Endocytosis--the process by which cells internalize macromolecules--plays an important role both in the normal metabolism of cells (through uptake of cellular nutrients like LDL or vitamin B12, and uptake of hormones like insulin and growth factors) and in pathological processes (infection by enveloped viruses). However, little is known of the molecules that catalyze the endocytic process. The long-term goal of this application is to discern the molecular mechanisms that govern the processes of endocytosis and recycling of endocytosed materials. A cell-free system detecting the fusion of endocytic vesicles, derived from tissue culture cells, has been devised.
The specific aim of this proposal is to isolate and characterize the components required for this fusion process to occur. Both soluble and membrane- associated proteins have been implicated in governing the cell- free fusion reaction. The soluble proteins will be purified by high resolution techniques of protein purification and characterized. The membrane vesicles involved in fusion will be identified morphologically, and isolated. The proteins of these vesicles important for the fusion reaction will be identified by making antibodies to the vesicles, and determining which antibodies affect the fusion process, thereby implicating the corresponding antigens in the fusion reaction. The mechanisms by which the proteins mediate fusion will be studied, with attention paid to the order of action of the proteins, their associations with other proteins or vesicles, and importance to the fusion process in vivo. The importance of pH or ionic gradients across vesicle membranes in promoting vesicle fusion will be examined. Finally, the fusion assay will be modified so that other processes in endocytosis--the budding of endocytic vesicles and the recycling of components to the cell surface-can be studied and the underlying molecular mechanisms promoting these reactions be discerned.
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