Abstract
Using the glycoprotein of the tsO45 mutant of vesicular stomatitis virus (VSV-G) as a marker, we have developed a system capable of measuring vesicular transport from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) in vitro. Movement from the ER to the cis Golgi compartment was assessed by the conversion of VSV-G from a totally endoglycosidase D (endo D)-resistant form to a species containing one endo D-resistant and one endo D-sensitive oligosaccharide (GD1). Similarly, delivery to the medial cisternae was measured by the appearance of the completely endo D-sensitive form of VSV-G (GD2) or by the acquisition of complete resistance to endoglycosidase H (endo H) (GHr) and delivery to the TGN by the appearance of an endo H-resistant form of VSV-G which was sensitive to digestion with neuraminidase and subsequently beta-galactosidase (GHt). Movement between each sequential compartment required ATP and soluble proteins (cytosol) and was inhibited by nonhydrolyzable analogues of GTP and by an antibody toward the N-ethylmaleimide-sensitive factor NSF. In contrast, fractionation of the cytosol by ammonium sulfate precipitation indicated that distinct proteins were required for movement between successive compartments. Similarly, inclusion of a mutant form of the small molecular weight GTP-binding protein rab1A inhibited movement between the ER and cis Golgi, and between the cis and medial cisternae, but did not affect transport from the medial Golgi to the TGN. Conversely, the protein kinase inhibitor staurosporine prevented movement between the medial Golgi and the TGN but did not influence transport between the ER and early Golgi compartments. This study provides the first demonstration that vesicular transport between the ER and TGN can be reconstituted in a cytosol-dependent fashion in vitro, allowing a direct analysis of the roles of individual components in multiple transport events.
Highlights
Using the glycoprotein of the ts045 mutant of vesic- transport remains to beelucidated
We further demonstrate that GTPyS-sensitive proteins are involved a t each step but show that a specific GTP- Terminal Glycosylation of vesicular stomatitis virus (VSV)-G in Semi-intact Normal ratkidney (NRK)
G to the cis Golgi compartment, we investigated the effects of the mutantprotein on transport as assessed by the acquisition of endoglycosidase D (endo D) sensitivity
Summary
Are involved a t each step but show that a specific GTP- Terminal Glycosylation of VSV-G in Semi-intact NRK binding protein, RablA, is only required for movement be- Cells-In mammalian cells most asparagine-linked oligosactween the ER andmedial Golgi. 30 "C under conditions shownpreviously to supportvesicular transport in semi-intact CHO cells (Davidson et al, 1992), supplemented with UTP tostabilize endogenous sugar nucleotides, produced an endo H-resistant form of VSV-G which migrated slower onSDS-PAGEthanthe undigested high mannose formof the glycoprotein (Fig. 1,trucks 1-4; compare buffer (Beckers et al, 1987). To those samples to be digested with tracks 1 and 3 (undigested) with tracks 2 and 4 (digested)). For digestion with endo D, cell pellets were resuspended in 50 pl of 0.1 M sodium citrate/sodium phosphate, p H 6.0, containing 0.2% Triton X-100, 5 mM EDTA,100 pg/ml leupeptin, 0.3 milliunit of endo D and 2 milliunits of @-N-acetylglucosaminidase,incubated a t 37 "Cfor 16 h, and terminatedby the additionof SDS-PAGE sample
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