The long-range objective of this proposal is an understanding of the events associated with synthesizing membrane components in eucaryotic cells and assembling these components into a functional membrane. We intend to reach this goal using the allantoin degradative system transport proteins of Saccharomyces cerevisiae as a model system. Our preliminary experiments indicate that at least four transport systems are associated with the allantoin pathway: one each for allantoin and allantoic acid and two for urea. The latter two systems have been studied in some detail. One is an inducible, repressible, energy-dependent, low Km active transport system with cytoplasmically generated energy, perhaps in association with an ion gradient, providing the driving force for uptake. The second system is a constitutive, non-repressible, energy independent facilitated diffusion system. Some mutants have been isolated that are defective in urea uptake. Using this work as a starting point we plan to pursue the following areas: (1) Genetic identification of the structural components associated with the allantoin pathway transport systems, (2) physiological and biochemical characterization of the allantoin pathway transport systems and their interactions with one another, (3) purification and structural characterization of the low Km urea permease and production of immune sera using this protein as antigen, (4) delineation of the events associated with assembly of the low Km urea permease transport system in the plasma membrane and (5) delineation of the roles played by the vacuole in the accumulation of metabolities and regulation of their intracellular concentrations.
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