Inhibition of the N-end rule pathway in living cells.

Abstract

The N-end rule relates the metabolic stability of a protein to the identity of its amino-terminal residue.Previous work, using amino acid derivatives such as dipeptides to inhibit N-end rule-mediated protein degradation in an extract from mammalian reticulocytes, has demonstrated the existence of specific N-end-recogniing proteins in this in vitro system. We now show that these nontoxic amino acid derivatives, when added to growing cells of the yeast Saccharomyces cerevisiae, are able to inhibit the degradation of proteins by the N-end rule pathway in vivo. Moreover, this inhibition is shown to be selective for the two distinct classes of destabilizing amino-terminal residues in substrates of the N-end rule pathway.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 ofone degradation signal is the protein's amino-terminal residue (1). This degradation signal is manifested as the N-end rule, which relates the metabolic stability of a protein to the identity of its amino-terminal residue (1-8).Similar but distinct versions of the N-end rule operate in mammals (2, 5, 8-11), yeast (1-5, 7, 12), and bacteria (J. Tobias and A.V., unpublished results). The N-end rule-based degradation signal in eukaryotes comprises a destabilizing amino-terminal residue and a specific internal lysine residue (1)(2)(3)(4)(5)8). The set of destabilizing amino-terminal residues is organized hierarchically. 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, aprimary destabilizing residue (1, 5-7). 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 (5).Previous work (1) has predicted the existence of "N-endrecognizing" proteins that select potential proteolytic substrates by binding to their amino-terminal residues. N-endrecognizing proteins have recently been detected in an in vitro ubiquitin-dependent proteolytic system derived from mammalian reticulocytes, and identified as the E3 proteins that were previously shown to bind proteolytic substrates prior to their ubiquitination by a subset of ubiquitinconjugating (E2) enzymes (5, 9, 10, 12, 13). Three distinct types of E3 activity have been detected in the reticulocytederived in vitro system by using assays based on selective inhibition of the degradation of specific proteins by dipeptides bearing different destabilizing amino-terminal residues. The type I E3 activity is specific for the positively charged destabilizing amino-terminal residues Arg, Lys, and His (5, 9). The type II activity is specific for the bulky hydrophobic destabilizing amino-terminal residues Phe, Trp, Tyr, and Leu (and lie in yeast) (5, 9). The type III activity is specif...

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

“…Conjugation of the primary destabilizing residue R to the secondary destabilizing amino-terminal residues D and E (7) is catalyzed by the arginyl-tRNA-protein transferase (R-transferase) encoded by the ATE] gene (10). The distinction between type 1 (R, K, H) and type 2 (L, F, Y, I, W) primary destabilizing amino-terminal residues is based on their binding to distinct sites on the UBRI-encoded N-recognin (7,11,13,14). If a substrate bears both determinants of the N-end rule-based degradation signal [the N-degron (2)], the UBR1-associated UBC enzyme encoded by the UBC2 gene (see main text) uses activated Ub, produced by the UBAI-encoded Ub-activating enzyme (15), to catalyze formation of a substrate-linked multiubiquitin chain.…”

“…3). Previous work (10,11,14) has shown that the steady-state level of an X-f8gal in yeast cells is a function of its metabolic stability; compare, for instance, the levels of (40) and BBY67 (a ubc2A:.LEU2 derivative ofYPH500) were transformed with the previously described (1) 2-Atm-based vectors expressing Ub-X-,gals from a galactose-inducible promoter. Exponential cultures (A600 < 1) growing at 30°C in SD-galactose medium (1, 6) were assayed for ,-galactosidase activity as described (41).…”

“…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 N‐end rule pathway of protein degradation

“…Hondermarck et al (1992) (see also Taban et al 1996) reported that differentiation of rat pheochromocytoma PC12 cells is inhibited by dipeptides bearing destabilizing N-terminal residues. (These compounds have been shown to inhibit the N-end rule pathway in S. cerevisiae (Baker & Varshavsky 1991); their efficacy as N-end rule inhibitors in mammalian cells remains to be evaluated.) Given the findings with Ga (Madura & Varshavsky 1994), one interpretation of these results (Hondermarck et al 1992) is that inhibitors of the N-end rule pathway may suppress cell differentiation through a metabolic stabilization of the relevant Ga subunits in PC12 cells.…”

“…This cleavage takes place regardless ᭧ Blackwell Science Limited Genes to Cells (1997) 2, 13-28 13 * Correspondence: E-mail: varshavskya@starbase1. caltech.edu a The in vivo degradation of many short-lived proteins, including the engineered N-end rule substrates, deviates from first-order kinetics (Baker & Varshavsky 1991). Therefore the term 'half-life', if applied to an entire decay curve, is a useful but often crude approximation.…”