Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein.
Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes in vitro require protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.
D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000502:physiology; D006023:Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008563:Membrane Lipids; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008566:Membranes / Q000502:physiology* / Q000648:ultrastructure; D008861:Microsomes / Q000502:physiology* / Q000648:ultrastructure; D010179:Pancreas / Q000201:enzymology; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D014908:Triticum / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus
D006023:Glycoproteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D012333:RNA, Messenger