Insertion of biologically active membrane proteins from rat liver into the plasma membrane of mouse fibroblasts.

Phospholipids, glucolipids, and total proteins were separated from a plasma membrane fraction of rat liver. Membrane glycoproteins were isolated from deoxycholate extracts of rat liver membranes and hepatoma tissue culture membranes by concanavalin A chromatography. The membrane glycoprotein on hepatocytes that acts as a receptor for serum glycoproteins have lost their terminal sialic acid was also purified from rat liver membranes. Closed membrane vesicles were reconstituted from mixtures of deoxycholate-solubilized phospholipids and proteins by dialysis and purified by isopycnic centrifugation. The orientation of the proteins and glycoproteins in these reconstituted vesicles was examined by their accessibility to trypsin and neuraminidase and by their ability to be released from the vesicle by different concentrations of detergent. Most of the proteins are embedded in a right-side-out orientation in the lipid bilayer. The reconstituted membrane vesicles can be fused to mouse L-cells with polyethylene glycol. The extent of fusion is a function of the phospholipid:protein ratio in the reconstituted vesicles. After fusion, the phospholipid component of the vesicles mixes relatively rapidly with cell membrane lipids as judged by the immunofluorescence pattern of cells fused with lipid vesicles containing trinitrophenylated lipids. In contrast, proteins transferred to L-cells show restricted diffusion as judged again by immunofluorescence techniques. The metabolic turnover of proteins and glycoproteins after transfer to the plasma membranes of mouse L-cells was examined by radioisotopic methods. Total rat liver membrane proteins are very stable after transfer to the L-cells. Some of these proteins may be involved in the formation of an exoskeleton at the cell surface. Hepatoma tissue culture cell glycoproteins after transfer to the L-cells are less stable in terms of turnover properties than are total liver membrane proteins. However, some of these proteins are released into the medium as large molecular weight material rather than being degraded to small molecular weight, acid-soluble component. The receptor for serum asialoglycoproteins is relatively stable after transfer in reconstituted vesicles to the membrane of L-cells. Most of this hepatocyte-specific membrane glycoprotein is degraded to acid-soluble material with a half-life in the L-cell of at least 50 h. Transfer of the purified receptor in reconstituted vesicles to L-cells confers upon the recipient cell the biological activities specified and initiated by these receptors in hepatocytes.