Membranes and Active Transport of Amino Acids

Ames, Giovanna

Abstract

This project is concerned with defining mechanisms by which substances are transported into and out of cells and with analyzing the biosynthesis and structure of bacterial membranes. The experiments will make use of microorganisms (Salmonella typhimurium) which can be manipulated genetically by isolating mutants defective in the various steps of active transport or of membrane biogenesis, which can then be analyzed biochemically. The histidine transport system, which we use as a modely system, is composed of three proteins: the histidine-binding protein J (periplasmic, already characterized), the P protein (membrane bound) and the Q protein. The P protein will be purified and characterized, which is an essential step in the understanding of the molecular mechanism of transport. The Q protein will be identified, purified, and characterized. To aid us in these efforts, we have cloned the histidine transport operon and will be able to identify missing components and produce large amounts of protein for purification purposes. The clone will also be used for sequencing the DNA of these genes in order to understand their regulation, the secretion mechanism of membrane and periplasmic proteins. We will attempt to reconstitute an active transport system in liposomes or spheroplasts. We will research the interaction of periplasmic proteins with membranes by cross-linking experiments. We are also starting a study of the energy coupling mechanisms functioning in our system: we essentially plan to test the hypothesis that acetylphosphate is the energy source for periplasmic systems (Hong) by isolating appropriate mutants in acetylphosphate biosynthsis and by assaying directly the level of acetylphosphate in the cell. The multiplicity of function of the membrane-bound component, P protein, will be investigated by studying its involvement with a lysine-, arginine-, ornithine-binding protein in vivo and in vitro.