Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates

J Biol Chem. 1994 Apr 1;269(13):9574-81.

Abstract

Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Drosophila
  • Electrophoresis, Polyacrylamide Gel
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Kinetics
  • Ligases / chemistry
  • Ligases / isolation & purification*
  • Ligases / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Rabbits
  • Reticulocytes / enzymology*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Ubiquitin-Conjugating Enzymes

Substances

  • Fungal Proteins
  • Peptide Fragments
  • Saccharomyces cerevisiae Proteins
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • RAD6 protein, S cerevisiae
  • Ubiquitin-Conjugating Enzymes
  • Ligases