Boudreau E, Takahashi Y, Lemieux C, Turmel M, Rochaix J D
Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211, Geneve 4, Switzerland.
EMBO J. 1997 Oct 15;16(20):6095-104. doi: 10.1093/emboj/16.20.6095.
The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41-52% and 64-78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C. reinhardtii, ycf4 and ycf3 are co-transcribed as members of the rps9-ycf4-ycf3-rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9-ycf4-ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild-type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex.
莱茵衣藻叶绿体基因ycf3和ycf4已被鉴定。推导的Ycf4（197个氨基酸残基）和Ycf3（172个氨基酸残基）氨基酸序列与来自藻类、陆地植物和蓝细菌的同源物分别显示出41%-52%和64%-78%的序列同一性。在莱茵衣藻中，ycf4和ycf3作为rps9-ycf4-ycf3-rps18多顺反子转录单元的成员被共同转录成8.0 kb和3.0 kb的RNA，分别对应于整个单元和rps9-ycf4-ycf3。利用生物枪法转化，用叶绿体选择标记盒破坏ycf4和ycf3。缺乏ycf4或ycf3的转化体不能进行光合自养生长，并且光系统I活性不足。蛋白质免疫印迹分析表明，光系统I（PSI）复合物在这些转化体的类囊体膜中不能稳定积累。Ycf4和Ycf3定位于类囊体膜上，但不与PSI复合物稳定结合，并且在缺乏PSI的突变体中积累到野生型水平。RNA印迹杂交表明，psaA、psaB和psaC的转录本在这些突变体中正常积累，并且嵌合报告基因的使用表明，Ycf3不是psaA和psaB mRNA翻译起始所必需的。我们的结果表明，Ycf3和Ycf4是PSI复合物稳定积累所必需的。
