This project explores the membranes of the thermoacidophilic archaebacteria and the acidophilic thiobacilli. These bacteria live in harsh environments including low pH (2-4) and high temperature (50-70C). Due to the unusual aspects of such environments it is hypothesized that they will have unusual membranes which allow them to survive at extremes of pH and temperature. In fact, it is known that the archaebacteria as a group do not have normal ester lipids but rather tetraether lipids. The foundation of the proposal is that the unusual nature of the membranes of these bacteria will provide lipids and ion transport proteins distinct from those in eukaryotic membranes. These differences will be informative in understanding both the cell biology of these bacteria and the function of eukaryotic lipids and ion transport proteins. This project proposes to use the expertise in two laboratories to study the lipids and proteins in the membranes of these bacteria. This project comprises two sub-projects, one on ion transport proteins in Dr. Smith's laboratory and the other on lipids and lipid protein interactions in Dr. Chong's laboratory as outlined below. Sub-project 1: Identification and characterization the membrane ATPases associated with transmembrane ion transport in acidophilic thiobacilli and thermoacidophilic archaebacteria. The goals of this sub- project are to: 1. Screen the membranes of various strains of archaebacteria and acidophilic thiobacilli for ion translocating ATPases using immunologic and enzymatic assays. 2. Characterize the ATPase activities found in specific aim 1, with regard to inhibitor specificity, pH optima, cation transport and kinetic properties. 3. Purify of some of the proteins associated with the ATPase activities described in specific aims 1 and 2. Particular emphasis will be placed on P-type ATPases. Sub-project 2: Characterization of the physical properties of membranes comprised of tetraether lipids isolated from thermoacidophilic archaebacteria. In this sub-project: 1. Steady-state and dynamic fluorometry will be employed to investigate the lateral motion of pyrene-labeled lipids and the rotational motion of diphenylhexatriene (DPH) and perylene in tetraether lipid membranes. 2. The lateral and rotational motions of small molecules in tetraether lipid membranes as a function of pressure will be studied using high pressure fluorometry. 3. Fluorometry and conventional biochemical methods will be used to study the topology of archaebacterial (Na+,K+)- type ATPase in tetraether lipid membranes.
