Institute of Molecular Biology, Academia Sinica, Taipei, 11529, Taiwan.
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, 10617, Taiwan.
Plant Cell Rep. 2024 Oct 9;43(11):257. doi: 10.1007/s00299-024-03347-z.
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.
LPAT1 的 N 端跨膜结构域穿过内膜，将 N 端置于膜间隙中，C 端酶结构域位于基质中。半乳糖脂单半乳糖基二酰基甘油和二半乳糖基二酰基甘油是光合膜的主要和重要脂质。它们由五种位于不同亚叶绿体位置的酶合成。然而，第二个作用酶，溶血磷脂酸酰基转移酶 1（LPAT1）的定位和拓扑结构，它酰化甘油-3-磷酸（G3P）的 sn-2 位以产生磷脂酸（PA），仍然不清楚。目前还不知道 LPAT1 位于外膜还是内膜，其酶结构域面向细胞质、膜间隙还是基质。甚至叶绿体中成熟 LPAT1 的大小也不清楚。更多的信息对于理解代谢物流动途径以及未来修饰甘油脂生物合成的工程努力是必要的。我们使用体外翻译的 LPAT1 前体蛋白进行导入测定，以确定成熟蛋白的确切大小，发现 LPAT1 转运肽至少 85 个残基长，比以前预测的要长得多。包含 LPAT1 融合到 Venus 荧光蛋白并由 LPAT1 启动子驱动的构建体被用于补充拟南芥 lpat1 敲除突变体。为了确定 LPAT1 的亚叶绿体定位和拓扑结构，我们使用含有体外导入的 LPAT1 的叶绿体和来自 LPAT1-Venus 互补转基因植物分离的叶绿体进行蛋白酶处理和碱性提取。我们表明，LPAT1 通过 N 端跨膜结构域穿过内膜，其 N 端突入膜间隙，C 端酶结构域位于基质中，因此与细菌同源物 PlsC 显示出不同的膜拓扑结构。
