Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity
Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity
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DOI：
10.1074/jbc.m608092200
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发表时间：
2006-11-10
影响因子：
4.8
通讯作者：
Obeid, Lina M.
中科院分区：
文献类型：
--
作者：
Spassieva, Stefka;Seo, Jae-Gu;Obeid, Lina M.
Lag1（长寿保证基因 1）同源物是所有真核生物中发现的跨膜蛋白家族，已被证明是（二氢）神经酰胺合成所必需的。所有 Lag1 同源物都包含一段高度保守的 52 个氨基酸，称为 Lag1p 基序。然而，保守的 Lag1p 基序对于（二氢）神经酰胺合成的功能意义目前尚不清楚。在这项工作中，我们通过在小鼠 LASS1（酵母 Lag1 的长寿保证同源物 1）的 Lag1p 基序中引入八个点突变来研究该基序的功能。使用微粒体在 HeLa 细胞和体外测试了突变体的（二氢）神经酰胺合酶活性。其中六种突变导致细胞内和体外活性丧失。此外，我们的结果表明，C18:0 脂肪酸 CoA（但不是顺式 C18:1 脂肪酸 CoA）是 LASS1 的底物，并且 HeLa 细胞中的 LASS1 对伏马菌素 B-1（一种（二氢）神经酰胺合酶的体外抑制剂）敏感。此外，我们还突变了另一个 Lag 同源物（人类 LASS5）的 Lag1p 基序。人 LASS5 中的氨基酸取代与小鼠 LASS1 中的相同，并且对 LASS5 的体外活性具有相同的影响，表明 Lag1p 基序似乎对所有 Lag1 同源物的酶活性至关重要。
Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro) ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro) ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro) ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B-1, an in vitro inhibitor of ( dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.