Department of Molecular Biology, Research Institute of Scripps Clinic, 92037, La Jolla, CA, USA.
Planta. 1991 Aug;185(1):105-10. doi: 10.1007/BF00194521.
By transformation with a cloned wild-type oee1 gene, which codes for the oxygen-evolving enhancer 1 (OEE1)protein, we have constructed a strain of Chlamydomonas reinhardtii containing multiple copies of this gene. A transformant (R1-K-50) containing four to five copies of the oee1 gene accumulated oee1 mRNA in approximately threefold excess of the wild type. The OEE1 protein accumulated in proportion to the oee1-mRNA levels in these cells. These data indicate that no apparant feedback mechanism is operating to reduce either transcription or translation of the introduced oee1 genes as a means to regulate OEE1-protein accumulation. The OEE1 protein in R1-K-50 was all of mature size, indicating that the transit peptide had been completely removed, and that all of the protein was located within the thylakoid lumen. Photosystem II reaction-center proteins D1 and D2 accumulated to wild-type levels, but not greater, in these cells, while there was no effect on accumulation of any of the PSII peripheral proteins such as OEE2 or LHCII. The OEE1 protein which accumulated in excess of wild-type levels was not bound to the thylakoid membranes, indicating that a limited number of binding sites for OEE1 exist on the thylakoid membranes. No difference in photosynthetic oxygen evolution was observed between wild-type and Rl-K-50 strains. These data show that whatever mechanisms are used to determine stoichiometry within the PSII complex they are not perturbed by overexpression of the OEE1 protein.
通过转化带有克隆野生型 oee1 基因的转化体，该基因编码氧释放增强子 1（OEE1）蛋白，我们构建了一株含有多个该基因拷贝的莱茵衣藻。含有四到五个 oee1 基因拷贝的转化体（R1-K-50）中，oee1 mRNA 的积累量比野生型高出约三倍。这些细胞中 OEE1 蛋白的积累与 oee1-mRNA 水平成正比。这些数据表明，没有明显的反馈机制可以降低引入的 oee1 基因的转录或翻译，以调节 OEE1 蛋白的积累。R1-K-50 中的 OEE1 蛋白全部为成熟大小，表明转运肽已完全去除，并且所有蛋白都位于类囊体腔中。光系统 II 反应中心蛋白 D1 和 D2 在这些细胞中积累到野生型水平，但不会更高，而对任何 PSII 外周蛋白如 OEE2 或 LHCII 的积累没有影响。在过量积累的野生型水平之上的 OEE1 蛋白未与类囊体膜结合，表明类囊体膜上 OEE1 的结合位点数量有限。在野生型和 Rl-K-50 菌株之间未观察到光合氧气释放的差异。这些数据表明，无论用于确定 PSII 复合物内化学计量的机制如何，它们都不会受到 OEE1 蛋白过表达的干扰。
