All cells depend on efficient targeting of specific proteins to their correct intracellular locations. In the secretory pathway this is carried out by vesicular transport. Little is known about the membrane components involved in vesicular transport primarily due to the difficulty of dissecting and reconstituting such a complex process. Recently, in vitro reconstitution of membrane transport steps have allowed the first insights into cytosolic components which are required for transport between Golgi cisternae. The objective of the research described in this proposal is the further development of cell-free systems to reconstitute the sorting of molecules and the formation of transport vesicles from the trans Golgi network and their correct targeting to the plasma membrane. Specifically, the components that are involved in the sorting, vesicle formation and vesicle targeting will be identified and characterized. The cell-free system we introduce to study vesicular transport from the trans Golgi network starts with an isolated organelle, a stacked Golgi fraction which is immobilized on the surface of a magnetic bead by an antigen/antibody interaction (immunoisolation). Transport vesicles are generated from the trans Golgi network under conditions where the recipient organelles are not present and the donor organelle can be rapidly removed since it is bound to a magnetic solid support. The intermediates in the traffic event, the transport vesicles, will be isolated from the budded fraction using specific antibodies. In a consequent set of experiments, recipient membranes will be added and the fusion process will be studied. Biochemical dissection and immunological reagents will be applied to analyze the two sub-cellular compartments involved in the transport to the cell surface: the trans Golgi network and the transport vesicles. Both cytosolic and membrane proteins involved in the traffic process will be sought and their interactions studied. Verification of the specificity of these proteins in the transport process will be accomplished by reintroducing them into our cell-free systems.
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