Multiple (α-NH-ubiquitin)Protein Endoproteases in Cells

Abstract

Ubiquitin is encoded as a variable, spacerless repeat of the gene terminating with an additional amino acid or as a gene coding for a single ubiquitin with a carboxyl-terminal extension of 52 to 80 amino acids. We report the identification and partial purification of enzymes that specifically hydrolyze the peptide bond between ubiquitin-ubiquitin conjugate (Ub-Ubase) or ubiquitin fusion proteins (Ub-Xase). The Ub-Ubase was separated from the Ub-Xase by dye-ligand-Sepharose chromatography. The Ub-Xase was purified further by affinity chromatography on ubiquitin-Sepharose. The fidelity of the endoprotease reaction was assessed by measuring the ability of the released ubiquitin to be activated by ubiquitin-activating enzyme (E1) which requires intact ubiquitin and by sequence analysis of the released carboxyl extension protein with 52 amino acids after endoproteolysis of human ubiquitin with 52-amino acid carboxyl extension. The failure of both Ub-Ubase and Ub-Xase to cleave a mutant ubiquitin-Gly-76----Ala-metallothionein showed that the endoproteases distinguish Gly-X from an Ala-X peptide bonds.