YEAST LIPID BINDING PROTEIN--IDENTIFICATION AND ANALYSIS

The long term goal of this proposal is an analysis of intracellular lipid trafficking, its mechanism of control, and the role of intracellular lipid binding proteins in facilitating lipid transfer within the cell. Within certain cells of mammalian tissues there exists a family of cytosolic low molecular weight, ca 15 kDa, proteins whose function is believed to be the solubilization and delivery of hydrophobic lipids to appropriate enzymes for metabolic activation and utilization. Intestinal FABP (fatty acid binding protein) may facilitate dietary adsorption of essential nutrient fatty acids. Heart FABP may deliver fatty acids derived from circulating stores to the mitochondrion for energy yielding beta- oxidation. Adipocyte lipid binding protein (ALBP) may shuttle fatty acids into and out of adipose tissue in response to insulin or epinephrine, respectively. Deficiencies or alterations in the regulation of these intracellular fatty acid carriers may affect clinically important processes such as nutrient uptake in the gut, obesity and its causal complications with NIDDM and related disorders. To test the hypothesis that the intracellular lipid binding proteins facilitate lipid trafficking it would be advantageous to have cell lines devoid of such proteins. However, there are no such lines available and attempts at genetic generation of mutants via antisense mRNA or oligonucleotides have proven unsuccessful. To circumvent these problems, the analysis of intercellular lipid trafficking in yeast will be undertaken. This proposal addresses fatty acid trafficking and the function of the intracellular lipid binding proteins by: A: Isolation of the yeast lipid binding protein gene (LBP) corresponding to the adipose member of the multigene family. B. Examine the development and nutritional regulation of the LBP gene using lac fusion and northern analysis. C. Determine the DNA sequence of the LBP gene that confers transcriptional regulation under conditions identified in B. above. D. Genetically generate LBP deficient yeast strains. Examine the metabolic consequences of such mutations, complement the deficiencies with the ALBP cDNA and the liver fatty acid binding protein cDNA. E. Create a tyr to phe mutation to examine phosphorylation in-vivo.