Molecular characterization of the thermosensitive E1 ubiquitin-activating enzyme cell mutant A31N-ts20. Requirements upon different levels of E1 for the ubiquitination/degradation of the various protein substrates in vivo.
According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin-protein ligase enzymes, transfer ubiquitin onto target proteins. A31N-ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat-inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein-ubiquitin conjugates when A31N-ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.
Salvat
C
C
Institut de Génétique Moléculaire, CNRS, Montpellier, France.
Acquaviva
C
C
Scheffner
M
M
Robbins
I
I
Piechaczyk
M
M
Jariel-Encontre
I
I
D000818:Animals; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D005347:Fibroblasts; D006657:Histones / Q000378:metabolism; D006358:Hot Temperature; D007976:Leupeptins / Q000494:pharmacology; D008025:Ligases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D006657:Histones; D007976:Leupeptins; D016755:Proto-Oncogene Proteins c-jun; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde