RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins.
The RAD6 (UBC2) gene of Saccharomyces cerevisiae which is involved in DNA repair, induced mutagenesis, and sporulation, encodes a ubiquitin-conjugating enzyme (E2). Since the RAD6 gene product can transfer ubiquitin directly to histones in vitro without the participation of a ubiquitin protein ligase (E3), it has been suggested that in vivo it also acts by the unassisted conjugation of ubiquitin to histones or to other target proteins. Here we show that the RAD6 protein can ligate ubiquitin in vitro to a hitherto unknown set of exogenous target proteins (alpha-, beta-, and kappa-casein and beta-lactoglobulin) when supplemented by a putative ubiquitin protein ligase (E3-R) from S. cerevisiae. RAD6 supplemented with E3-R ligates 1 or, sometimes, 2 ubiquitin molecules to the target protein molecule. UBC3 (CDC34) protein in the presence of E3-R has barely detectable activity on the non-histone substrates. Other ubiquitin-conjugating enzymes tested (products of the UBC1 and UBC4 genes) do not cooperate with E3-R in conjugating ubiquitin to the same substrates. Thus, E3-R apparently interacts selectively with RAD6 protein. These findings suggest that some of the in vivo activities of the RAD6 gene may involve E3-R.
Sharon
G
G
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Raboy
B
B
Parag
H A
HA
Dimitrovsky
D
D
Kulka
R G
RG
D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005822:Genetic Vectors; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases