title: Creation of a pluripotent ubiquitin-conjugating enzyme.
abstract: We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Ptak
authors.forename: C
authors.initials: C
authors.lastname: Gwozd
authors.forename: J T
authors.initials: JT
authors.lastname: Huzil
authors.forename: T J
authors.initials: TJ
authors.lastname: Gwozd
authors.forename: G
authors.initials: G
authors.lastname: Garen
authors.forename: M J
authors.initials: MJ
authors.lastname: Ellison
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D002455:Cell Division; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C577449:CDC34 protein, S cerevisiae; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C063109:ubiquitin-conjugating enzyme UBC4; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases