Dahlin C, Sundqvist C, Timko M P
Dept. of Plant Physiology, Botanical Institute, Göteborg University, Sweden.
Plant Mol Biol. 1995 Oct;29(2):317-30. doi: 10.1007/BF00043655.
The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.
NADPH-原叶绿素酸酯氧化还原酶（原叶绿素酸酯还原酶，EC 1.6.99.1）是黄化质体原片层体（PLB）中的主要蛋白质，在高等植物叶绿素合成过程中，它催化原叶绿素酸酯在光依赖下还原为叶绿素酸酯。在光照生长植物的叶绿体中，该酶在亚细胞器内的定位尚不清楚。我们进行了体外试验，以表征豌豆叶绿体中原叶绿素酸酯还原酶蛋白的组装过程。使用原叶绿素酸酯还原酶放射性标记前体蛋白的导入反应表明，该蛋白能高效导入豌豆完全成熟的绿色叶绿体中。导入反应后的分级分离试验表明，导入的蛋白靶向类囊体膜。导入后，在基质或包膜区室中未检测到放射性标记蛋白。在叶绿体裂解物中进行的组装反应表明，当试验在可水解ATP和NADPH存在下进行时，最大量的放射性标记蛋白以抗嗜热菌蛋白酶的构象与类囊体膜结合，但在NADH存在下则不然。此外，膜组装在pH 7.5和25℃时最为理想。然而，组装反应后用NaOH对类囊体进行进一步处理，去除了大部分与膜相关的蛋白。用缺乏转运肽的原叶绿素酸酯还原酶成熟形式进行的组装试验表明，膜组装不需要前序列。这些结果表明，原叶绿素酸酯还原酶是一种位于膜基质侧的外周蛋白，该蛋白的前体和成熟形式均可作为膜组装的底物。
