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.
Mesh Terms:
Amino Acid Sequence, Animals, Base Sequence, Chromosome Mapping, Chromosomes, Human, Pair 3, Cloning, Molecular, DNA, Complementary, Humans, Ligases, Mice, Molecular Sequence Data, Recombinant Fusion Proteins, Recombinant Proteins, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Ubiquitin-Activating Enzymes, Ubiquitins
J. Biol. Chem.
Date: Apr. 23, 1999
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