Lipids with Photosensitive Groups as Chemical Probes for the Structural Analysis of Biological Membranes. On the Localization of the G- and M-Protein of Vesicular Stomatitis Virus

Abstract

16-Azido-[9,10-3H2]palmitic acid was used as a chemical probe in the elucidation of the topography of proteins of the rhabdovirus VSV (vesicular stomatitis virus) interacting in the lipid bilayer of the envelope. VSV was grown in BHK-21 cells (baby hamster kidney) in monolayer cultures, the complex lipids of which were prelabelled with 16-azido-[9,10-3H2]palmitic acid. Virions were purified by gradient centrifugation and the nitrene generated by UV irradiation from the azido lipids in the virus membrane. The viral proteins were separated into 4 main bands corresponding to the L- (polymerase), G- (glycoprotein), N- (nucleocapsid) and M-protein (membrane protein). After UV irradiation, cross-linking of the nitrene group attached to the .omega.-methylene group of [3H]palmitic acid in the envelope lipids occurred only with the G-protein but not with the M-protein. The G-protein band was highly radioactive, but its staining with Coomassie Brilliant Blue decreased in favor of new radioactive bands appearing between stacking and separation gel, due to the crosslinks of radioactive lipids with glycoprotein molecules. The same experiment was carried out with radioinactive 16-azidopalmitic acid, but with 3H-labeled glucosamine to label the carbohydrate groups of the G-protein. The same radioactive high MW bands occurred, now radioactive due to the carbohydrate label. The localization of the reactive azido group in the center of the lipid bilayer of the virus envelope shows that the G-protein is anchored at least at a depth reaching this area of the bilayer. The role of photosensitive azido groups substituting the alkyl chain of fatty acids of complex lipids in different positions as chemical probes and rulers in topographic studies of membrane components is discussed.