MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION OF A UBIQUITIN CONJUGATION ENZYME (E2(17KB)) HIGHLY EXPRESSED IN RAT TESTIS

Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-media ted proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa c onjugating enzyme [E2(14k); Wing, Dumas and Banville (1992) J. Biol. C hem. 267, 6495-6501] that efficiently supported ubiquitination and pro tein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support m uch of the ubiquitination of yeast proteins. This suggested that mamma lian homologues of UBC4/UBC5 remained to be identified. Using oligonuc leotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% ide ntical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquit ination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14k). Examination of RNA from diffe rent tissues indicated that this type of E2 was expressed in a broad s pectrum of tissues but at particularly high levels in the testis. Frac tionation of a testis extract by anion-exchange chromatography identif ied several putative ubiquitin protein ligase activities with which th is E2 could interact in promoting conjugation of ubiquitin to proteins . One of these activities supported conjugation of ubiquitin to histon e H2A, a substrate degraded in the ubiquitin system by a non-N-end rul e mechanism. This paper reports the first cloning of an apparent mamma lian homologue of S. cerevisiae UBC4/ UBC5. Its high expression in tes tis and ability to efficiency support conjugation to testis proteins s uggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.