Membrane assembly in vitro : Synthesis, glycosylatio, and asymmetric insertion of a transmembrane protein

Abstract

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 glycoprotein (G protein) of vesicular stomatitis virus (VSV) is well suited for such studies. The G protein is found in the plasma membrane of the infected cell, and after budding of the virus from the cell becomes the spike of the mature virion (4-7). Most of the G protein is located external to the viral (8)

Mechanisms for the incorporation of proteins in membranes and organelles.

“…1 e and f) is made apparent by the fact that, in in vitro reconstruction experiments with exogenous membranes, polysomes synthesizing the G protein of VSV can compete with polysomes synthesizing a secretory polypeptide and prevent its processing and vectorial discharge (124) . The essential difference between them is that the membrane protein remains associated with the ER membrane after cleavage of the cotranslational insertion signal and achieves a transmembrane disposition in the ER that is maintained as the polypeptide is transferred to the plasma membrane and is incorporated into viruses by the budding process (106,107).…”

“…These include the Ca"-ATPase of the sarcoplasmic reticulum (36,79), cytochrome P-450 (5,64,147), epoxide hydrolase (76), and NADPH cytochrome P-450 reductase (75,112,114,151), integral proteins of the ER that are present in both rough and smooth portions of this organelle . Integral membrane proteins of the plasma membrane that have been shown to be synthesized in bound polysomes include not only the well-studied envelope glycoproteins of vesicular stomatitis (VSV) (106,107), Sindbis, and Semliki Forest viruses (20,21,67,246) but also cellular plasma membrane proteins such as the hepatocyte 5'-nucleotidase (11), band 3, the major glycoprotein of the erythrocyte membrane (190), and the glycoprotein subunit of the Na+,K+-ATPase (201) .…”

The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

“…Most integral proteins of the membranes in the exo-and endocytotic pathway are asymmetrically integrated into the ER by signal-and stop-transfer sequence-mediated mechanisms (14,17). After integration into the ER membrane, sorting determinants specify retention within the ER or various downstream membranes of the exo-and endocytotic pathways (3).…”

Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation.

“…The two main families are those having oligosaccharide chains linked N-glycosidically from N-acetyl-D-glucosamine to the amide nitrogen of asparagine and those possessing oligosaccharide chains O-glycosidically linked from N-acetyl-D-galactosamine (GaINAc)' to the hydroxyl groups of serine and/or threonine (22,28,35,36,72). The biosynthesis of Nlinked glycoproteins is currently considered to be a single pathway involving the assembly ofa lipid linked oligosaccharide (1,41,42), the en bloc transfer of the oligosaccharide from the lipid carrier to the nascent peptide chain (31,32,55), the processing of the oligosaccharide chains by glycosidases (12,18,24,27,38,(63)(64)(65)(66) and the addition of terminal sugars by glycosyltransferases (5,44,57). The establishment of the carbohydrate to protein linkage in N-glycosylation seems to be a cotranslational process located in the rough endoplasmic reticulum and later steps of the assembly are thought to occur mainly in the Golgi apparatus (2,22,28).…”