IN-VIVO NUCLEAR TRANSPORT KINETICS IN SACCHAROMYCES-CEREVISIAE - A ROLE FOR HEAT-SHOCK PROTEIN-70 DURING TARGETING AND TRANSLOCATION

The transport of proteins into the nucleus is a receptor-mediated proc ess that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assa y in Saccharomyces cerevisiae for the nuclear transport of green fluor escent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-G FP nuclear import rates in wild-type and mutant cells under various ph ysiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NL S-cargo in the cytoplasm and accompanies it into the nucleus. When com pared to SRP1(+) cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS pep tide binding. We show that the kinetics of NLS-directed nuclear transp ort in wildtype cells is stimulated by the elevated expression of SSA1 , which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions with in the nuclear pore complex subsequent to docking. These results provi de genetic evidence that Hsp70 acts during both targeting and transloc ation phases of nuclear transport, possibly as a molecular chaperone t o promote the formation and stability of the Srp1p-NLS-cargo complex.