A Novel Very High Molecular Mass Protein Located in the Membrane Skeleton

Abstract

A monoclonal antibody generated from a mouse immunized with L6 rat myoblast cells was found to react with a major 700-kDa band and a minor 500-kDa band in immunoblots. Immunofluorescence microscopy demonstrated a submembranous location in tissue sections and an exclusion from stress fiber regions in cultured cells. Further, permeabilization of cultured cells with nonionic detergent prior to fixation changed the diffuse pattern of fluorescence to a web. These findings are characteristic of membrane skeletal proteins. In muscle tissue, the protein was much more abundant in fast twitch fibers and was found in internal locations as well as at the membrane. The protein could be solubilized in the absence of detergents and, hence, is not transmembrane. Although initially discovered in myoblast cells, the protein is present in a variety of tissue types, including brain, kidney, heart, liver, and lung. Pulse-chase labeling of the two bands suggested that the 500-kDa band was not a breakdown product of the 700-kDa protein. The protein appears to be a previously undiscovered membrane skeletal constituent for which the name "endossin" is proposed.