Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments

We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PIsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-clihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PIsEtn, we investigated its subcellular localization. PIsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PIsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PIsEtn was not essential for lipid-raft architecture in CHO cells. (c) 2008 Elsevier B.V. All rights reserved.