70-KDA HEAT-SHOCK COGNATE PROTEIN COLOCALIZES WITH KARYOPHILIC PROTEINS INTO THE NUCLEUS DURING THEIR TRANSPORT INVITRO
70-KDA HEAT-SHOCK COGNATE PROTEIN COLOCALIZES WITH KARYOPHILIC PROTEINS INTO THE NUCLEUS DURING THEIR TRANSPORT INVITRO
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DOI：
10.1006/excr.1993.1129
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发表时间：
1993-05-01
影响因子：
3.7
通讯作者：
YONEDA, Y
中科院分区：
文献类型：
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作者：
OKUNO, Y;IMAMOTO, N;YONEDA, Y
最近，我们发现抗70-kDa热休克同源蛋白（hsc 70）的抗体在体内抑制嗜核蛋白的核转运。在这项研究中，我们使用毛地黄皂苷透化的无细胞转运系统研究了hsc 70参与核转运的情况。通过与抗hsc 70抗体孵育来耗尽hsc 70的核运输所需的胞质提取物显著降低了核运输活性，并且将纯化的hsc 70添加到耗尽的提取物中恢复了运输活性。我们用间接免疫荧光法检测了hsc 70在体外核转运过程中的定位。当牛血清白蛋白（BSA）与SV 40大T抗原核定位信号（NLS）肽（T-BSA）或核质蛋白（nucleoplasmin）偶联时，Hsc 70在细胞核中积累，但当BSA与转运缺陷点突变的NLS肽偶联时，Hsc 70不在细胞核中积累。这种亲核蛋白依赖性积累的hsc 70依赖于胞质提取物，温度和ATP，并对麦胚凝集素敏感。加入过量的未标记的T-BSA的胞质提取物竞争性抑制荧光标记的T-BSA或核质蛋白的核积累，但不影响积累的hsc 70进入细胞核。这些结果表明，hsc 70是核转运所必需的，并且在亲核蛋白主动输入细胞核的过程中，hsc 70与亲核蛋白共定位。
Recently, we showed that antibodies against 70-kDa heat-shock cognate protein (hsc70) inhibit nuclear transport of karyophilic proteinsin 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 transportin vitroby 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 nucleusin vitro.