Ubiquitin/ATP-Dependent Protease

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.

Ubiquitin, a 76 residue protein, occurs in eucaryotic cells either free or covalently joined to a variety of protein species. Previous work suggested that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. We show that the mouse cell line ts85 , a previously isolated cell cycle mutant, is temperature-sensitive in ubiquitin-protein conjugation, and that this effect is due to the specific thermolability of the ts85 ubiquitin-activating enzyme (E1). From E1 thermoinactivation kinetics in mixed (wild-type plus ts85 ) extracts, and from copurification of the determinant of E1 thermolability with E1 in ubiquitin-affinity chromatography, we conclude that the determinant of E1 thermolability is contained within the E1 polypeptide. ts85 cells fail to degrade otherwise short-lived intracellular proteins at the nonpermissive temperature (accompanying paper), demonstrating that degradation of the bulk of short-lived proteins in this higher eucaryotic cell proceeds through a ubiquitin-dependent pathway. We discuss possible roles of ubiquitin-dependent pathways in DNA transactions, the cell cycle, and the heat shock response.