Rab proteins are small GTPases which function as molecular switches. The GTPase cycle of Rab proteins plays a critical role in the sequential events dictating the formation, targeting and fusion of carrier vesicles mediating protein transport through the constitutive and regulated secretory pathways of eukaryotic cells. Rab1 is critical for the transport of protein between the endoplasmic reticulum (ER) and the cis Golgi compartment, and between cis and medial Golgi compartments. We have identified and partially characterized proteins which interact with Rab1 at different steps in its GTPase cycle. These effectors include: (1) guanine nucleotide dissociation inhibitor (RabGDI) which serves as a cytosolic escort protein, (2) guanine nucleotide exchange protein (Rab1- GEF) which promotes GTP exchange during Rab1 during vesicle formation, and (3) guanine nucleotide activating protein (Rab1-GAP) which promotes GTP hydrolysis, an event likely to be essential for vesicle targeting and fusion. In addition, we have characterized a putative vesicle targeting receptor (syntaxin 5) which links Rab1 function to a late Ca2+-dependent step involved in the docking and fusion of ER-derived carrier vesicles. Highly enriched, ER-derived carrier vesicles have now been generated in vitro and will provide new insight into the function, distribution and composition of Rab1 and its effector proteins. Through the biochemical analysis of specific protein interactions controlled by the Rab1 GDP/GTP switch we will elucidate the functional role of each of these components in the recruitment of vesicle coats during budding from the ER and the assembly of a docking/fusion complex at the target (cis Golgi) acceptor membrane. Given the ubiquitous role of Rab proteins in membrane traffic through the exocytic and endocytic pathways, we anticipate that these studies will provide general insight into the function of the Rab GTPase molecular switch in membrane interactions. Our studies will have important implications for understanding a wide range metabolic disorders affecting the constitutive secretory pathway including cystic fibrosis, alpha-1- anti-trypsin deficiency, and familial hypercholesterolemia.
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