Soltys B J, Gupta R S
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Exp Cell Res. 1996 Jan 10;222(1):16-27. doi: 10.1006/excr.1996.0003.
The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Chinese hamster ovary cells, human fibroblasts, B-SC-1 kidney cells, Daudi Burkitt's lymphoma cells, and rat liver, by immunogold electron microscopy employing six different monoclonal and polyclonal antibodies that are specific for Hsp60. In cryosections or LR Gold sections of different cultured cells, intense labeling of mitochondria was obtained, typically 200-500 gold particles per mitochondrion and accounting for 80-85% of the total gold particles. In addition, however, in all cell types and using all of the antibodies, about 15-20% of the labeling due to Hsp60 was seen at discrete extramitochondrial sites. Such sites included those in close proximity to mitochondrial outer membranes, foci on endoplasmic reticulum, on the cell surface, and in unidentified vesicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellular components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the labeling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation and Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodies show that under normal conditions no detectable precursor Hsp60 protein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precursor form of Hsp60 accumulates, providing evidence that at least partial mitochondrial import of Hsp60 is necessary for its maturation. These results also provide evidence that no other 60-kDa protein other than mitochondrial Hsp60 is recognized by the antibodies used for electron microscopy. These findings raise interesting questions concerning the possible role of Hsp60 at extramitochondrial sites.
利用六种不同的对60 kDa热休克蛋白（Hsp60）具有特异性的单克隆和多克隆抗体，通过免疫金电子显微镜技术，在多种哺乳动物细胞和组织中检测了Hsp60的亚细胞分布，这些细胞和组织包括中国仓鼠卵巢细胞、人成纤维细胞、B-SC-1肾细胞、Daudi伯基特淋巴瘤细胞以及大鼠肝脏。在不同培养细胞的冰冻切片或LR Gold切片中，线粒体呈现强烈标记，每个线粒体通常有200 - 500个金颗粒，占总金颗粒的80 - 85%。然而，除此之外，在所有细胞类型中，使用所有抗体时，约15 - 20%由Hsp60引起的标记出现在离散的线粒体外位点。这些位点包括紧邻线粒体外膜的位点、内质网上的病灶、细胞表面以及不明囊泡中的位点。在大鼠肝脏的冰冻切片中，过氧化物酶体内也观察到了Hsp60抗体引起的特异性标记。用纯化的重组线粒体Hsp60进行预吸附可阻止这些抗体对所有细胞成分的标记，表明该标记对Hsp60具有特异性。通过免疫沉淀和蛋白质印迹分析，细胞表面蛋白的生物素标记导致Hsp60的生物素化，这为Hsp60存在于质膜上提供了进一步证据。用Hsp60抗体进行的免疫沉淀实验表明，在正常条件下细胞中不存在可检测到的前体Hsp60蛋白。然而，在用阻断线粒体导入的钾离子载体无活菌素处理的细胞中，只有Hsp60的前体形式积累，这证明Hsp60至少部分的线粒体导入对其成熟是必要的。这些结果还证明，用于电子显微镜的抗体不会识别除线粒体Hsp60以外的其他60 kDa蛋白。这些发现引发了关于Hsp60在线粒体外位点可能作用的有趣问题。
