Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport.
Frozen, thin sections of baby hamster kidney (BHK) cells were incubated with either concanavalin A (Con A) or Ricinus communis agglutinin I (RCA) to localize specific oligosaccharide moieties in endoplasmic reticulum (ER) and Golgi membranes. These lectins were then visualized using an anti-lectin antibody followed by protein A conjugated to colloidal gold. All Golgi cisternae and all ER membranes were uniformly labeled by Con A. In contrast, RCA gave a uniform labeling of only half to three-quarters of those cisternae on the trans side of the Golgi stack; one or two cis Golgi cisternae and all ER membranes were essentially unlabeled. This pattern of lectin labeling was not affected by infection of the cells with Semliki Forest virus (SFV). Infected cells transport only viral spike glycoproteins from their site of synthesis in the ER to the cell surface via the stacks of Golgi cisternae where many of the simple oligosaccharids on the spike proteins are converted to complex ones (Green, J., G. Griffiths, D. Louvard, P. Quinn, and G. Warren. 1981. J. Mol. Biol. 152:663-698). It is these complex oligosaccharides that were shown, by immunoblotting experiments, to be specifically recognized by RCA. Loss of spike proteins from Golgi cisternae after cycloheximide treatment (Green et al.) was accompanied by a 50% decrease in the level of RCA binding. Hence, about half of the RCA bound to Golgi membranes in thin sections was bound to spike proteins bearing complex oligosaccharides and these were restricted to the trans part of the Golgi stack. Our results strongly suggest that complex oligosaccharides are constructed in trans Golgi cisternae and that the overall movement of spike proteins is from the cis to the trans side of the Golgi stack.
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003208:Concanavalin A; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D037102:Lectins; D009844:Oligosaccharides / Q000032:analysis; D037121:Plant Lectins; D011499:Protein Processing, Post-Translational; D012672:Semliki forest virus; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism*
D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D014764:Viral Proteins; D003208:Concanavalin A; D003513:Cycloheximide; D005690:Galactose