Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.

Abstract

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by onedimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.For several years, work in our laboratory has been concerned with the characterization of an abundant cytoplasmic protein migrating with an apparent Mr of 85,000 (85K protein) in sodium dodecyl sulfate (SDS)-polyacrylamide gels. We described the purification and preliminary characterization of the denatured protein (22) and established that its synthesis and turnover are regulated when L929 cells are maintained in the absence of glucose (14,21). Based on mobility in SDS-polyacrylamide gels and one-dimensional partial proteolytic peptide maps, we concluded that this protein is identical with one of the pp60src-associated proteins (23). Subsequently, we showed that synthesis of this protein is induced, along with several others, by exposure to elevated temperatures (15,21,24), i.e., that it is a heat shock protein (hsp85).The heat shock proteins are synthesized by a wide variety of cells after exposure to elevated temperatures. Although it has been suggested that these highly conserved proteins may participate in opposing the lethal (12,22,26,35) or metabolic (10, 21) effects of hyperthermia, their function is still essentially unknown. Since certain functions might be excluded or suggested by a unique intracellular location, e...

The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

“…The number of known Hsp90 substrates is continuing to grow very rapidly (for an updated list, see http://www.picard.ch/downloads/Hsp90interactors.pdf) as could be expected for a molecular chaperone that may be the most abundant cytosolic protein even in unstressed cells [24]. It is still difficult to provide a comprehensive list of the substrates that account for the essential functions of Hsp90 for viability of both unicellular and metazoan organisms.…”

Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

“…A rabbit polyclonal antibody to hsp4o was prepared using recombinant human hsp4o as immunogen (Nagata et al, manuscript in preparation). A rabbit polyclonal antibody to hsp9o was used for immunoprecipitation of nondenatured samples (W.J.W., unpublished observations) and was supplemented with the rat monoclonal antibodies 16F1 and 9D2 (obtained from Stressgen; Lai et al, 1984) for the analysis of denatured samples.…”

Low concentration of GA activates a preconditioning response in HepG2 cells during oxidative stress—roles of Hsp90 and vimentin

“…Heat shock responses include a transient blockage of protein synthesis, overexpression of Hsps, and induction of thermotolerance. Hsp90 is the most abundant soluble cytosolic protein (∼1% of total protein, 10-150 μM) even before heat shock (Lai et al 1984;Nollen and Morimoto 2002), and it is poorly induced. Hsp90 is a dimer and binds to several cellular proteins including steroid receptors and protein kinases (Jakob and Buchner 1994;Kimmins and MacRae 2000;Soti et al 1998).…”