Cytosolic pool of Rsp5 ubiquitin ligase is sufficient for proper intracellular tRNA distribution in Saccharomyces cerevisiae.
Piotr Cholbinski (1), Malgorzata Lewandowska (1), Anita K. Hopper (2), Teresa Zoladek (1)
(1) Department of Genetics, IBB PAS, Warsaw, Poland; (2) The Pennsylvania State University College of Medicine, Hershey, PA, USA
The Rsp5p ubiquitin ligase regulates many cellular processes at almost every subcellular compartment, including the nucleus. Many rsp5 mutants reveal nuclear tRNA accumulation, but the mechanism by which Rsp5p affects tRNA transport is unknown. Rsp5p localizes in the cytoplasm as uniformly distributed punctuate complexes. However, we identified potential nuclear localization sequence NLS RSP5 477-PEDLKKRL-484 and showed that it is able to direct reporter protein, β -galactosidase, to the nucleus indicating that Rsp5p might be a shuttling protein. Mutations causing substitutions of asparagines for the basic amino acids in NLS RSP5 (477-PEDLNNNL-484) prevented the nuclear localization of β -galactosidase. Nuclear pool of Rsp5p, however, seems not to be involved in intracellular tRNA movement as determined by FISH analysis; no nuclear tRNA accumulation in rsp5-nls strain bearing Rsp5p with inactivated NLS RSP5 was observed. This data suggest that the cytosolic pool of Rsp5p provides all required activities for efficient cytosol-nucleus tRNA exchange.