Identification of a high molecular weight trans-membrane protein in mouse L cells.
We have developed a new method for identifying proteins which span the plasma membrane ("trans-membrane" proteins) of mammalian cells grown in tissue culture. The method involves labeling proteins exposed on the cell surface with 125I by the lactoperoxidase technique and then preparing sealed, "inside-out" membrane vesicles (phagosomes) from the labeled cells using the polystyrene latex bead procedure. These inside-out vesicles are then treated briefly with trypsin and analyzed by SDS-polyacrylamide gel electrophoresis for the presence of 125I-labeled protein species which were degraded by proteolytic attack. Such proteins must be exposed on both the outer and inner membrane surfaces and, therefore, they must pass through the lipid barrier. This method is a general one in the sense that it is suitable for use with a wide variety of cell types, and here we show how it has been employed to prove that a particular high molecular weight polypeptide, called band 1, spans the plasma membrane of mouse L cells. Further studies of the band 1 polypeptide have demonstrated that it is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of band 1 to iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of cell proliferation.