Nucleocytoplasmic trafficking of the molecular chaperone Hsp104 in unstressed and heat-shocked cells.
Hsp104 is a molecular chaperone in yeast that restores solubility and activity to inactivated proteins after severe heat shock. We investigated the mechanisms that influence Hsp104 subcellular distribution in both unstressed and heat-shocked cells. In unstressed cells, Hsp104 and a green fluorescent protein-Hsp104 fusion protein were detected in both the nucleus and the cytoplasm. We demonstrate that a 17-amino-acid sequence of Hsp104 nuclear localization sequence 17 (NLS17) is sufficient to target a reporter molecule to the nucleus and is also necessary for normal Hsp104 subcellular distribution. The nuclear targeting function of NLS17 is genetically dependent on KAP95 and KAP121. In addition, wild-type Hsp104, but not an NLS17-mutated Hsp104 variant, accumulated in the nucleus of cells depleted for the general export factor Xpo1. Interestingly, severe, nonlethal heat shock enhances the nuclear levels of Hsp104 in an NLS17-independent manner. Under these conditions, we demonstrate that karyopherin-mediated nuclear transport is impaired, while the integrity of the nuclear-cytoplasmic barrier remains intact. Based on these observations, we propose that Hsp104 continues to access the nucleus during severe heat shock using a karyopherin-independent mechanism.
Tkach
Johnny M
JM
Department of Biochemistry, University of Toronto, Room 5302, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
Glover
John R
JR
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D021381:Protein Transport; D004718:Saccharomycetales / Q000378:metabolism* / Q000502:physiology
D004271:DNA, Fungal; D005656:Fungal Proteins; D006360:Heat-Shock Proteins