A NOVEL RAT HOMOLOG OF THE SACCHAROMYCES-CEREVISIAE UBIQUITIN-CONJUGATING ENZYMES UBC4 AND UBC5 WITH DISTINCT BIOCHEMICAL FEATURES IS INDUCED DURING SPERMATOGENESIS

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal pro teins, We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely ex pressed in rat tissues, consistent with a fundamental cellular functio n for these E2s, We now report a new isoform, 8A, which despite having > 91% amino acid identity with the other isoforms, shows several nove l features, Expression of the 8A isoform appears restricted to the tes tis, is absent in early life, but is induced during puberty, Hypophyse ctomy reduced expression of the 8A isoform. In situ hybridization stud ies indicated that 8A mRNA is expressed mainly in round spermatids. Im munoblot analyses showed that 8A protein is found not only in subfract ions of germ cells enriched in round spermatids but also in subfractio ns containing residual bodies extruded from more mature elongated sper matids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homolog s of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is u nique in possessing an acidic pi, The small differences in sequence be tween the 8A isoform and other rat isoforms conferred differences in b iochemical function. The 8A isoform was less effective than an isoform with a basic pi or ineffective in conjugating ubiquitin to certain fr actions of testis proteins, Thus, although multiple isoforms of a spec ific E2 may exist to ensure performance of a critical cellular functio n, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoform s and that subtle differences in E2 primary structure can dictate conj ugation of ubiquitin to different subsets of cellular proteins.