DYNAMIN LIKE PROTEIN IN HEPATOCELLULAR TRANSPORT

This is a proposal to study the molecular mechanisms by which hepatocytes transport proteins and lipids through the cytoplasm. It is well known that a major function of the hepatocytes is the active secretion and removal of numerous proteins and ligands to and from the blood sinusoid. This process is supported by a complex vesicular trafficking machinery which is comprised of cytoskeletal filaments, vesicle targeting and docking proteins, and enzymes which participate in the budding and transport of nascent endocytic and secretory vesicles. One of these enzymes, called dyanamin, is a large 100-kDa GTPase which is required for endocytosis in all eukaryotic cells and is believed to act as a "molecular pinchase" to sever nascent vesicles from a donor compartment. Recently, I have identified, cloned, and sequenced a novel member of dynamin superfamily from rat liver. Further, I have demonstrated that this dynamin-like protein, DLP1, is expressed in hepatocytes where it associates with numerous motile cytoplasmic vesicles, tubules of the endoplasmic reticulum (ER), and mitochondria. Based on known function of dynamin and the cytoplasmic localization of this newly identified DLP1, I will test the CENTRAL HYPOTHESIS that DLP1 participates in the formation of nascent vesicles from the endoplasmic reticulum and in the transport of protein and lipid to multiple membranous organelles from the ER in the hepatocyte. This hypothesis will be tested using state-of-the-art biochemical, molecular and cell biological methods in the laboratories of my two co-sponsors, Dr. Richard Pagano, Ph.D., an expert in lipid synthesis and transport, and Dr. Mark McNiven, Ph.D., an authority on the dynamin family of proteins. I will pursue three focused, hypothesis driven, SPECIFIC AIMS. First, I will further define the cytoplasmic location of DLP1 in the hepatocyte and test if specific isoforms of DLP1 show distinct distribution. Second, I will test if inhibition of DLP1 function in hepatocytes alters organelle morphology. Third, I will test if DLP1 function is essential for the transport of protein and lipid to and from the ER. This study will enable me to explore and define totally novel vesicular pathway that will expand our understanding of vesicle and organelle biogenesis, and protein and lipid trafficking in the hepatocyte. Further, from the experimentation and training described here, I will greatly expand my intellectual and technical expertise toward the goal of becoming a fully independent investigator.