Asymmetric distribution of plasma membrane proteins in mouse L-929 cells

Abstract

The distribution of plasma membrane-associated proteins was studied by using latex-filled phagolysosomes prepared from cultured mouse L-929 cells as a model of "inside-out" membrane. Proteins from 131I/lactoperoxidase-labeled phagolysosomes, phagolysosomes derived from 131I/lactoperoxidase-labeled cells, and phagolysosomes prepared from [35S]methionine metabolically labeled cells were analyzed by high-resolution two-dimensional gel electrophoresis. The gel patterns of iodinated proteins showed specific differences in the availability of membrane proteins to lactoperoxidase labeling between inside-out and right-side-out membranes. However, at least two prominent [35S]methionine-labeled proteins of approximately 60,000 and 100,000 daltons were available for iodination at both sides of the membrane. Partial proteolysis of the 100,000-dalton protein revealed that different peptides were iodinated when the iodination was performed on intact cells or on phagolysosomes, consisent with the idea that this protein spans the plasma membrane.