A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis

L. Gregori, M. S. Poosch, G. Cousins, V. Chau

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The proteolytic targeting function of ubiquitin was investigated by a combination of site-specific mutagenesis and covalent modification. Lys48 was replaced by a cysteine via mutagenesis of a synthetic ubiquitin gene to generate the mutant Ub-C48. The single cysteine residue in Ub-C48 can be converted into a lysine analog by modification with the sulfhydryl-specific reagent, aminoethyl-8 (N-(iodoethyl)trifluoroacetamide). The resulting protein, Ub-(S-aminoethyl)C48, is equivalent to a wild type ubiquitin except for the substitution of a sulfur atom at the γ carbon of Lys48. We have tested the ability of these two modified ubiquitins to target the degradation of an engineered β-galactosidase substrate protein in ubiquitin-depleted reticulocyte lysates. Ub-C48 was unable to stimulate the degradation of this protein substrate although a monoubiquitinated β-galactosidase was formed. In contrast, Ub-(S-aminoethyl)C48 appears to be as effective as wild type ubiquitin in targeting this substrate protein's degradation as well as the formation of multiply ubiquitinated β-galactosidase intermediates. In conjuction with the cysteine substitution and modification, we have also examined the effects of blocking the amino groups in ubiquitin with reductive methylation. The methylation of either Lys48 in ubiquitin or its S-aminoethylcysteine counterpart abolished its proteolytic function while the blockage of the remaining six lysines in Ub-(S-aminoethyl)C48 did not alter its competence. Thus, of the seven lysine residues in ubiquitin, only Lys48 is essential. These results established unambiguously that a uniform multiubiquitin chain with ubiquitin-ubiquitin linkage solely at Lys48 is sufficient to target the degradation of a substrate protein in ubiquitin-mediated proteolysis.

Original languageEnglish (US)
Pages (from-to)8354-8357
Number of pages4
JournalJournal of Biological Chemistry
Issue number15
StatePublished - 1990

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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