Genetic Analysis of Membrane Traffic in Mammalian Cells

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95-13244 Draper Eukaryotic cells consist of various endomembranous structures, each with a unique complement of proteins and lipids. Despite a dynamic membrane traffic involving transfer of membrane components from one compartment to another, the membranes maintain their distinct composition. This dynamic membrane traffic is presumed to occur by membrane vesicles that originate at donor membranes, migrate to acceptor membranes, and fuse to the acceptor compartment. The mechanisms by which vesicles are formed and by which vesicles identify and fuse to the acceptor compartment are poorly understood. This proposal is concerned with gaining insights into the molecular events in membrane trafficking. Dr. Draper has isolated temperature-sensitive mutants of CHO cells that comprise five complementation groups, End1 through End5, that are defective in certain aspects of membrane trafficking. The focus of this proposal is to gain insights into the nature of the mutants of the End4 complementation group and to isolate new mutants. Mutants of the End4 group have a block at the non-permissive temperature in endoplasmic reticulum to Golgi transport that correlates with a dissipation of the latter. The objectives of the proposal are to establish an in vitro system to biochemically analyze the End4 mutants, to clone the affected gene by complementation, and to isolate and characterize mutants that are resistant to protein toxins and are also defective in membrane trafficking. %%% Eukaryotic cells consist of various endomembranous structures, each with a unique complementation of proteins and lipids. Despite a dynamic membrane traffic involving transfer of membrane components from one compartment to another, the compartments maintain their distinct membrane composition. This dynamic membrane traffic is presumed to occur by the formation of membrane vesicles at the donor compartment, migration of these to the acceptor compartment, and fusion to the acceptor compartment. The mechanisms by which vesicles are formed, by which vesicles identify and fuse to the acceptor compartment are poorly understood. This proposal is concerned with gaining insights into the molecular events of membrane trafficking. ***