Rajakumari Sona, Rajasekharan Ram, Daum Günther
Institute of Biochemistry, Graz University of Technology, Graz, Austria.
Biochim Biophys Acta. 2010 Dec;1801(12):1314-22. doi: 10.1016/j.bbalip.2010.08.004. Epub 2010 Aug 19.
Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction [K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using [(3)H]inositol, [(32)P]orthophosphate, [(3)H]palmitate and [(14)C]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids.
我们实验室之前的研究表明,编码主要酵母三酰甘油(TAG)脂肪酶的TGL3缺失会导致TAG动员减少、孢子形成缺陷以及脂肪酸模式改变,尤其是TAG组分中C22:0和C26:0极长链脂肪酸的含量增加[K. 阿滕施泰特和G. 道姆,《生物化学杂志》278 (2003) 23317 - 23323]。为了研究TAG脂解与膜脂生物合成之间可能的联系,我们用野生型以及在酵母三种主要TAG脂肪酶Tgl3p、Tgl4p和Tgl5p中存在缺陷的缺失菌株进行了代谢标记实验。使用[³H]肌醇、[³²P]正磷酸盐、[³H]棕榈酸酯和[¹⁴C]乙酸作为复合脂质的前体,我们证明tgl突变体的鞘脂和甘油磷脂水平低于野生型。电喷雾串联质谱(ESI - MS/MS)分析证实,这些突变细胞中TAG的积累导致磷脂和鞘脂的含量减少。体外和体内实验表明,TAG脂解显著影响鞘脂和甘油磷脂合成所需的长链脂肪酸和极长链脂肪酸的代谢通量。由于TAG脂解减少,脂肪酸延长酶Elo1p和Elo2p的活性和表达水平增强。最后,在tgl缺失菌株中,磷脂酰胆碱、磷脂酰乙醇胺和磷脂酰丝氨酸分子种类的模式发生了改变,这突出了TAG周转在维持这些化合物的库大小以及复合膜脂重塑中的重要作用。