This project is directed at an understanding of the assembly of glial plasma membrane ion transport proteins. The experiments are designed to examine the translational and post translational events mediating the insertion of 1) membrane-bound carbonic anhydrase and 2) the two plasma membrane proteolipid subunits associated with the formation of cation specific channels, into glial membranes. The cotranslational insertion of these proteins into the endoplasmic reticulum(ER) will be analyzed with cell free systems utilizing membrane-bound polysomes isolated from rat brain. Appropriate controls have been incorporated into the experimental design to help define specific interactions of nascent proteins with the ER membrane. Analyses will be carried out using well characterized immunoadsorbants for each protein. The peptide segment of membrane-bound carbonic anhydrase, that is imbedded in the membrane, will be identified by post- translational proteolysis of the ER and identification of the peptide by immune adsorption. The nature of this peptide in the nascent protein will be compared to that found in the native protein in isolated plasma membrane. The post translational events intervening between the translation and the insertion into plasma membrane of membrane-bound carbonic anhydrase will be studied in tissue slices while the assembly of the plasma membrane proteolipids will be studied in cultured glioma cells. These separate systems have been deliberately chosen so as to optimize the analyses of each protein. Inhibitors of golgi function, de novo lipid synthesis and microtubular function will be employed to help define the role of these cellular processes in the assembly of these proteins. The possibility that these proteins are acylated will also be examined. We feel that membrane-bound carbonic anhydrase and the plasma membrane proteolipids are integral to ion dynamics in the central nervous system and the elucidation of the mechanisms controlling their assembly should yield valuable data on the factors controlling the ionic milieu of the brain. It is therefore reasonable to assume that the basic information obtained in this study will not only contribute to our understanding of the biogenesis of ion transport proteins but to factors that could contribute to the pathophysiologic states of edema and epilepsy.
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