The Golgi complex consists of a series of subcompartments that constitute the hub for intracellular membrane traffic. It serves as the main sorting station where biosynthetic products and recycling membrane traffic are sorted for delivery to multiple destinations. Little is known at present about how its compartmentalization is maintained, how this traffic is controlled and what signals and signal transduction processes are involved. Recently attention has focused on the role of several families of GTP-binding proteins including members of the ras supergene family (rab ARF) as well as heterotrimeric GTP binding proteins in these processes. The overall goal of Project 1 is to understand the role of GTP-binding proteins in maintaining Golgi organization and function. Three systems have been developed for use in these studies: an exocrine pancreatic lobule system, a permeabilized murine erythroleukemia cell (MEL) cell system, and (in collaboration with J. Feramisco) the REF 52 cell studied in vivo by microinjection. Problems we intend to pursue include: 1) To determine the distribution of ARF, beta-COP and the alpha subunit of Gi3 in BFA-treated and ATP-depleted pancreatic acinar cells (PAC). This will be done by immunocytochemistry and by biochemical assay of Golgi fractions. The goals of these experiments are a) to further investigate the effects of these perturbants on the distribution of proteins which are assumed to be required for maintenance of Golgi organization and trafficking, and b) to further characterize what appear to be different pools of beta-COP. 2) To purify and characterize Golgi remnants from BFA-treated pancreas and generate antibodies to proteins of interest. 3. To utilize the permeabilized MEL cell system we have developed to investigate the sites of synthesis of O-linked oligosaccharides. The purpose of these experiments is to attempt to identify the specific compartment among preGolgi and Golgi subcompartments where O- glycosylation is initiated (by addition of GalNAc to ser/thr residues) and the sites where subsequent sugars (gal, sialic acid) are added. 4. To explore the role of heterotrimeric G proteins in Golgi assembly/disassembly and Golgi functions. The approaches to be used include microinjection of antibodies and anti-sense RNA for Gialpha3 and other Ga subunits into living cells (REF53 or CHO cells). Particular attention will be paid to assessing the effect of Gialpha3 on ER to Golgi and intra-Golgi transport. To assess their role on the Golgi microenvironment, we will determine if Gialpha3 or other Ga subunits control the uptake of nucleotide sugars and/or cations into isolated Golgi vesicles.