Membranes play a key role in many cellular regulatory and biosynthetic processes. It is a distinct possibility that certain human diseases result from incorrect assembly of cellular membranes. However, we know very little about how membranes and membranes proteins are synthesized and assembled. Consequently, since the microorganism Neurospora is a bona fide eukaroyte and offer the advantages of 1) haploid genetics, 2) easily obtaining mass quantities, and 3) easily isolating the various subcellular fractions. It is felt that Neurospora will provide an excellent system for studying biosynthesis of membranes and membrane proteins. The principal investigator's main objective is to understand the mechanism of biosynthesis of one particular Neurospora membrane protein, the plasma membrane H+-ATPase. The approach is the following: first, to determine the site of synthesis of the H+-ATPase, ribonucleic acid from various subcellular fractions will be extracted, isolated, and translated in an in vitro protein synthesis system using nuclease-pretreated rabbit reticulocyte lysate. The in vitro synthesized H+-ATPase will be collected by specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gels. Secondly, to locate putative component(s) required for recognition and insertion of the H+-ATPase into the lipid bilayer, the cytosol fraction or salt or protease-salt extracts from various membrane fractions will be fractionated by gel-filtration chromatography; the various fractions combined with nuclease and EDTA or salt or protease-salt pretreated membrane fractions in the aforementioned in vitro system to assay their capacity to facilitate incorporation of H+-ATPase into the lipid bilayer. The most active fractions from one of these various methods in this assay will be further purified by ion-exchange or gel-filtration chromatography or a combination thereof. Alternatively, isolated membranes will be solubilized by a detergent, the solubilized extract fractionated, the various fractions reconstituted into liposomes and assayed for in vitro incorporation of H+-ATPase; the most active fractions further purified as aforementioned. This approach will be used to locate putative processing protease(s); the various fractions will be assayed for their ability to convert precursor(s) of H+-ATPase. Finally, the various isolated components will be reconstituted in a well-defined chemical system; this system will be used to study the mechanism of these components in the maturation of the plasma membrane H+-ATPase.
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