Functional analysis of the Fe-S cluster containing chloroplast J-domain proteins CDJ3-5

含有叶绿体 J 结构域蛋白 CDJ3-5 的 Fe-S 簇的功能分析

• 批准号: 427947477
• 负责人: Professor Dr. Michael Schroda
• 金额: --
• 依托单位: Fachgebiet Molekulare Biotechnologie und Systembiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427947477?language=en
• 关键词: Functional; analysis; Fe; cluster; containing

Hsp70 chaperone systems are highly conserved and exist in the cytosol, the ER, mitochondria, and chloroplasts of eukaryotic cells. The chloroplast Hsp70 system is derived from the bacterial one and consists of an Hsp70, a GrpE-type nucleotide exchange factor, and so-called J-domain proteins. The latter bind specific substrate proteins and hand them over to Hsp70 for further processing. CDJ3-5 are three out of six J-domain proteins in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. In addition to the J domain and a domain of unknown function, they harbor a bacterial ferredoxin domain. For CDJ3 and 4 we could show that this domain binds a redox-active 4Fe-4S cluster. Members of the CDJ3-5 proteins exist in the chloroplasts of all Viridiplantae (plants and algae) as well as in the Thaumarchaeota, who have acquired it via horizontal gene transfer. We could show that CDJ3 and 4 interact ATP-dependently with chloroplast Hsp70 and stimulate its ATPase activity. However, they do not support the folding of denatured proteins and therefore appear not to play a role in protein homeostasis. About CDJ3 we know that its expression is inducible by light and that it forms a complex with RNA. CDJ3-5 all accumulate at very low levels. Nothing else is known about the function of CDJ3-5. The goal of this project is to shed light onto the functions of CDJ3-5 using the model system Chlamydomonas reinhardtii. With the help of a new toolkit for synthetic biology in Chlamydomonas, we want to generate constructs for the overexpression of CDJ3 wild-type and mutated forms. In the latter we will change the conserved HPD motif in the J domain into AAA in order to obtain dominant negative effects. These constructs will also code for C-terminal extensions that will allow us to i) identify stably interacting proteins via affinity purification and LC-MS/MS; ii) identify transient interaction partners via proximity labeling; iii) identify bound RNA species, also with the help of RIP-Chip and CLIP; iv) determine the suborganellar localization of CDJ3-5 in the chloroplast. Further, we want to employ the CRISPR/Cpf1 system to generate cdj3-5 knock-out mutants. These, existing cdj3/4 mutants, and the overexpressor lines will then be analyzed phenotypically. For this, we will first determine survival rates under diverse stress conditions. Next, we want to identify proteins differentially accumulating in the mutants compared to wild type by quantitative shotgun proteomics based on the 15N stable isotope. Finally, we want to analyze the Fe-S cluster in CDJ5 by Mösbauer spectroscopy to see, whether this cluster is distinct from those harbored by the phylogenetically more distant CDJ3/4 proteins.

HSP70分子伴侣系统高度保守，存在于真核细胞的胞浆、内质网、线粒体和叶绿体中。叶绿体HSP70系统起源于细菌系统，由HSP70、GRPE型核苷酸交换因子和所谓的J结构域蛋白组成。后者结合特定的底物蛋白，并将它们移交给Hsp70进行进一步处理。CDJ3-5是单细胞绿藻衣藻叶绿体中6个J结构域蛋白中的3个。除了J结构域和一个功能未知的结构域外，它们还含有细菌铁氧还蛋白结构域。对于CDJ3和CDJ4，我们可以证明该结构域结合了氧化还原活性的4Fe-4S簇。CDJ3-5蛋白的成员存在于所有病原菌(植物和藻类)的叶绿体中，也存在于通过水平基因转移获得CDJ3-5蛋白的塔姆考古塔中。我们可以证明CDJ3和CDJ4与叶绿体Hsp70的相互作用依赖于ATP，并刺激其ATPase活性。然而，它们不支持变性蛋白质的折叠，因此似乎不在蛋白质动态平衡中发挥作用。关于CDJ3，我们知道它的表达是受光诱导的，它与RNA形成复合体。CDJ3-5的累积水平都很低。CDJ3-5的功能尚不清楚。该项目的目标是使用模式系统莱茵衣藻阐明CDJ3-5的功能。在衣藻属合成生物学新工具包的帮助下，我们希望生成CDJ3野生型和突变形式过表达的结构。在后者中，我们将J结构域中保守的HPD基序改为AAA，以获得显性的负面效应。这些结构还将编码C-末端延伸，使我们能够：1)通过亲和纯化和LC-MS/MS鉴定稳定相互作用的蛋白质；2)通过邻近标记识别瞬时相互作用伙伴；3)识别结合的RNA物种，也借助RIP-Chip和CLIP；4)确定CDJ3-5在叶绿体中的细胞器下定位。此外，我们希望使用CRISPR/Cpf1系统来产生cdj3-5基因敲除突变体。然后将对这些现有的cdj3/4突变体和过表达品系进行表型分析。为此，我们将首先确定在不同压力条件下的存活率。接下来，我们希望通过基于15N稳定同位素的定量猎枪蛋白质组学来鉴定与野生型相比在突变体中积累的差异蛋白质。最后，我们想用MöSBAuer光谱分析CDJ5中的Fe-S簇，看看这个簇是否与系统发育上更远的CDJ3/4蛋白所包含的簇不同。