Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera
Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera
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DOI：
10.1074/jbc.m609365200
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发表时间：
2006-12-29
影响因子：
4.8
通讯作者：
Dunn, Teresa M.
中科院分区：
文献类型：
--
作者：
Han, Gongshe;Gable, Kenneth;Dunn, Teresa M.
球石病毒属是最近发现的一组病毒，感染全球重要的海洋钙化微藻Emiliania huxleyi。令人惊讶的是，病毒基因组包含一簇在其他病毒属中未发现的推定鞘脂生物合成基因。为了解决这些基因在病毒发病机制中的作用，ehv 050基因预测编码丝氨酸棕榈酰转移酶（SPT），鞘脂生物合成的第一和限速酶，在酿酒酵母中表达和表征。我们表明，编码的蛋白质确实是一个功能齐全，内质网定位，单链SPT。在真核生物中，SPT是由长链碱基1（LCB 1）和LCB 2亚基组成的异源二聚体。序列比对和突变分析表明，该病毒蛋白的N-末端结构域最接近LCB 2亚基和C-末端结构域最接近LCB 1亚基。无论病毒蛋白是作为单一多肽还是作为两个独立的结构域表达，它都表现出对肉豆蔻酰辅酶A而不是棕榈酰辅酶A的不寻常的偏好。这种偏好反映在表达病毒蛋白的酵母细胞中C16-鞘氨醇碱的存在增加。单链SPT的出现向我们表明，有可能创造出具有独特性质的其他融合SPT。值得注意的是，当酵母SPT的两个亚基如此表达时，单链嵌合体是功能性的，并显示出新的底物偏好。这表明，其他多亚基膜蛋白作为单链嵌合体的表达可以提供一个强大的方法来表征的完整的膜蛋白。
The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Surprisingly, the viral genome contains a cluster of putative sphingolipid biosynthetic genes not found in other viral genus. To address the role of these genes in viral pathogenesis, the ehv050 gene predicted to encode a serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid biosynthesis, was expressed and characterized in Saccharomyces cerevisiae. We show that the encoded protein is indeed a fully functional, endoplasmic reticulum-localized, single-chain SPT. In eukaryotes SPT is a heterodimer comprised of long chain base 1 (LCB1) and LCB2 subunits. Sequence alignment and mutational analysis showed that the N-terminal domain of the viral protein most closely resembled the LCB2 subunit and the C-terminal domain most closely resembled the LCB1 subunit. Regardless of whether the viral protein was expressed as a single polypeptide or as two independent domains, it exhibited an unusual preference for myristoyl-CoA rather than palmitoyl-CoA. This preference was reflected by the increased presence of C16-sphingoid bases in yeast cells expressing the viral protein. The occurrence of a single-chain SPT suggested to us that it might be possible to create other fusion SPTs with unique properties. Remarkably, when the two subunits of the yeast SPT were thus expressed, the single-chain chimera was functional and displayed a novel substrate preference. This suggests that expression of other multi-subunit membrane proteins as single-chain chimera could provide a powerful approach to the characterization of integral membrane proteins.