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

N. Blumenfeld, H. Gonen, A. Mayer, C. E. Smith, N. R. Siegel, A. L. Schwartz, Aaron Ciechanover

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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(14 kDa) 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 NH 2 terminus) and to the ubiquitination and degradation of certain N-α-acetylated proteins such as histone H2A, actin, and α-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

Original languageEnglish
Pages (from-to)9574-9581
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number13
StatePublished - 1 Jan 1994

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Purification
Ubiquitination
Proteolysis
Ubiquitin
Degradation
Substrates
Enzymes
Ubiquitinated Proteins
Ubiquitin-Conjugating Enzymes
Anion Exchange Resins
Tumor Suppressor Protein p53
Crystallins
Glyceraldehyde-3-Phosphate Dehydrogenases
Proteins
Ubiquitin-Protein Ligases
Reticulocytes
Ligases
Sulfhydryl Compounds
Histones
Sequence Analysis

Cite this

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title = "Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-'N-end rule' protein substrates",
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(14 kDa) 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 NH 2 terminus) and to the ubiquitination and degradation of certain N-α-acetylated proteins such as histone H2A, actin, and α-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.",
author = "N. Blumenfeld and H. Gonen and A. Mayer and Smith, {C. E.} and Siegel, {N. R.} and Schwartz, {A. L.} and Aaron Ciechanover",
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Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-'N-end rule' protein substrates. / Blumenfeld, N.; Gonen, H.; Mayer, A.; Smith, C. E.; Siegel, N. R.; Schwartz, A. L.; Ciechanover, Aaron.

In: Journal of Biological Chemistry, Vol. 269, No. 13, 01.01.1994, p. 9574-9581.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-'N-end rule' protein substrates

AU - Blumenfeld, N.

AU - Gonen, H.

AU - Mayer, A.

AU - Smith, C. E.

AU - Siegel, N. R.

AU - Schwartz, A. L.

AU - Ciechanover, Aaron

PY - 1994/1/1

Y1 - 1994/1/1

N2 - 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(14 kDa) 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 NH 2 terminus) and to the ubiquitination and degradation of certain N-α-acetylated proteins such as histone H2A, actin, and α-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

AB - 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(14 kDa) 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 NH 2 terminus) and to the ubiquitination and degradation of certain N-α-acetylated proteins such as histone H2A, actin, and α-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

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