Posible participación de la GTPasa Npa3 en la síntesis de proteínas en la levadura Saccharomyces cerevisiae

Abstract

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between mutants lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3∆C) and mutants lacking Bud site selection protein 27 (bud27∆). As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3C, Npa3K16R, and Npa3G70A). The Npa3C mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially support RNAPII nuclear targeting and exhibit a slower growth rate due to a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A>npa3K16R>npa3∆C>NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3∆C, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3∆C cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and8 previously unrecognized role in translation initiation. This finding provides a rationale for the transcriptional co-regulation of NPA3 and genes involved directly in translation or in ribosome biogenesis