Membrane biogenesis is required for growth, differentiation and maintenance of the steady state of cells. In eukaryotic cells newly synthesized membrane components must be transported from the sites of synthesis to the multiple membrane species found in these cells. Although the mechanisms by which proteins are translocated in cells is fairly well understood, the mechanism of phospholipid movement within cells remains one of the major unsolved problems of cell biology. This grant proposal addresses this problem in eukaryotic cells by using a novel genetic and biochemical approach in yeast. Favored but unproven participants in phospholipid translocation are the phospholipid transport proteins found in the cytoplasmic fraction of all eukaryotic cells. The structural genes which code for several of these proteins will be cloned by several approaches using yeast genomic DNA libraries; many of these approaches will rely on specific antibodies directed against these proteins. Once the cloned structural genes are available, they will be mutated by deletion or insertion of DNA in vitro. These interrupted genes will be used to replace the normal wild type genomic copies by integrative transformation of a diploid strain of yeast. After meiotic segregation the spores will be analyzed for phenotype and viability to assess the role of phospholipid transfer proteins in cells. If the mutated spores are not viable, they will be rescued prior to sporulation by transformation of the diploid with plasmids carrying wild type copies of the transfer proteins whose expression can be regulated by the growth medium. By using this direct approach of obtaining mutants in the phospholipid transfer proteins, the requirement of these proteins for normal cell function and their involvement in phospholipid movement will be established. The understanding of how phospholipids are translocated in cells is important to our understanding of the processes of normal and abnormal cell growth and development.
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