70-kDa heat-shock cognate protein colocalizes with karyophilic proteins into the nucleus during their transport in vitro.
Recently, we showed that antibodies against 70-kDa heat-shock cognate 66otein (hsc70) inhibit nuclear transport of karyophilic proteins in vivo. In this study, we examined the involvement of hsc70 in nuclear transport using a digitonin-permeabilized cell-free transport system. Depletion of the cytosolic extract required for nuclear transport of hsc70 by incubation with anti-hsc70 antibodies reduced the nuclear transport activity significantly, and addition of purified hsc70 to the depleted extract restored the transport activity. We examined the localization of hsc70 during nuclear transport in vitro by indirect immunofluorescence studies. Hsc70 accumulated in the nucleus when bovine serum albumin (BSA) conjugated to SV40 large T-antigen nuclear localization signal (NLS) peptides (T-BSA) or nucleoplasmin was added exogenously to the cytosolic extract, but not when BSA conjugated to transport-incompetent point-mutated NLS peptide was added. This karyophilic protein-dependent accumulation of hsc70 was dependent on the cytosolic extract, temperature, and ATP and was sensitive to wheat germ agglutinin. Addition of excess unlabeled T-BSA to the cytosolic extract competitively inhibited the nuclear accumulation of fluorescently labeled T-BSA or nucleoplasmin, but did not affect accumulation of hsc70 into the nucleus. These results show that hsc70 is required for nuclear transport and that it is colocalized with karyophilic proteins during their active import into the nucleus in vitro.
Okuno
Y
Y
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Imamoto
N
N
Yoneda
Y
Y
D000255:Adenosine Triphosphate / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D004173:Dipodomys; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D056489:Nucleoplasmins; D010750:Phosphoproteins; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013696:Temperature
D006360:Heat-Shock Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D012710:Serum Albumin, Bovine; D000255:Adenosine Triphosphate