Newly-synthesized membrane and secretory proteins are transported to the surface of epithelial cells, via the constitutive pathway, in a series of vesicles which move between sequential compartments. Some of the mechanisms by which vesicles bud from, and fuse with, membranes within the Golgi have recently been elucidated by studies using in vitro Golgi transport systems. These studies have shown that GTP is required for vesicle trafficking. Several monomeric GTP-binding proteins (G proteins) have now been found throughout the exocytic and endocytic pathways where they are believed to be involved in the regulation and targeting of vesicle trafficking. Different monomeric G proteins are found on distinct vesicle populations and membranes. The rab 6 protein, for instance is found on medial and trans-Golgi cisternae membranes. We have recently shown that a heterotrimeric G protein, Galphai-3, is also present on Golgi membranes. Our studies show that Galphai-3 regulates Golgi trafficking of a constitutively secreted heparin sulfate proteoglycan (HSPG) in LLC-PK1 renal epithelial cells. Overexpression of this subunit in transfected cells and uncoupling of alphai-3 with pertussis toxin-induced ADP- ribosylation were found to alter the rate of secretion and intra-Golgi transport of the HSPG. The goals of current proposal are to gain insight into the regulation of intra-Golgi vesicle trafficking by G proteins and to elucidate the functional and structural interactions of heterotrimeric G protein (alphai-3), a monomeric G protein (rab 6), and """"""""effector"""""""" proteins on the Golgi membranes. We will take advantage of our LLC-PK1 cell transfection model to examine the effects of Golgi trafficking when mutated Galphai-3 or chimeras of the Galphai-3/alphai-2 subunits are expressed on the Golgi. We will also investigate the roles of Galphai-3 and rab 6 in the Golgi trafficking of another group of vesicle passengers, MHC-I membrane proteins through the Golgi. Galphai-3 on the Golgi is likely to interact with """"""""effector"""""""" proteins. We will identify potential alphai-3- binding and forskolin-binding proteins on the Golgi which may be effector proteins in this regulation system. Studies on the function and interaction of the rab 6 protein will follow from the cloning of rab 6 in LLC-PK1 cells with the production of antibodies and transfected cells for the overexpression of rab 6. The proposed studies of Golgi trafficking in renal epithelial cells will bring together two major cell biological processes, vesicular transport and regulation by major families of signal transducers, G proteins. These studies will thereby contribute to our understanding of processes that are essential to all cells.
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