Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

Rothman, James E.; Fries, Erik

doi:10.1083/jcb.89.1.162

Cited by 69 publications

(44 citation statements)

References 21 publications

(9 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Membranes from VSV-infected CHO clone 15B cells [the cell line employed as donor of G protein in vitro (11)(12)(13)] were centrifuged to equilibrium in a sucrose density gradient, and the distributions of enzymes believed to catalyze steps in the two stages of Golgi-associated oligosaccharide processing were determined ( Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Four confluent 10-cm plates of VSV-infected L cell clone 6 were pulse-labeled for 30 min with 2 ml per plate ofJoklik's minimal essential medium (with 10% normal glucose level) containing nonessential amino acids (GIBCO) and [2-3H] (24), modified as described (13). Sialyltransferase assays, based on a described procedure (19), contained (in 20 6 glycopeptide during a 90-min incubation at 37°C, essentially as described by Tabas and Komfeld (14).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Dunphy¹,

Fries²,

Urbani³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

198

View full text Add to dashboard Cite

Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, a-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl-and sialyltransferases) that act in the later, terminal stage ofglycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack. The stack of cisternae composing the Golgi apparatus is markedly asymmetric (1-7), its cis [or entry (8, 9)] and trans [or exit (9, 10)] faces differing in morphological and histochemical properties. This cis-trans polarity could signify a fundamental division of the stack into functionally distinct compartments between which proteins can be transported vectorially. Alternatively, this overall asymmetry could reflect a continuous gradient ofcomposition, the result ofa sorting process operating within a stack composed of multiple copies of the same kind of compartment.We recently reported (11) a dramatic change in the behavior in a cell-free assay system (12, 13) of a plasma membrane-type glycoprotein [the G protein encoded by vesicular stomatitis virus (VSV)] that occurred as a result of passage through the Golgi. Specifically, the assay distinguished two intracellular pools through which G protein passed in rapid succession: an earlier "transferable" pool that can be donated by membranes from infected cells to exogenous Golgi for oligosaccharide processing in vitro and a later "nontransferable" pool that cannot. The transferable pool appeared to reside in Golgi membranes (11). If the other pool also resided in Golgi membranes, but in a different region, then the change in properties of G protein (transferable-versus nontransferable) in going from one region ofthe Golgi to the other would signify a compartment boundary in between.Asparagine-linked oligosaccharides (such as those ofthe VSV G protein) are processed in two major stages (14-16). First, the precursor oligosaccharide is "trimmed" by the removal of four a-1,2-linked mannose units. Later, terminal glycosyl...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Dunphy¹,

Fries²,

Urbani³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

198

View full text Add to dashboard Cite

show abstract

“…We have described a cell-free system in which a transport-coupled glycosylation is used to monitor movement of a glycoprotein between two Golgi membrane populations (1,4,6,7,22). One population ofGolgi elements (from a mutant cell) contains the glycoprotein to be transported but lacks the needed glycosyltransferase .…”

mentioning

confidence: 99%

Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

Rothman¹,

Urbani²,

Brands³

1984

The Journal of Cell Biology

Self Cite

View full text Add to dashboard Cite

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed . Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with ["S]methionine) or Golgi complex (labeled with [3 H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion ; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis à vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J . E . Rothman . 1981 . J. Cel l Biol., 90: 697-704) . Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex . The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein -5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays . Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements . The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system .

show abstract

“…10 gradient fractions (1.3 ml each) were collected. Portions of each fraction were assayed for the following marker enzymes: fl-N-acetylhexosaminidase (43), galactosyltransferase (44), and ouabain-sensitive Na+K'ATPase (42). A 400-id portion of each gradient fraction was centrifuged at 100,000 g to remove Percoll and concentrate the membranes; the resultant membrane pellets were subjected to Western blotting using antibody against rab5b.…”

Section: Methodsmentioning

confidence: 99%

Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

Wilson¹,

Wilson²

1992

J. Clin. Invest.

View full text Add to dashboard Cite

Members of the mammalian rab family of GTPases are associated with specific subcellular compartments, where these proteins are postulated to function in vesicular transport. By screening a human umbilical vein endothelial cell library with degenerate oligonucleotide probes, we have isolated a 1.6-kb cDNA clone encoding a 215-amino-acid protein belonging to the rab family of GTPases. This newly identified rab protein is 81% identical to human rab5, the canine counterpart of which has been localized to the plasma membrane and early endosomes. In light of this homology, we have named this new member of the GTPase superfamily "rab5b." Northern analysis using the rab5b cDNA as a probe revealed a 3.6-kb mRNA in a variety of cell types, including human umbilical vein endothelial cells, K562 erythroleukemia cells, U937 monoblastic cells, and HeLa cells. A fusion protein between glutathione-Stransferase (GST) and rab5b was expressed in bacteria and purified to homogeneity. The recombinant protein was shown to bind GTP and GDP. As is typical of other recombinant rab proteins, the rab5b-GST fusion protein displayed a low intrinsic rate of GTP hydrolysis (0.005/min). An antiserum to rab5b was prepared and used to determine the apparent molecular size and subcellular distribution of the protein. Western blotting with this antibody revealed a 25-kD protein in COS cells transfected with rab5b and in nontransfected HeLa cells. Indirect immunofluorescence and subcellular fractionation showed that rab5b localizes to the plasma membrane. We speculate that rab5b plays a role in vesicular trafficking at the plasma membrane in various cell types. (J. Clin. Invest. 1992Invest. .89:996-1005

show abstract

Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

Cited by 69 publications

References 21 publications

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

Contact Info

Product

Resources

About