The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme.

Abstract

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operate in all organisms examined, from mammals to bacteria (refs. 1-12; J. Tobias, T. Shrader, G. Rocap, and A.V., unpublished data).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).If a substrate bears both determinants of the N-degron, the binding of N-reco...

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“…The N-terminal 15 amino-acid sequence is nearly identical among all Rad6 homologs [33][34][35][36]; deletion of the first 9 amino acids from Rad6 (rad6 ∆1-9 ) abolishes sporulation, reduces cell survival after UV treatment, but surprisingly increases spontaneous and UV-induced mutagenesis [37]. Furthermore, the N-terminus of Rad6 is also required for N-end rule protein degradation [29, 37,38]: while the full-length Rad6 interacts with the E3 protein Ubr1, the Rad6 ∆1-9 protein is unable to form a complex with Ubr1 [37]. Rad6 is known to form a stable complex with Rad18 [39], and this complex displays Ub conjugation (from Rad6), ssDNA-binding and ATPase (from Rad18) activities [40].…”

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