Transport of vesicular stomatitis virus glycoprotein in a cell-free extract.

Abstract

We describe a cell-free system in which the membrane glycoprotein of vesicular stomatitis virus is rapidly and efficiently transported to membranes of the Golgi complex by a process resembling intracellular protein transport. Transport in vitro is energy dependent and is accompanied by terminal glycosylation of the membrane glycoprotein (dependent upon UDP-lcNAc and resulting in resistance to endo-f-Nacetylglucosaminidase H). The elucidation of the mechanisms of assembly of cellular membranes and of the organelles that these membranes define poses a major challenge to cell biologists. In particular, it will be important to understand how distinct sets of proteins are delivered to the different subcellular organelles so as to confer upon them their distinct functions. How is this highly specific intracellular transport and sorting of proteins accomplished?An essential step towards obtaining a molecular description of these complex cellular events will be to achieve conditions under which the same transport of nascent proteins can proceed in cell-free extracts, for only then can the tools of biochemistry be fruitfully applied. We describe here experiments demonstrating that it is possible to obtain transfer of protein between specific organelles in a cell-free extract by a reaction resembling that of intracellular protein transport. For these studies we have utilized Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) as a well-defined system in which to investigate the transport of a membrane protein destined for the plasma membrane (1).The viral glycoprotein (G protein) that will reside in the membrane of the mature virion is the only glycoprotein synthesized in VSV-infected cells. Like cellular surface glycoproteins, G is synthesized in the endoplasmic reticulum (ER), is transported to the Golgi complex,* and is then transported to the plasma membrane (1). Clathrin-coated vesicles appear to mediate both of these transport steps (6). The small genome of VSV (encoding only five proteins, all found in virions) provides assurance that the maturation of G follows host-specific pathways.During its synthesis in the ER, G acquires two mannose-rich oligosaccharides that are subsequently processed as G passes through the Golgi complex (2,3,7). This change in oligosaccharide structure provides an indirect means for assaying the arrival of G at the Golgi complex. Endo-H has proved useful in this regard, because this enzyme will cleave the mannose-rich precursor oligosaccharides of G, causing a marked reduction of apparent molecular weight, but will not attack the Golgiprocessed oligosaccharides (7).The general approach we have taken to obtain transport of

Glycosylation of measles virus haemagglutinin protein in infected cells

“…A similar shift in the electrophoretic mobility of the H protein has been observed by several investigators (Graves, 1981 ;Bellini et al, 1983;Young et al, 1985;Kohama et al, 1985). We examined the effect of CCCP, an inhibitor of oxidative phosphorylation which blocks the transport of secretory or membrane proteins from the RER to the Golgi apparatus (Fries & Rothman, 1980) and monensin, a sodium ionophore which is known to disrupt the functioning of the transGolgi (Tartakoff, 1983), on the glycosylation of the H protein. In the presence of CCCP the intensity of the 74K H protein band increased with chase time (Fig.…”

Golgi Glycosylation

“…This approach was pioneered by James Rothman using the CHO mutant 15B (or Lec1) in mammalian cells (Fries and Rothman 1980). These mutants lack GlcNAcT-I activity in the medial Golgi, and thus by mixing Golgi membranes from mutant with membranes from wild type in vitro, glycoprotein transfer between them could be determined by the acquisition of the product of GlcNAcT-I activity only present in wild type Golgi membranes.…”

Les gènes et les protéines qui contrôlent la voie de sécrétion

“…Pourtant, quelques miles plus loin dans son nouveau labo de Stanford, Jim Rothman avait réussià obtenir une réaction qui semblait mesurer une portion significative du trafic golgien reconstitué dans un lysat de cellules de mammifères (Fries & Rothman, 1980). Mes propres efforts restaient en attente jusqu'à ce que je trouve unétudiant courageux pour endosser le défi.…”

“…Si l'accumulation des substrats dans le RE n'était pas bloquée, le test du trafic devrait reposer sur un niveau faible de glycoprotéines immatures, radiomarquées pendant un temps bref lors de la biosynthèse. Rothman avait réussi avec exactement cette stratégie (Fries & Rothman, 1980), mais le temps de transit des glycoprotéines chez la levure est beaucoup plus rapide que dans les cellules de mammifères.…”