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Evidence that the 90-Kilodalton Heat Shock Protein is Associated with Tubulin-Containing Complexes in L Cell Cytosol and in Intact PtK Cells

Department of Pharmacology, The University of Michigan Medical School Ann Arbor, Michigan 48109
Department of Anatomy and Cell Biology, The University of Michigan Medical School Ann Arbor, Michigan 48109

Address requests for reprints to: William B. Pratt, Department of Pharmacology, Medical Science Building I, The University of Michigan Medical School, Ann Arbor, Michigan 48109.

Abstract

It has been established that the 90-kilodalton murine heat shock protein, hsp90, is associated with the untransformed, non-DNA-binding form of the glucocorticoid receptor in L cell cytosol. In this work, we show that incubation of L cell cytosol with Affi-Gelcoupled monoclonal antibodies directed against either -tubulin alone or both - and β-tubulin results in the immune-specific adsorption of hsp90 identified by Western blotting with the AC88 monoclonal antibody. Similarly, the AC88 antibody, which is specific for hsp90, causes the immune-specific isolation of both - and β-tubulin from hypotonic cytosol. The distribution of hsp90 in cultured Potorous tridactylis kidney cells was examined by indirect immunofluorescence using the AC88 monoclonal as primary antibody. In interphase cells, AC88-dependent fluorescence was distributed like antitubulin antibodydependent fluorescence in a fibrillar array located in the cytoplasm and around the periphery of the nucleus. In cells undergoing mitosis, AC88 fluorescence was located in the mitotic spindle. These observations suggest that a significant portion of hsp90 is associated with a tubulin-containing complex both in a hypotonic cytosol preparation from mouse fibroblasts and in intact marsupial kidney epithelial cells. The distribution of AC88 fluorescence in interphase Potorous tridactylis kidney cells is similar to the distribution of glucocorticoid receptor demonstrated by Wikstrom, A. C, Bakke, O., Okret, S., Bronnegard, M., and Gustafsson, J. A in rat hepatoma and human uterine cells.

FOOTNOTES

This investigation was supported by Grant CA-28010 from the NCI (to W.B.P.) and Grant GM-33980 from the NIH (to M.J.W.).

Received for publication March 15, 1988. Accepted for publication April 28, 1988.

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