Nixon P J, Trost J T, Diner B A
Central Research and Development Department, E.I. Du Pont de Nemours & Company, Wilmington, Delaware 19880-0173.
Biochemistry. 1992 Nov 10;31(44):10859-71. doi: 10.1021/bi00159a029.
The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.
光系统II（PSII）反应中心的D1多肽最初作为前体多肽合成，随后在羧基末端进行翻译后加工。在菠菜中已表明，这种加工会去除九个氨基酸，使丙氨酸344成为C末端残基[高桥，M.，白石，T.，& 浅田，K.（1988）欧洲生物化学学会联合会快报。240，6 - 8；高桥，Y.，中根，H.，小岛，H.，& 佐藤，K.（1990）植物细胞生理学。31，273 - 280]。我们在此表明，在集胞藻6803中，丙氨酸344羧基侧的加工也会发生，导致16个氨基酸被去除。通过构建缺失编码D1的psbA基因三个拷贝的集胞藻6803缺失菌株，我们开发了一种生成psbA突变体的系统。利用该系统，我们构建了集胞藻6803的突变体，这些突变体在D1多肽的C末端区域有所改变。对这些突变体的表征表明：（1）当切割位点后的残基从丝氨酸变为脯氨酸（突变体Ser345Pro）时，D1多肽的加工被阻断，结果锰簇无法正确组装；（2）16个氨基酸残基的C末端延伸可以被删除，而对D1插入类囊体膜或D1组装成完全活性的PSII复合物几乎没有影响；（3）仅再去除一个残基（突变体Ala344stop）会导致锰簇组装丧失；（4）去污剂溶解的PSII核心复合物（缺乏锰簇）通过二级供体Z +结合和氧化外源Mn2 +的能力在加工突变体（集胞藻6803的Ser345Pro突变体和斜生栅藻的LF - 1突变体）和截短突变体Ala344stop中基本不受影响。我们的结果与加工在调节光合锰簇组装中的作用以及成熟D1多肽的游离羧基末端在簇中一个或多个锰离子连接中的作用一致。
