Purification of a HeLa cell high molecular weight actin binding protein and its identification in HeLa cell plasma membrane ghosts and intact Hela cells

Abstract

The high MW protein (HMWP) which was previously observed to be a major component of the actin-based gels formed by incubating cytoplasmic extracts of HeLa cells at 25.degree. C was now purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred .mu.g of HMWP, which is .apprx. 90% pure, can be isolated from 4 .times. 109 cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit MW is 250,000, its Stokes radius is 125 .ANG., and its sedimentation coefficient is 9 S. A native MW of 480,000 was calculated by using the latter 2 parameters, and therefore, the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. Apparently, HMWP is homologous to the latter proteins. HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high MW polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells; it did not react with rabbit myosin H chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed; however, the generalized fluorescence was only slightly reduced. Unpermeabilized cells were not stained. Evidently HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.