Structure and Function of Membrane Proteins

University Of Texas Hlth Sci Ctr Houston, Houston TX

Investigators

Linked publications & trials

Abstract

DESCRIPTION: (Adapted from the Investigator's abstract): Several members or the Major Facilitator Supertamily (MFS) are detective in active solute transport when expressed in mutants of Escherichia co/i lacking phosphatidylethanolamine (PE). The molecular basis for dysfunction in lactose permease (LacY) was established to be a requirement for PE as a molecular chaperone in the conformational maturation of LacY after membrane insertion. A large cytoplasmic domain in the middle of LacY when assembled in PE-lacking cells is topologically mis-assembled and exposed to the periplasm. A major aim of this proposal is to establish the molecular determinants within polytopic membrane proteins that in cooperation with membrane lipid composition and the protein assembly machinery dictate the topological organization of membrane proteins. The primary molecular probe will be accessibility of single cysteine replacements within a cysteine-lacking derivative of LacY. The topological organization of LacY and its interaction with PE will be studied using both in vivo and in vitro assembly of LacY in the presence and absence of PE. The structural and functional properties of LacY will be studied in proteoliposomes reconstituted from defined lipid components. Interaction between lipids and LacY will be studied in detergent-lipid mixed micelles. Site directed mutagenesis aimed at putative topogenic signals within LacY will be used to define elements within LacY that determine its topology. Second site suppressors of dysfunction of LacY will be isolated to identify elements of LacY and other components that determine protein topology. The phenylalanine permease and the aromatic amino acid permease are dysfunctional in PE-lacking cells. The same approaches will be used to study the molecular basis for the dysfunction of these transporters and to establish the generality of the involvement of PE in assembly of members of the MFS. The development of strains lacking FE has provided versatile biological reagents to probe the role of lipids in cell function. To broaden the scope of reagents available to study the role of lipids, E. co/i strains will be developed that either contain or replace native lipids with lipids (monoglucosyl diacylglycerol, phosphatidylcholine, phosphatidylinositol) found in other organisms. The proposed experiments are expected to define at the molecular level the role of PE and other lipids in the assembly, organization, and function of polytopic membrane proteins.