title: Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis.
abstract: Ubiquitin carrier proteins (E2s) are involved in the covalent attachment of ubiquitin to a variety of cellular target proteins in eukaryotes. Here, we report the cloning of genes from wheat and Arabidopsis thaliana that encode 16-kDa E2s and a domain analysis of E2s by in vitro mutagenesis. The genes for E216kDa, which we have designated wheat and At UBC1, encode proteins that are only 33% identical (58% similar) with a 23-kDa E2 from wheat (encoded by the gene now designated wheat UBC4), but are 63% identical (82% similar) with the E2 encoded by the Saccharomyces cerevisiae DNA repair gene, RAD6. Unlike the proteins encoded by RAD6 and wheat UBC4, the UBC1 gene products lack acidic C-terminal domains extending beyond the conserved core of the proteins and are incapable of efficient in vitro ligation of ubiquitin to histones. From enzymatic analysis of the UBC1 and UBC4 gene products mutagenized in vitro, we have identified several domains important for E2 function, including the active site cysteine and N-terminal and C-terminal domains. Cysteine residues 88 and 85 in the UBC1 and UBC4 gene products, respectively, are necessary for formation of the ubiquitin-E2 thiol ester intermediate. Whereas the UBC1 gene product does not require its additional cysteine residue at position 116 for thiol ester formation, alteration of cysteine 143 in the UBC4 gene product greatly diminishes this ability. The N terminus of UBC1 contains two domains that affect activity: a proximal region containing hydroxylated and uncharged residues whose removal increases the rate of thiol ester formation and a distal tract rich in basic residues. Deletion or substitution of these basic residues with neutral residues diminishes the rate of thiol ester formation. We have demonstrated also that C-terminal extensions can function to confer substrate specificity to E2s. When the acidic extension was deleted from UBC4, the protein was unable to efficiently conjugate ubiquitin to histones in vitro. Furthermore, fusion of the UBC4 acidic extension to the C terminus of UBC1 resulted in a chimeric protein capable of efficient histone conjugation, as did fusion of short tracts of alternating aspartate and glutamate residues. This result suggests that the target protein specificity of E2s can be altered by the addition of appropriate C-terminal extensions, thus providing a way to modify the selectivity of the ubiquitin system.
authors.affiliation: Department of Horticulture, University of Wisconsin, Madison 53706.
authors.forename: M L
authors.initials: ML
authors.lastname: Sullivan
authors.forename: R D
authors.initials: RD
authors.lastname: Vierstra
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008025:Ligases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000235:genetics* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D006657:Histones; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases