Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway.
NEDD8 is a ubiquitin-like molecule that can be covalently conjugated to a limited number of cellular proteins, such as Cdc53/cullin. We have previously reported that the C terminus of NEDD8 is efficiently processed to expose Gly-76, which is required for conjugation to target proteins. A combination of data base searches and polymerase chain reaction cloning was used to identify a cDNA encoding human UBA3, which is 38% identical to the yeast homologue, 22% identical to human UBA2, and 19% identical to the C-terminal region of human UBE1. The human UBA3 gene is located on chromosome 3p13 and gave rise to a 2.2-kilobase pair transcript that was detected in all tissues. Human UBA3 could be precipitated with glutathione S-transferase (GST)-NEDD8, but not with GST-ubiquitin or GST-sentrin-1. Moreover, human UBA3 could form a beta-mercaptoethanol-sensitive conjugate with NEDD8 in the presence of APP-BP1, a protein with sequence homology to the N-terminal half of ubiquitin-activating enzyme. We have also cloned human UBC12 and demonstrated that it could form a thiol ester linkage with NEDD8 in the presence of the activating enzyme complex. Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway should allow for a more detailed study of the role of NEDD8 modification in health and disease.
Gong
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Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, Department of Internal Medicine, University of Texas-Houston Health Science Center, Houston, TX, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D000075687:NEDD8 Protein; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D044764:Ubiquitin-Activating Enzymes; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C513172:Nedd8 protein, mouse; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C112594:UBA3 protein, S cerevisiae; C491805:Ube1c protein, mouse; D014452:Ubiquitins; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human