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 (G(H1)) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (G(D)), 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-acetylglucosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (G(H2)) contains a second wave of vesicular transport. Processing of G(H1) to G(H2) in vitro occurs only after a lag period following the appearance of G(H1); 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 G(H2) 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.