Dewez David, Park Sungsoon, García-Cerdán Jose Gines, Lindberg Pia, Melis Anastasios
Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.
Plant Physiol. 2009 Sep;151(1):88-99. doi: 10.1104/pp.109.140798. Epub 2009 Jul 2.
The function of the REP27 protein (GenBank accession no. EF127650) in the photosystem II (PSII) repair process was elucidated. REP27 is a nucleus-encoded and chloroplast-targeted protein containing two tetratricopeptide repeat (TPR) motifs, two putative transmembrane domains, and an extended carboxyl (C)-terminal region. Cell fractionation and western-blot analysis localized the REP27 protein in the Chlamydomonas reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for amino- and C-terminal regions as well as the two TPRs. A REP27 gene knockout strain of Chlamydomonas, termed the rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild-type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a dual role in the regulation of D1 protein turnover by facilitating cotranslational biosynthesis insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process.
阐明了REP27蛋白（GenBank登录号：EF127650）在光系统II（PSII）修复过程中的功能。REP27是一种核编码且定位于叶绿体的蛋白，含有两个四肽重复（TPR）基序、两个假定的跨膜结构域以及一个延伸的羧基（C）末端区域。细胞分级分离和蛋白质印迹分析将REP27蛋白定位在莱茵衣藻叶绿体类囊体中。REP27的折叠模型表明其氨基末端区域、C末端区域以及两个TPR基序定位于叶绿体基质。使用莱茵衣藻的REP27基因敲除菌株（称为rep27突变体）进行互补研究。rep27突变体在PSII修复过程中异常，其D1蛋白水平显著低于野生型。构建了截短的REP27 cDNA构建体用于rep27的互补，其中删除了TPR1、TPR2、TPR1 + TPR2或C末端结构域。缺失TPR基序的rep27互补菌株类囊体中D1水平升高，与野生型相当，但这些菌株的PSII光化学效率未恢复，这表明在没有TPR基序的情况下PSII反应中心的功能无法恢复。提示TPR基序在PSII反应中心新整合的D1蛋白的功能激活中起作用。缺失C末端结构域的rep27互补菌株类囊体中D1蛋白水平低，PSII光化学效率也低，与rep27突变体相当。因此，C末端结构域是光损伤PSII模板中D1从头生物合成和/或组装所必需的。我们得出结论，REP27在PSII修复过程中通过促进新生D1的共翻译生物合成插入（C末端结构域）和激活（TPR基序）在D1蛋白周转调节中起双重作用。
