotypes of the ubrlA ubc2A BBY68 strain (see the legend to Fig. 4). Coimmunoprecipitation. Exponential yeast cultures (A6w < 1) were labeled with [35S]methionine (0.3 mCi/ml; 1 Ci = 37 GBq) for 30 min at 30°C in synthetic medium (11) lacking methionine. The cells were pelleted by centrifugation, resuspended in DB buffer (50 mM NaCI/1 mM Na2EDTA/50 mM Na Hepes, pH 7.5) containing protease inhibitors leupeptin, pepstatin A, antipain, chymostatin, and aprotinin (each at 20 Abbreviations: Ub, ubiquitin; UBC, Ub-conjugating; Ub-X-,3gal, Ub-X-.8-galactosidase. 7351 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7352 Biochemistry: Dohmen et al. tertiary destabilizing