BASOLATERAL MEMBRANE-ASSOCIATED 27-KDA HEAT-SHOCK-PROTEIN AND MICROFILAMENT POLYMERIZATION

The in vivo activity of the 27-kDa heat shock protein, a barbed-end mi crofilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells exp ressing the human wild type or a mutant nonphosphorylatable 27-kDa hea t shock protein were subjected to subcellular fractionation and immuno blot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane component s, indicating that phosphorylation is not required for membrane associ ation. Phorbol ester treatment resulted in phosphorylation of only mem brane-associated 25-kDa and wild type 27-kDa heat shock protein and di d not induce redistribution. In a second fractionation protocol, strep tavidin-agarose precipitation of extracts prepared from cells biotinyl ated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stim ulation of transfectants expressing the wild type 27-kDa heat shock pr otein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein d ecreased the amount of F-actin precipitated. These data suggest that m embrane-associated 25- and 27-kDa heat shock proteins inhibit the gene ration of basolateral microfilaments and that phosphorylation releases this inhibition.