Molecular characterization of the thermosensitive E1 ubiquitin-activating enzyme cell mutant A31N-ts20 - Requirements upon different levels of E1 forthe ubiquitination/degradation of the various protein substrates in vivo

According to our current knowledge, protein ubiquitination involves three s teps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin ont o E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combinatio n 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-in activatable in vitro; and (b) a major mechanism responsible for E1 inactiva tion in vivo consists of accelerated destruction. Surprisingly, >90% reduct ion in E1 abundance little alters the formation of the bulk of protein-ubiq uitin conjugates when A31N-ts20 cells are grown at the nonpermissive temper ature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surpri singly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, in dicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitinati on of the various cell proteins in vivo, and for providing criteria of reli ability when developing in vitro ubiquitination assays for specific protein s.