In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue

Abstract

When a chimeric gene encoding a ubiquitin-β-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae , ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated β-galactosidase (βgal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of βgal at the ubiquitin-βgal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical βgal proteins. The βgal 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 βgal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on βgal 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.