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 process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared 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 peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type 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 within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.
Shulga
N
N
Department of Biology, University of Rochester, New York 14627, USA.
Roberts
P
P
Gu
Z
Z
Spitz
L
L
Tabb
M M
MM
Nomura
M
M
Goldfarb
D S
DS
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D013539:Simian virus 40; D013997:Time Factors; D028901:alpha Karyopherins
D000957:Antigens, Viral, Tumor; D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase