Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme

Patrick Sung, Erica Berleth, Cecile Pickart, Satya Prakash, Louise Prakash

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein-its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian EZ14k in E3 dependent ubiquitin - protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S.cerevisiae E230k, product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.

Original languageEnglish
Pages (from-to)2187-2193
Number of pages7
JournalEMBO Journal
Volume10
Issue number8
StatePublished - 1991
Externally publishedYes

Fingerprint

Ubiquitin-Conjugating Enzymes
Ubiquitin
Yeast
Proteolysis
Yeasts
Degradation
Enzymes
Proteins
Saccharomyces cerevisiae
Genes
Ubiquitin-Specific Proteases
Mutagenesis
Ubiquitin-Protein Ligases
Reticulocytes
DNA
Mutant Proteins
Eukaryota
DNA Repair
DNA Damage
Conservation

Keywords

  • DNA repair
  • E3
  • Proteolysis
  • RAD6
  • Sporulation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme. / Sung, Patrick; Berleth, Erica; Pickart, Cecile; Prakash, Satya; Prakash, Louise.

In: EMBO Journal, Vol. 10, No. 8, 1991, p. 2187-2193.

Research output: Contribution to journalArticle

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AB - The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein-its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian EZ14k in E3 dependent ubiquitin - protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S.cerevisiae E230k, product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.

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