Chun-Wei Yu, Van C Nguyen, Niña Alyssa M Barroga, Yuki Nakamura, Hsou-Min Li
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It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. 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We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. 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引用次数: 0
摘要
关键信息:LPAT1 的 N 端跨膜结构域穿过内膜,将 N 端置于膜间隙,将 C 端酶结构域置于基质。半乳糖脂一半乳糖基和二半乳糖基二酰甘油是光合膜的主要重要脂质。它们由五种酶合成,分别位于叶绿体下的不同位置。然而,第二作用酶溶血磷脂酸酰基转移酶 1(LPAT1)的定位和拓扑结构仍不清楚,该酶将甘油-3-磷酸(G3P)的 sn-2 位酰化,生成磷脂酸(PA)。目前尚不清楚 LPAT1 位于外包膜还是内包膜,其酶域是面向细胞膜、膜间隙还是基质。甚至叶绿体中成熟的 LPAT1 的大小也不清楚。更多的信息对于了解代谢物流动的途径以及未来改造甘油酯生物合成的工程努力至关重要。我们使用体外翻译的 LPAT1 前蛋白进行导入试验,以确定成熟蛋白的精确大小,结果发现 LPAT1 过境肽的长度至少为 85 个残基,大大长于之前的预测。由 LPAT1 与金星荧光蛋白融合并由 LPAT1 启动子驱动的构建体被用来补充拟南芥 lpat1 基因敲除突变体。为了确定 LPAT1 的亚叶绿体位置和拓扑结构,我们使用含有体外导入的 LPAT1 的叶绿体和从 LPAT1-Venus 互补转基因植物中分离的叶绿体进行了蛋白酶处理和碱性提取。我们发现 LPAT1 通过 N 端跨膜结构域穿过内膜,其 N 端突出于膜间隙,C 端酶结构域位于基质中,因此显示出与其细菌同源物 PlsC 不同的膜拓扑结构。
Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.
Key message: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.
期刊介绍:
Plant Cell Reports publishes original, peer-reviewed articles on new advances in all aspects of plant cell science, plant genetics and molecular biology. Papers selected for publication contribute significant new advances to clearly identified technological problems and/or biological questions. The articles will prove relevant beyond the narrow topic of interest to a readership with broad scientific background. The coverage includes such topics as:
- genomics and genetics
- metabolism
- cell biology
- abiotic and biotic stress
- phytopathology
- gene transfer and expression
- molecular pharming
- systems biology
- nanobiotechnology
- genome editing
- phenomics and synthetic biology
The journal also publishes opinion papers, review and focus articles on the latest developments and new advances in research and technology in plant molecular biology and biotechnology.