Cloning and characterization of a 20-kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro.
Recent evidence indicates that the commitment to degrade cellular proteins by the ubiquitin proteolytic pathway is dependent on the covalent attachment of multiubiquitin chains to the target protein [Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K. & Varshavsky, A. (1989) Science 243, 1576-1583]. We have isolated a 20-kDa ubiquitin carrier protein [E2(20 kDa)] from wheat by using ubiquitin covalent affinity chromatography and anion-exchange HPLC that catalyzes multiubiquitin chain formation in vitro. This reaction is blocked by the addition of a mutant ubiquitin in which arginine has been substituted for lysine at residue 48, demonstrating that the coupling of ubiquitin to ubiquitin is likely to be through an isopeptide linkage between the C-terminal glycine and Lys48 of ubiquitin. By immunoscreening a wheat cDNA expression library with anti-E2(20 kDa) antibodies, a cDNA encoding the complete protein was isolated. The clone (designated UBC7) was confirmed as encoding E2(20 kDa) by comparison of the derived amino acid sequence with peptide sequences of E2(20 kDa) tryptic fragments. The encoded protein contains a single cysteine at position 91, which is presumably the active site, and has regions of amino acid sequence similarity to other known E2s from plants and yeast. Expression of this cDNA in Escherichia coli produced an active E2 capable of catalyzing multiubiquitin chain formation in vitro. By virtue of its activity, E2(20 kDa) may have a pivotal role in protein degradation by the ubiquitin-dependent proteolytic pathway.
Van Nocker
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Department of Horticulture, University of Wisconsin, Madison 53706.
Vierstra
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D008025:Ligases; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D002352:Carrier Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases