Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation
Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation
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DOI：
10.1074/jbc.m708993200
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发表时间：
2008-04-25
影响因子：
4.8
通讯作者：
Igarashi, Yasuyuki
中科院分区：
文献类型：
--
作者：
Kihara, Akio;Sakuraba, Hiroko;Igarashi, Yasuyuki
酵母Phs1是一种3-羟基酰辅酶A脱水酶，催化超长链脂肪酸(VLCFA)四步反应中的第三步反应。在酵母中，神经鞘脂脂的疏水骨架神经酰胺由一个长链碱基和一个酰胺连接的C26 VLCFA组成。因此，VLCFA合成中的缺陷有望极大地影响鞘磷脂的合成。事实上，在这项研究中，我们发现Phs1水平的降低导致神经酰胺向肌醇磷化神经酰胺的转化显著受损。Phs1蛋白在真核生物中高度保守，构成了一个新的蛋白质家族。Phs1家族成员与其他脱水酶家族成员没有序列相似性，因此它们的活性部位序列和催化机制一直是完全未知的。在这里，通过突变Phs1家族成员之间保守的22个氨基酸残基，我们确定了六个在Phs1功能中重要的氨基酸残基，其中两个(Tyr-149和Glu-156)是必不可少的。我们还使用N-糖基化报告实验检测了Phs1的膜拓扑结构。我们的结果表明，Phs1是一种跨膜蛋白，它穿过细胞膜6次，N端和C端面向胞浆。重要的氨基酸集中在所提出的六个跨膜区中的两个或附近。因此，我们还提出了Phs1的催化机制，该机制与其他水合酶在脂质合成中所用的机制没有什么不同。
Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.