An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1
An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1
复制标题
DOI：
10.1242/jcs.223016
复制
发表时间：
2019-03-01
影响因子：
4
通讯作者：
Fullekrug, Joachim
中科院分区：
文献类型：
--
作者：
Exner, Tarik;Romero-Brey, Ines;Fullekrug, Joachim
脂肪酰基辅酶A还原酶1（Fart）是一种广泛表达的过氧化物酶体膜蛋白，其产生醚脂质生物合成所需的脂肪醇。在组织培养细胞中甘油三酯合成增加的条件下，通过荧光显微镜观察外源性表达和内源性人Far 1的脂滴定位。相关的光电子显微镜和亚细胞分级进一步支持了这一意外的发现。选择性透化，蛋白酶敏感性和N-糖基化标记表明，Far 1能够承担两种不同的膜拓扑结构，不同的方向短的亲水性C-末端朝向管腔或细胞质，分别。两个紧密间隔的疏水结构域包含在C-末端区域内。当单独分析时，第二结构域足以将荧光报告分子定位到脂滴。在Pex 19或ASNA 1（也称为TRC 40）CRISPR/Cas9敲除细胞中，Far 1靶向脂滴的能力没有受损。总之，我们的数据表明，Far 1是一个新的成员，而独特的双拓扑结构的膜蛋白。与此同时，Far 1显示出依赖于脂质代谢的差异亚细胞定位于过氧化物酶体和脂滴。
Fatty acyl-CoA reductase 1 (Fart) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.