Metabolic regulation and intracellular transport of phospholipids in mammalian cells

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1)Study on the metabolic regulation of phospholipidsPhosphatidylserine (PS) in mammalian cells is synthesized through the action of ER membrane enzymes, PS synthase (PSS) 1 and 2, activities of which are post-translationally regulated by PS-mediated inhibition.(1)We purified Chinese hamster PSS 2 to near homogeneity. The kinetic analysis of the purified enzyme suggested that PSS 1 was inhibited by the binding of PS to a putative regulatory site of the enzyme.(2)To identify amino acid residues crucial for activity and/or regulation of PSS 1, we systematically introduced alanine replacement mutations into a Chinese hamster PSS 1 cDNA clone. On analysis of CHO cells transfected with each of the mutant clones, we identified 8 and 6 amino acid residues, respectively, as those crucial for the enzyme activity and regulation.(3)Membrane topology analysis of PSS 1 suggested that PSS 1 contained ten transmembrane segments, which placed the N and C termini in the cytosol. Furthermore, it was suggested that all of the eight amino acid residues important for the activity were placed from the center to luminal side of ER membrane, and that all of six amino acid residues important for enzyme regulation were exposed to the cytosol.2)Study on the intracellular transport of phospholipids(1)We identified a novel mammalian protein, MSBP1 involved in the PS transport from ER to mitochondria.(2)We isolated 〜40 candidates for yeast mutants that were defective in the intracellular transport of PS.