Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.
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.
Schwaninger
R
R
Department of Cell, Biology Scripps Research Institute, La Jolla, California 92037.
Beckers
C J
CJ
Balch
W E
WE
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008562:Membrane Glycoproteins; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D005033:Ethylmaleimide; D002118:Calcium