Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

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Abstract
A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts tranformed with avian sarcoma viruses. Cellular fractionation and immunofluorescence was used to locate the 36 kilodalton (kd) protein in virus-transformed and uninfected chicken fibroblasts. The 36 kd protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36 kd protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36 kd protein that otherwise was pelletable with high g forces. Evidently, this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36 kd protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36 kg staining. The 36 kd protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kd antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36 kd protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.