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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM024035-13A3
Application #
3272040
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1977-07-01
Project End
1996-05-31
Budget Start
1992-06-01
Budget End
1993-05-31
Support Year
13
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Kansas
Department
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Li, Hong; Tremblay, Jacqueline M; Yarbrough, Lynwood R et al. (2002) Both isoforms of mammalian phosphatidylinositol transfer protein are capable of binding and transporting sphingomyelin. Biochim Biophys Acta 1580:67-76
Yoder, M D; Thomas, L M; Tremblay, J M et al. (2001) Structure of a multifunctional protein. Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine. J Biol Chem 276:9246-52
Tremblay, J M; Li, H; Yarbrough, L R et al. (2001) Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity. Biochemistry 40:9151-8
Tremblay, J M; Voziyan, P A; Helmkamp Jr, G M et al. (1998) The C-terminus of phosphatidylinositol transfer protein modulates membrane interactions and transfer activity but not phospholipid binding. Biochim Biophys Acta 1389:91-100
Voziyan, P A; Tremblay, J M; Yarbrough, L R et al. (1997) Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species. Biochemistry 36:10082-8
Voziyan, P A; Tremblay, J M; Yarbrough, L R et al. (1996) Truncations of the C-terminus have different effects on the conformation and activity of phosphatidylinositol transfer protein. Biochemistry 35:12526-31
Tremblay, J M; Helmkamp, G M; Yarbrough, L R (1996) Limited proteolysis of rat phosphatidylinositol transfer protein by trypsin cleaves the C terminus, enhances binding to lipid vesicles, and reduces phospholipid transfer activity. J Biol Chem 271:21075-80
Dickeson, S K; Helmkamp Jr, G M; Yarbrough, L R (1994) Sequence of a human cDNA encoding phosphatidylinositol transfer protein and occurrence of a related sequence in widely divergent eukaryotes. Gene 142:301-5
Borror, C A; Helmkamp Jr, G M (1991) Transport of phosphatidylinositol to rat hepatocyte plasma membrane catalyzed by phosphatidylinositol transfer protein. Biochim Biophys Acta 1068:52-60
Khan, Z U; Helmkamp Jr, G M (1990) Stimulation of cholinephosphotransferase activity by phosphatidylcholine transfer protein. Regulation of membrane phospholipid synthesis by a cytosolic protein. J Biol Chem 265:700-5

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