Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase.
Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase.
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鞘磷脂翻转酶的定向进化揭示了 P4-ATP 酶区分底物主链的机制。
DOI：
10.1073/pnas.1525730113
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发表时间：
2016
影响因子：
11.1
通讯作者：
Graham,ToddR
中科院分区：
文献类型：
--
作者：
Roland,BartholomewP;Graham,ToddR
IV 型 P 型 ATP 酶 (P4-ATP 酶) 家族中的磷脂翻转酶可建立膜不对称性，并在囊泡运输、细胞极性、信号转导和神经发育中发挥关键作用。所有表征的 P4-ATP 酶都会将甘油磷脂翻转穿过双层至膜的胞质小叶，但这些酶如何区分甘油磷脂和鞘脂尚不清楚。我们使用定向进化方法来检查 P4-ATP 酶区分底物骨架的分子机制。对酵母酿酒酵母的诱变筛选发现了 P4-ATP 酶 Dnf1 中的几个功能获得性突变，这些突变促进了新型脂质底物鞘磷脂的运输。我们发现第一个跨膜片段中高度保守的天冬酰胺（N220）是甘油磷脂选择的关键执行者，并且该位点的特定取代允许鞘磷脂的运输。
Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeastSaccharomyces cerevisiaehas identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.