RECONSTITUTION OF THE TRANSPORT OF PROTEIN BETWEEN SUCCESSIVE COMPARTMENTS OF THE GOLGI MEASURED BY THE COUPLED INCORPORATION OF N-ACETYLGLUCOSAMINE

Transport of the VSV[vesicular stomatitis virus]-encoded glycoprotein (G protein) between successive compartments of the Golgi was reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transproted during mixed incubations from the donor compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive acceptor compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay were extensively purified and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.