Takahashi Y, Utsumi K, Yamamoto Y, Hatano A, Satoh K
Graduate School of Natural Science and Technology, Okayama University, Japan.
Plant Cell Physiol. 1996 Mar;37(2):161-8. doi: 10.1093/oxfordjournals.pcp.a028927.
The D1 protein (D1) of photosystem II (PSII) reaction center is synthesized as a precursor (pD1) and then processed at its carboxyl terminus to establish the function of water cleavage. The amino acid sequence of the carboxyl terminal extension excised by this process is poorly conserved except for a residue after the cleavage site at position of 345. We have constructed a vector for site-directed mutagenesis of the chloroplast psbA gene encoding D1 of the green alga, Chlamydomonas reinhardtii. The vector enables one to transform the chloroplasts of a psbA deletion mutant (Fud7) and directly select transformants for resistance to spectinomycin. Using this transforming vector, we have substituted Ser345 to Gly, Cys, Val and Phe in order to investigate effects of the amino acid side chain at this position on the processing rate. All of the resulting transformants exhibited the PSII activity as wild type and grew normally under photoautotrophic conditions even under strong light where rapid turnover of D1 protein is expected to occur. Western blotting analysis demonstrated that mature D1 accumulates in these transformants at wild type level. Pulse and chase labeling of chloroplast-encoded proteins using [35S] sulfate revealed that the processing of D1 precursor protein occurs in all four transformants as efficiently as in wild type, at least under the experimental conditions examined. The results suggest that either the amino acid side chain at position of 345 (+1 position) is not crucial to the enzymatic cleavage of pD1 in vivo or the apparent rate of processing in vivo is not limited by the enzymatic cleavage.
光系统II（PSII）反应中心的D1蛋白（D1）最初以前体形式（pD1）合成，随后在其羧基末端进行加工以建立水裂解功能。此加工过程切除的羧基末端延伸部分的氨基酸序列，除了在345位裂解位点后的一个残基外，保守性很差。我们构建了一个用于对编码莱茵衣藻绿色藻D1的叶绿体psbA基因进行定点诱变的载体。该载体能使psbA缺失突变体（Fud7）的叶绿体转化，并直接筛选出对壮观霉素有抗性的转化体。利用这个转化载体，我们将345位的丝氨酸分别替换为甘氨酸、半胱氨酸、缬氨酸和苯丙氨酸，以研究该位置氨基酸侧链对加工速率的影响。所有得到的转化体都表现出与野生型一样的PSII活性，即使在预期D1蛋白快速周转的强光下，也能在光合自养条件下正常生长。蛋白质免疫印迹分析表明，成熟的D1在这些转化体中以野生型水平积累。使用[35S]硫酸盐对叶绿体编码蛋白进行脉冲追踪标记显示，至少在所检测的实验条件下，所有四个转化体中D1前体蛋白的加工效率与野生型一样高。结果表明，要么345位（+1位）的氨基酸侧链对体内pD1的酶切裂解并不关键，要么体内明显的加工速率不受酶切裂解的限制。
