We have developed a new model system to access directly intracellular organelles of the eucaryotic cell. The plasma membrane of the eucaryotic cell can be selectively perforated to produce semi-intact cells, a cell population in which organelles of the secretory pathway (nucleus, endoplasmic reticulum (ER), Golgi, and plasma membrane) are retained in an intact, functional form and directly accessible to a wide range reagents and macromolecules. It is the broad objective of the proposal to use this model system to study in vitro the transport of protein between the ER and the cis Golgi compartment. Transport of protein from the ER to the Golgi occurs by the budding and fusion of carrier vesicles. Vesicle formation requires ATP and as yet unspecified soluble and membrane-associated components. Fusion of carrier vesicles to the Golgi compartment occurs through a series of intermediate transport steps which require GTP hydrolysis and Ca2+. We propose to specifically examine the role of the rab gene family of synthesis of peptide reagents encoding functional domains of the rab gene family. Peptides will be used as chemical analogs to inhibit transport in order to elucidate rab protein function and to prepare domain specific polyclonal and monoclonal antibodies to inhibit ER to Golgi transport in vitro. These antibodies will be used as reagents to study the role of low-molecular weight GTP binding proteins in ER to Golgi transport and to identify potentially new members of this family. In addition, recombinant rab protein will be prepared using both procaryotic and eucaryotic expression vectors for analysis of the role of known rab gene products in transport in vivo and in vitro. Site-directed binding, hydrolysis and membrane-association in transport. We will also explore the role of a newly discovered Ca2+-dependent step(s) in vesicular transport which is likely to require novel Ca2+-binding protein(s) for vesicle fusion to the Golgi compartment. These studies will include purification and characterization of 3 sequential vesicular intermediates which accumulate transported protein under 3 conditions: (1) reduced temperature (15degreesC), (2) in the absence of GTP hydrolysis, and (3) in the absence of free Ca2+. Many medically important diseases result from defects in transport between intracellular organelles and the cell surface. These include lysosomal storage diseases, familial hypercholesterolemia, cystic fibrosis, and cancer. Establishing a model in vitro system for the study of transport events occurring along the secretory pathway will have a major impact on our understanding of the biochemistry of these diseases as a result of understanding the basic mechanisms fundamental to trafficking of protein between organelles of the eucaryotic cell.
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