Abstract
The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.
Highlights
In order to explore transport of VSV-G protein from the ER through additionaGl olgi compartments, we examined the capacity of wild-type CHO semi-intacct ells to transporVt SV
In addition to CHOcells, we have found that awide range Temporally Identical to the Appearaonfctehe Endo D-sensitive of cell lines including COS, VERO, NRK, and BHKcells can (G,)Form-Reconstitution of processing of VSV-G protein be perforated toform semi-intact cells which efficientlytrans- to the Endoglycosidase D (endo D)- and endo H-resistant forms in vitro provides port VSV-G protein to Golgi compartments in a cytosol and the opportunity to compare, using the same preparation of ATP-dependent manner, conferring endo H resistai n cveitro semi-intact cells, the kinetics of appearance of the G, and in thepresence of UDP-GlcNAc
These results suggest that processing of VSVG protein to theGH2 form required a separate and temporally distinct fusion event from that required for the transport of VSV-G protein from the ER to the cis Golgi (Go and GH1 processing)
Summary
Cells duringintra-Golgitransportinsemi-intact cells requires trimmed man5form through the activiotyf a-1,2-mannosidase physiological concentrations of free Ca2+,a property which I (Mann I), a resident cis Golgi enzyme (Beckers et al, 1987, differsmarkedly fromthat observedusing enriched Golgi 1990;Beckers and Balch,1989;Plutner et al.,1990). Incubation Conditionsand Analysis of Transport-The ER to Golgi (endo H)-sensitive forms found in EthRe toendo H-resistant transport assays using semi-intact Chinese hamster ovary cells (CHO) infected with ts VSVwere performed as described previously (Beckers et al, 1987, 1990; Beckers and Balch, 1989; Plutner et al, 1990).For assays using wild-type VSV, cells were radiolabeled in vivo as described previously for ts except that the pulse of [35S]Met (200 pCi) was reduced to 3 min at 37 "C to generate a population of structures in early Golgi compartments (Balch and Keller, 1986). Endo D and H digestions were transportfromtheERthroughthe cis and medial Golgi compartments could not be successfully reconstituted using wild-type semi-intact cells.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
