title: Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70.
abstract: Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone. We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70. In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions. GFP-Hsc70 is localized in both the cytoplasm and the nucleus at 37 degrees C and accumulated into the nucleolus/nucleus after heat shock, whereas GFP-Hsc54 always remained exclusively in the cytoplasm under these conditions. Mutation studies indicated that 20 amino acid residues of nuclear localization-related signals, which are missing in Hsc54 but are retained in Hsc70, are required for proper nuclear localization of Hsc70. We further found that Hsc54 contains a functional leucine-rich nuclear export signal (NES, (394)LDVTPLSL(401)) which is differently situated from the previously proposed NES in Saccharomyces cerevisiae Ssb1p. The cytoplasmic localization of Hsc54 was impaired by a mutation in NES as well as by a nuclear export inhibitor, leptomycin B, suggesting that Hsc54 is actively exported from the nucleus to the cytoplasm through a CRM1-dependent mechanism. In contrast, the nucleocytoplasmic localization of Hsc70 was not affected by the same mutation of NES or leptomycin B. These results suggest that the nuclear localization-related signal could functionally mask NES leading to prolonged retention of Hsc70 in the nucleus. An additional mechanism for unmasking the NES may regulate nucleocytoplasmic trafficking of Hsc70.
authors.affiliation: Department of Pharmacology, Tokyo Women's Medical University, School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. fuji@research.twmu.ac.jp
authors.forename: Fujiko
authors.initials: F
authors.lastname: Tsukahara
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Maru
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins; D009154:Mutation; D019913:Nuclear Localization Signals; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins