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 eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-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 molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities.
Oughtred
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R
Department of Medicine, Polypeptide Laboratory, McGill University, Montreal, Quebec H3A 2B2, Canada.
Bédard
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N
Vrielink
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A
Wing
S S
SS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D006657:Histones / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013737:Testis / Q000201:enzymology; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D006657:Histones; D007527:Isoenzymes; D009687:Nuclear Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C489270:UBC5 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D008025:Ligases