BOTH SPHINGOLIPIDS AND CHOLESTEROL PARTICIPATE IN THE DETERGENT INSOLUBILITY OF ALKALINE-PHOSPHATASE, A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN, IN MAMMALIAN MEMBRANES
BOTH SPHINGOLIPIDS AND CHOLESTEROL PARTICIPATE IN THE DETERGENT INSOLUBILITY OF ALKALINE-PHOSPHATASE, A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN, IN MAMMALIAN MEMBRANES
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DOI：
10.1074/jbc.270.11.6254
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发表时间：
1995-03-17
影响因子：
4.8
通讯作者：
PAGANO, RE
中科院分区：
文献类型：
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作者：
HANADA, K;NISHIJIMA, M;PAGANO, RE
SPB-1 是一种中国仓鼠卵巢细胞变体，其鞘脂合成的丝氨酸棕榈酰转移酶活性存在缺陷，为研究鞘脂和/或胆固醇剥夺对细胞功能和膜特性的影响提供了一个有用的系统。为了研究生物膜中鞘脂、胆固醇和糖基磷脂酰肌醇 (GPI) 锚定蛋白之间是否存在相互作用，我们通过稳定转染将人胎盘碱性磷酸酶 (FLAP) 引入 SPB-1 和野生型细胞中，并检查鞘脂和/或胆固醇剥夺对 FLAP 在 Triton X-100 中溶解度的影响。尽管从 Triton X-100 中的对照细胞中分离出的膜的 FLAP 溶解度仅为 10%，但鞘脂和胆固醇的剥夺进一步增强了溶解度，当鞘脂和胆固醇都被剥夺时，溶解度达到 50%。通过与外源性鞘氨醇和胆固醇的代谢互补，溶解度的增加被抑制至对照水平。分离膜的鞘脂和胆固醇含量独立变化，消除了鞘脂剥夺导致细胞胆固醇减少和 FLAP 溶解度增强的可能性，反之亦然。溶解度的增加也不太可能是由于 FLAP 分子的结构变化所致，因为无论鞘脂和胆固醇的缺乏如何，几乎所有 FLAP 都具有 GPI 锚定部分，并且经去污剂处理的膜的上清液和沉淀部分中蛋白质的表观分子量没有差异。此外，分离膜中小窝蛋白的表达水平并未受到鞘脂和/或胆固醇消耗的显着影响。这些结果表明，鞘脂和胆固醇均与 FLAP 不溶性有关，并表明这些脂质在 Triton X-100 抗性复合物的形成中协同发挥作用。
SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (FLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of FLAP in Triton X-100. Although the FLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced FLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in FLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all FLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the FLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.