IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THE 60-KDA HEAT-SHOCK CHAPERONIN PROTEIN (HSP60) IN MAMMALIAN-CELLS

The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Ch inese hamster ovary cells, human fibroblasts, B-SC-I kidney cells, Dau di Burkitt's lymphoma cells, and rat liver, by immunogold electron mic roscopy employing six different monoclonal and polyclonal antibodies t hat are specific for Hsp60. In cryosections or LR Gold sections of dif ferent 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 du e to Hsp60 was seen at discrete extramitochondrial sites. Such sites i ncluded those in close proximity to mitochondrial outer membranes, foc i on endoplasmic reticulum, on the cell surface, and in unidentified v esicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellu lar components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the lab eling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation a nd Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodie s show that under normal conditions no detectable precursor Hsp60 prot ein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precur sor form of Hsp60 accumulates, providing evidence that at least partia l 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. (C) 1996 Academic Press, Inc.