genetic and biochemical study on intracellular lipid transport and lipid functions in microdomain

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1.We isolated CHO cell mutants defectivein PSS II from PSA-3 cells, and found that the pssB gene product, PSS II,functions as the principal enzyme in the PS formation from PE in CHO-K1 cells.2.A partial human cDNA encoding a PSS I -related peptide has been found during a homology search of DNA datavases, and then the full-length cDNA of the CHO homolog has been isolated [15]. Introduction of this CHO cDNA,designated pssB,into CHO-K1 cells results in striking increases in both serine and ethanolamine base-exchange activities. In contrast to the pssA cDNA,the pssB cDNA is incapable of elevating the choline base-exchange activity. Expression of the pssB gene in insect cells also leads to elevation of serine and ethanolamine base-exhange activities. These findings indicate that the pssB gene encodes PSS II catalyzing the serine and ethanolamine base-exchange but not the choline base-exchange.3.We showed that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of … More a normal PtdSer levelin CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for tha inhibition.We found that both sphingolipids and cholesterol were involved in insolubility of PLAP,a GPI-anchored protein, and suggested that these lipids coordinately playd a role in formation of Triton X-100-resistant complexes.5.We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells, and found that the CHO LCB1 homolog encodes a component of SPT.6.Phosphatidylglycerophosphate (PGP) synthase catalyzes the committed step in cardiolipin (CL) biosyntheses, and is thought to be a key enzyme for regulating cellularphosphatidylglycerol (PG) and CL content. We purified PGP synthase from Chines hamster ovary (CHO) cells. We also isolated a homologue of yeast PGS1, which encodes PGP synthase, from CHO cells. This cDNA,designated as CPGS1, encodes a protein which exhibits 28% amino acid identity with yeast PGS1, and its calculated molecular weight is 62,329 Da.7.We found that exposure of mouse macrophage-like J774.1 cells to lipopolysaccharide (LPS) induces rapid production of the cellular dialglycerol (DAG) from phosphatidylcholine (PC). We also found that LPS stimulus activated PLD toward PC,resulting in the formation of PA which was then conerted to DAG by PAP,and that the DAG production from PC by the PLD/PAP pathway was upstream of the NF-kB activation in response to LPS in J774.1 cells. Less