Limited Proteolysis in the Study of Membrane Proteins

A substantial proportion of glycoproteins of mammalian cells is found associated with the surface membrane. Several modes of association are known. For example, transmembrane or integral proteins are linked with the membrane by specific stretches of the protein consisting predominantly of hydrophobic amino acids that function as membrane anchors and span the membrane at least once. Some other proteins are covalently attached to a phospholipid in the membrane through an oligosaccharide (denoted glycophosphatidyl inositol-(GPI)-anchored proteins). Association of glycoproteins with the cell surface and even the types of these associations could be determined by treatment of intact cells or vesicular membranes with limited dilutions of proteolytic enzymes, such as trypsin. For example, the cell surface expression of recombinant pro-lactase-phlorizin hydrolase (pro-LPH) in transfected COS-1 cells (Nairn et al., 1991) or the hemagglutinin of influenza virus (HA) in infected CV-1 cells (Doyle et al., 1985; Naim and Roth, 1993) has been assessed by the susceptibilty of these proteins to limited dilutions of trypsin. The appearance of specifically cleaved molecular species of pro-LPH and HA is indicative for the presence of these molecules at the cell surface.