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

TLDR

The N‑end rule predicts that long‑lived intracellular proteins possess stabilizing N‑terminal residues, and post‑translational addition of single amino acids may also be governed by this rule. A chimeric ubiquitin‑β‑galactosidase fusion expressed in yeast is processed by ubiquitin cleavage to expose the β‑galactosidase N‑terminus. Cleavage of the fusion occurs for all but one N‑terminal residue, allowing β‑galactosidase variants with N‑termini that confer half‑lives ranging from over 20 h to under 3 min, thereby establishing an order of residues by their destabilizing effect and linking N‑terminal identity to protein stability.

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.