Chinese Education Ministry's Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University, China P.R.229 North Taibai Road, Xi'an, Shaanxi, 710069, China.
Chinese Education Ministry's Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University, China P.R.229 North Taibai Road, Xi'an, Shaanxi, 710069, China.
J Plant Physiol. 2021 Sep;264:153487. doi: 10.1016/j.jplph.2021.153487. Epub 2021 Jul 30.
AtCYP38, a thylakoid lumen localized immunophilin, is found to be essential for photosystem II assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen. To identify potential interacting proteins of AtCYP38, we first adopted ATTED-II and STRING web-tools, and found 12 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with different domain of AtCYP38, and five thylakoid lumen-localized targets were identified. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we generated a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumenal fractions of thylakoid membrane proteins, and we obtained six thylakoid membrane proteins and nine thylakoid lumenal proteins as interacting proteins of AtCYP38. The interactions between AtCYP38 and several potential targets were further confirmed via pull-down and co-immunoprecipitation assays. Together, a couple of new potential candidate interacting proteins of AtCYP38 were identified, and the results will lay a foundation for unveiling the regulatory mechanisms in photosynthesis by AtCYP38.
在叶绿体中，定位于类囊体腔的免疫亲和素 AtCYP38 对于光系统 II 的组装和维持是必不可少的，但 AtCYP38 在叶绿体中的功能尚不清楚。基于先前的功能研究及其晶体结构，我们假设 AtCYP38 应该通过在类囊体腔中结合其靶标或辅助因子来发挥作用。为了鉴定 AtCYP38 的潜在相互作用蛋白，我们首先采用了 ATTED-II 和 STRING 网络工具，发现了 12 种与 AtCYP38 功能相关的蛋白。然后，我们筛选了一个包含拟南芥全基因组 cDNA 的酵母双杂交文库，文库中的 cDNA 具有不同的 AtCYP38 结构域，鉴定到了 5 个定位于类囊体腔的靶标。为了在类囊体腔中特异性地搜索 AtCYP38 的相互作用蛋白，我们构建了一个酵母双杂交 mini 文库，包含类囊体腔蛋白和类囊体膜蛋白的腔室部分，获得了 6 种类囊体膜蛋白和 9 种类囊体腔蛋白作为 AtCYP38 的相互作用蛋白。通过下拉和共免疫沉淀实验进一步证实了 AtCYP38 与几种潜在靶标的相互作用。总之，鉴定到了几个 AtCYP38 的新的潜在候选相互作用蛋白，研究结果将为揭示 AtCYP38 参与光合作用的调控机制奠定基础。
