IDENTIFICATION OF AMINO-ACID-RESIDUES IN A CLASS-I UBIQUITIN-CONJUGATING ENZYME INVOLVED IN DETERMINING SPECIFICITY OF CONJUGATION OF UBIQUITIN TO PROTEINS

The ubiquitin pathway is a major system for selective proteolysis in e ukaryotes, However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoform s of a rat ubiquitin-conjugating enzyme (E2) homologous to the Sacchar omyces cerevisiae class I E2 genes, UBC4/ UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitous ly, whereas UBC4-testis is expressed in spermatids, Interestingly, alt hough these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubi quitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an Ii-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecul e responsible for the observed difference in substrate specificity. Fo ur residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfac es away from the active site appeared necessary and sufficient for UBC 4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or sub strates, These findings demonstrate that a limited number of amino aci d substitutions in E2s can dictate conjugation of ubiquitin to differe nt proteins and indicate a mechanism by which small E2 molecules can e ncode a wide range of substrate specificities.