Supported by this grant, we have developed a cell-free system in which the VSV G protein (initially present in Golgi-derived membranes from an infected mutant CHO cell) is glycosylated by an exogenous Golgi (derived from a wild-type uninfected cell). Because the mutant (CHO clone 15B) lacks the Golgi-associated glycosyltransferase (UDP-G1cNAc Transferase I) needed for this glycosylation, the incorporation of G1cNAc into G protein in this cell-free system results either from (vesicular) transport between the two Golgi-like membranes or from their direct but specific fusion. These events require ATP and at least two distinct factors present in the high speed supernatant. Based on several lines of evidence, our working hypothesis is that we may have reconstituted a segment of the intracellular transport pathway concerned with shuttling among Golgi elements. This assay represents a first step that may open the door for the first time to a biochemical analysis of the pathway of intracellular protein transport. We hope now to: 1. Resolve and purify the needed cytosol factors. 2. Study their mechanisms of action using classical enzymological approaches. 3. Evaluate the physiological significance of the in vitro system and its required factors by microinjecting anti-factor antibodies to examine their effects, and by demonstrating a similar inter-Golgi transport of G to in vivo following fusion of VSV-infected 15B cells with wild-type CHO cells. 4. Study the morphology of G protein processing in vitro by EM autoradiography and EM immunocytochemistry to help locate the donor and acceptor sites whose activity we study in vitro. And, in future years, we hope to work towards the identification, purification, and in situ localization of membrane-associated factors needed for transport in vitro.
