The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum of UT-1 cells (original) (raw)
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Journal of Cell Biology, 1992
We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the G...
Cell, 1983
We have altered the structure of the COOH-terminus of the vesicular stomatitis virus (VSV) glycoprotein (G) by introducing deletions into a cDNA clone encoding G protein. We examined the effects of these deletions on intracellular transport of G protein after expression of the deleted genes in eucaryotic cells under control of the SV40 late promoter. To prevent readthrough of translation into vector sequences, we introduced synthetic DNA linkers containing translation stop codons at the site of the deletion. G proteins that lacked the cytoplasmic domain and most of the transmembrane domain were secreted slowly from the cells. Deletion mutants affecting the structure of the cytoplasmic domain fell into two classes. The first class completely arrested transport of the protein to the cell surface at a stage prior to acquisition of complex oligosaccharides. The second class showed severely reduced rates of complex sugar addition although the proteins were eventually transported to the cell surface. Indirect immunofluorescence microscopy suggested that mutant proteins in both classes may accumulate in the rough endoplasmic reticulum.
Transport of vesicular stomatitis virus glycoprotein in a cell-free extract
Proceedings of the National Academy of Sciences, 1980
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-GlcNAc and resulting in resistance to endo-beta-N-acetylglucosaminidase H).
Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes
The Journal of cell biology, 1981
In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30%...
The Journal of cell …, 1988
With the availability of a reliable assay to quantitatively determine the oligomeric state of VSV G protein (Doms et al., 1987), it is now possible to address the relationship between folding, oligomerization, and transport in more detail. In this study we have examined 12 VSV G proteins with mutations in the ectodomain, transmembrane domain, or cytoplasmic domain. The mutant proteins display a variety of transport phenotypes, ranging from complete block in the ER to near normal transport to the cell surface. Three general observations emerged. (a) Mutations in the cytoplasmic domain did not generally affect folding of the ectodomain or subsequent trimerization, but they did slow down or prevent exit from the ER. (b) Mutations in the ectodomain blocked initial folding and therefore trimerization and transport. These mutant proteins accumulated in the ER in the form of aggregates. (c) Although rather tolerant to changes in the transmembrane domain, the G protein's folding and trimerization was affected by a drastic shortening of the transmembrane sequence. Materials and Methods Cell Lines and Viruses COS-i cells were maintained in DME supplemented with 5% FCS as described (Rose and Bergmann, 1982). The Chinese hamster ovary (CHO) cell line clone 15B, which lacks the Golgi-associated carbohydrate-pro
Journal of virology, 1977
Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model ...
Experimental Cell Research, 1996
cretory cell lines show that clusters of small vesicles, Vero cells were infected with the ts-045 strain of ve-short tubules, and pleomorphic structures, designated sicular stomatitis virus, and the cells were incubated IC 2 or ERGIC [7-9; the term IC will be used throughat 39ЊC to accumulate the mutant G glycoprotein in out this paper], are involved in the anterograde export the ER as a misfolded aggregate. Cycloheximide was pathway (ER to Golgi complex). However, it is still deadded to the culture medium 3.5 h after infection to bated whether these structures represent a new comprevent further protein synthesis, and the temperapartment or whether they are a subregion of either the ture was lowered to 10, 15, or 31ЊC. At these tempera-ER or the Golgi complex. The IC could be related to the tures, the mutant G glycoprotein correctly folds and classical transitional elements described in specialized oligomerizes. Immunofluorescence light microscopy secretory cells [2, 10]; however, there is evidence that showed that the G glycoprotein was exported to the the IC is part of the forming, cis-face of the Golgi com-Golgi complex at 31ЊC and to the intermediate complex, hence the term cis-Golgi network [11]. On the partment (IC) at 15ЊC, but no export was observed at other hand, several studies suggest that IC structures 10ЊC. However, incubations at 10ЊC followed by shift are connected by convoluted thin tubules to the ER, to 15 or 31ЊC resulted in the normal transfer of the and represent the exit sites where transport vesicles glycoprotein to the IC and the Golgi, respectively. Imdestined for the Golgi complex are formed 12]. Other munoelectron microscopical analysis confirmed all data indicate that the IC is not in physical contact with these results, but showed also that the glycoprotein either the ER and or the Golgi complex and is present was frequently clustered in the ER at 10ЊC. Conveneven very far from the Golgi stacks 13, 14]. Results tional electron microscopy showed that the morpholobtained with a cell-free system support the existence ogy of the ER, IC, and Golgi complex remained essenof an intermediate step in the anterograde transport tially unchanged at all temperatures. The only signifi-[15, 16]. No enzymatic activities have yet been localized cant difference detectable in cells incubated at 10ЊC
Molecular biology of the cell, 2000
The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition...
Biochemistry, 1981
The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in the time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group 111, which contain lesions in the M protein. At the permissive temperature, a pronounced