title: Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.
abstract: The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.
authors.affiliation: Department of Molecular Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Wiborg
authors.forename: Charlotte
authors.initials: C
authors.lastname: O'Shea
authors.forename: Karen
authors.initials: K
authors.lastname: Skriver
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases