Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments
Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments
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DOI：
10.1016/j.bbamcr.2008.05.018
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发表时间：
2008-10-01
影响因子：
5.1
通讯作者：
Fujiki, Yukio
中科院分区：
文献类型：
--
作者：
Honsho, Masanori;Yagita, Yuichi;Fujiki, Yukio
我们在此分离了血浆原缺乏的中国仓鼠卵巢（CHO）突变体ZPEG251，其表型为正常输入过氧化物酶体基质和膜蛋白。在ZPEG251中，plasmenylethanolamine （PIsEtn）被严重还原。通过对plasmalogen biogenesis相关基因表达的互补分析表明，催化plasmalogen biogenesis第二步的烷基- cli羟基丙酮磷酸合成酶（alkyl- cli羟基acetonepphosphate synthase， ADAPS）在ZPEG251中缺乏。Northern blot和逆转录- pcr分析证实，ADAPS mRNA几乎检测不到。免疫印迹法检测ADAPS表达缺陷。作为描述PIsEtn功能作用的一步，我们研究了它的亚细胞定位。PIsEtn定位于后高尔基腔室，并在耐洗涤剂膜中富集。PIsEtn向后高尔基区室的运输明显受到细胞ATP降低的影响，而不受微管组装和囊泡运输抑制剂的影响。胆固醇和鞘磷脂（脂筏的典型特征）的分配在质原缺乏的细胞（包括过氧化物酶体组装缺陷突变体）中没有受损，因此表明PIsEtn对CHO细胞中的脂筏结构不是必需的。(c) 2008 Elsevier B.V.版权所有
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.