D1蛋白成熟过程的分子机制及生理意义

D1 is a core subunit of the photosystem II complex. Initially, it is synthesized as a precursor protein (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase (Ctp) to remove the C-terminal extension and form active protein, and the process is so called as D1 maturation. D1 maturation is essential for PSII function，however, little is known about its molecular process and regulatory mechanism,especially in high plants.. It is also very puzzled as to the presence and significance of the C-terminal peptide tail in the pD1 sequence. We plan to address these questions in Arabidopsis and tobacco. Recently, three Ctp homologues, AtCtpA, AtCtpB and AtCtpC, were identified in Arabidopsis, and only AtCtpA has been demonstrated to be responsible for D1 maturation. To understand how AtCtpA functions in chloroplast, we firstly plan to apply structure biology strategies such as CD, NMR etc to study the conformational change of AtCtpA in the liquid phase under various environmental conditions. We further plan to use in vitro and in vivo site-directed mutagenesis approaches to create changes to putative key amino acid residues in AtCtpA sequence, and dissect the affects on its structure and function due to the replacement of those amino acid residues. At the same time, We will carry out yeast two hybrid, protein pull-down, and co-immuno-precipitation strategies to identify interacting proteins associated with AtCtpA, in order to elucidate the possible regulatory mechanisms of AtCtpA activity during the process of D1 maturation. In addition, we are going to uncover why AtCtpB and AtCtpC are not involved in D1 maturation by analyzing their sub-cellular localization and enzyme activity. Result from this study should be helpful for better understanding the specificity and high efficiency of D1 carboxyl-terminal peptidase. Finally, we are aiming to test the significance of the C-terminal tail in pD1 sequence. Protein pD1 is encoded by a chloroplast gene psbA; thus, we will interrupt the psbA in tobacco chloroplast and replace it with DNA fragments encoding D1 without C-terminal extension or with different forms of C-terminal extension through the chloroplast transformation method. The resulting mutants with different forms of psbA gene will be examined for their photosynthetic activities to evaluate their effects on PSII. We believe that studies proposed above will systematically elucidate the molecular process, the regulatory mechanism, and physiological roles of D1 maturation. It will strengthen our knowledge as to the complex process of photosynthesis, especially the PSII assembly and repair.

在合成初始，光合系统II的核心亚基D1在其碳末端有一小肽，必须被D1碳末端剪切酶（Ctp）去除，才形成成熟的D1。D1的成熟是PSII发挥功能的前提条件，但目前对于D1成熟的分子过程及调节机制、以及碳末端小肽的功能不十分了解。最近在拟南芥中发现了Ctp的三种同源形式：AtCtpA、AtCtpB及AtCtpC，只有AtCtpA负责D1的成熟。本项目拟：1）用拟南芥为材料，结合圆二色谱、核磁共振等结构生物学方法与定点突变技术解析AtCtpA作用机制；并用酵母双杂交、蛋白Pull-down和免疫共沉降等手段鉴定AtCtpA的互作蛋白，探讨AtCtpA的调节机制；再用分子生物学手段分析三种AtCtp功能的差异，进一步阐明Ctp的专一性与高效性的机制；2）用烟草为材料，用叶绿体基因转化的办法改变叶绿体中D前体碳末端小肽的长度，分析碳末端小肽的生理意义。通过以上研究，获得对D1成熟过程深入细致的了解。

D1蛋白的成熟是PSII的组装和光损伤修复过程的必经之路, PSII 发挥功能的前提条件。对于D1 成熟的分子过程及调节机制、以及碳末端小肽的功能不十分了解。. 我们发现从低等的原核蓝藻到高等的被子植物，都至少存在着D1 碳末端剪切酶Ctp 的三种同源形式：CtpA、CtpB 及CtpC。我们建立了一个快速高效的检测D1 碳末端剪切酶活性的方法，测试了低等的原核蓝藻到高等的被子植物的各种Ctp，发现只有CtpA具有在体外剪切D1碳末端小肽的功能，CtpB和CtpC则没有D1 碳末端剪切酶的酶活性。用分子遗传学的方法发现，我们发现缺失CtpA拟南芥的突变体植株体内积累带碳末端小肽的D1蛋白(pD1)，不产生成熟的D1蛋白，植物无法进行光合作用而死亡。CtpB和CtpC的缺失突变体的表型和野生型植株无异，进行正常的D1 碳末端剪切和光合作用。不同物种的CtpA在拟南芥CtpA缺失的突变体植株中表达，可以使突变体恢复表型，和野生型植株相似；而不同物种的CtpB和CtpC则没有这种功能。证实CtpA 是光合生物细胞中唯一负责D1 成熟的酶。. CtpA的结构有很强的特异性，用CtpB和CtpC蛋白中的某段序列去取代CtpA的相应片断，会导致CtpA失去D1 碳末端剪切酶的活性。CtpA和pD1的碳末端小肽特异性相结合，而CtpB和CtpC则不能和pD1的碳末端小肽结合。CtpA由丝氨酸类蛋白酶进化而来，然而丝氨酸蛋白酶抑制剂并不能抑制CtpA蛋白的活性，说明CtpA已经特化出来成为一种专门切除pD1的碳末端小肽的酶。. 在CtpA/ctpa杂合植株中转入野生型的mD1基因，再进行繁殖下一代，得到ctpa/ctpa纯合背景的mD1转基因植株。发现CtpA缺失且表达mD1的转基因植株可以正常生长，和野生型无异，在各种胁迫条件下的测试，也表明转基因植物的生长发育和野生型相似，没有明显的不同。这说明pD1的碳末端小肽可能不是光合作用所必需，核基因编码的mD1蛋白可以进入叶绿体中行驶正常的功能。同时也说明CtpA的唯一功能可能就是剪切pD1的碳末端小肽，如果植物有mD1，则CtpA不是叶绿体正常功能所必需。这是一个比较重大的发现，不支持D1蛋白必须在叶绿体基因组中表达的观点。