Protein-mediated transport of phospholipids between membranes has been described in all eukaryotic cells investigated. The cytosolic proteins responsible for this unusual type of transport have been purified from a variety of sources and differ in chemical properties, substrate specificity, and catalytic mechanism. One of the most widely distributed and highly conserved phospholipid transfer proteins binds and transports phosphatidylinositol and, with less affinity, phosphatidylcholine. The goal of proposed research is to identify and explore in detail the substrate binding domain and membrane interaction domain of phosphatidylinositol transfer proteins from different eukaryotic sources and relate these domains to biological function. In one series of experiments the substrate binding and membrane interaction structural domains will be identified and characterized through the use of protein modifications and catalytic activity measurements. Experimental approaches include: (1) photoaffinity labelling of the protein regions or specific amino acids involved in substrate binding and membrane interaction; (2) proteolytically digesting the protein to localize structural domains; (3) using antibodies against specific peptides and phospholipids to probe structural domains and catalytic activity; and (4) designing new spectroscopic and biological membrane assay systems to compare the activity of native and mutated proteins. In a complementary series of experiments the substrate binding and membrane interaction structural domains will be investigated by the use of comparative protein sequence analysis, molecular genetics, and expression systems. Experimental approaches include: (1) comparing the protein sequences of evolutionarily divergent species, Homo sapiens - Drosophila melanogaster, to identify conserved regions which may represent critical structural and functional domains; (2) developing systems for the expression of cloned, biologically active proteins; (3) altering protein structure at the genetic level by truncation or cassette mutagenesis to identify the substrate binding and membrane interaction domains; and (4) evaluating the ability of mammalian phosphatidylinositol transfer protein cDNA to complement mutations in the Saccharomyces cerevisiae PIT1 (SEC14) phosphatidylinositol transfer protein gene. These proposed experiments will allow a test of the hypothesis that specific protein domains are involved with binding phospholipid molecules and with interacting with membrane surfaces; both domains are essential to the function of phosphatidylinositol transfer protein in cellular lipid metabolism, membrane integrity, and vesicular trafficking.
Showing the most recent 10 out of 18 publications
