Normal cell function requires the compartmentalization of biochemical pathways into different membrane-bond compartments. The acquisition and maintenance of functionally distinct compartments requires sorting machines that selectively retain permanent resident proteins while allowing the removal of transient resident proteins during dynamic membrane exchange. Our long term goal is to understand mechanisms involved in regulating protein sorting in these dynamic membrane systems. A candidate sorting machine is the spectrin-based membrane skeleton, a submembransous, spatially limited, two-dimensional lattice that sequesters a subset of proteins into domains in the membrane. In preliminary studies we have shown that homologs of erythrocyte beta- spectrin and ankyrin co-localize with marker proteins of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin and ectopically expressed ankyrin co-distribute with Golgi membranes. Significantly, we have found that disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin/ankyrin from Golgi membranes and their dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin/ankyrin with the dispersed Golgi membranes. Our results indicate that the association of the membrane skeletal proteins B-spectrin and ankyrin with Golgi membranes is coupled to Golgi function. We propose 3 specific aims to define the structural organization of, and the membrane proteins that interact with, the Golgi membrane skeleton: (1). Define Golgi membrane skeletal protein complexes; (2). Investigate mechanisms of Golgi membrane skeletal protein complex assembly dynamics; and (3) . Examine Golgi membrane skeleton function in situ. The significance of these studies is that they will elucidate mechanisms involved in the structural and functional organization of the Golgi complex, and define protein interactions involving the spectrin-based membrane skeleton that regulate retention and sorting of proteins in the secretory pathway.
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