In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii
In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii
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DOI：
10.1007/s11120-017-0370-2
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发表时间：
2017-09-01
影响因子：
3.7
通讯作者：
Ifuku, Kentaro
中科院分区：
文献类型：
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作者：
Nishimura, Taishi;Sato, Fumihiko;Ifuku, Kentaro
PsbP蛋白是光系统II （PSII）的一个外在亚基，专门在绿色植物物种中发育，包括陆地植物和绿藻。利用分离的PSII膜在体外研究了涉及PsbP的蛋白-蛋白相互作用及其对氧释放的影响。然而，这些相互作用的重要性需要在细胞水平上进行检验。为此，我们在衣藻BF25突变体缺乏原生PsbP的背景下建立了一个表达外源PsbP的系统。表达his标记的PsbP成功地恢复了突变体的进化氧活性和光自养生长，而PsbP-a dagger 15缺乏对菠菜PSII体外进化氧活性至关重要的n端15残基，只是部分恢复了突变体的进化氧活性。这证明了PsbP n端序列对体内光合活性的重要性。此外，PSII-LHCII超复合体可以用金属亲和层析从具有his标记的PsbP的衣藻细胞中特异性纯化。本研究不仅为体内PsbP的功能分析提供了平台，也为绿藻中PSII-LHCII超复合体的结构分析提供了平台。
The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-a dagger 15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII-LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII-LHCII supercomplex from green algae.