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

Hsp 70分子伴侣系统是高度保守的，存在于真核细胞的胞质溶胶、内质网、线粒体和叶绿体中。叶绿体Hsp 70系统来源于细菌系统，由Hsp 70、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，并将其交给Hsp 70进行进一步处理。CDJ 3 -5是单细胞绿色衣藻叶绿体中六个J结构域蛋白中的三个。除了J结构域和未知功能的结构域之外，它们还具有细菌铁氧还蛋白结构域。对于CDJ 3和4，我们可以表明该结构域结合氧化还原活性的4Fe-4S簇。CDJ 3 -5蛋白的成员存在于所有绿色植物（植物和藻类）的叶绿体中，以及通过水平基因转移获得的Thaumarchaeota中。结果表明，CDJ 3和4与叶绿体Hsp 70的相互作用是ATP依赖的，并能刺激叶绿体Hsp 70的ATP酶活性。然而，它们不支持变性蛋白质的折叠，因此似乎在蛋白质稳态中不起作用。关于CDJ 3，我们知道它的表达可以被光诱导，并且它与RNA形成复合物。CDJ 3 -5都以非常低的水平积累。关于CDJ 3 -5的功能还不清楚。本项目的目标是阐明CDJ 3 -5的功能使用模型系统莱茵衣藻。在衣原体合成生物学的新工具包的帮助下，我们希望生成用于CDJ 3野生型和突变形式的过表达的构建体。在后者中，我们将J结构域中保守的HPD基序改变为AAA，以获得显性负效应。这些构建体还将编码C-末端延伸，这将允许我们i）通过亲和纯化和LC-MS/MS鉴定稳定相互作用的蛋白质; ii）通过邻近标记鉴定瞬时相互作用配偶体; iii）也在RIP-Chip和CLIP的帮助下鉴定结合的RNA种类; iv）确定叶绿体中CDJ 3 -5的亚细胞器定位。此外，我们希望使用CRISPR/Cpf 1系统来产生cdj 3 -5敲除突变体。然后对这些现有的cdj 3/4突变体和过表达株系进行表型分析。为此，我们将首先确定不同压力条件下的存活率。接下来，我们希望通过基于15 N稳定同位素的定量鸟枪蛋白质组学来鉴定突变体中与野生型相比差异积累的蛋白质。最后，我们想通过穆斯堡尔光谱分析CDJ 5中的Fe-S簇，看看这个簇是否与那些在遗传学上更遥远的CDJ 3/4蛋白所包含的簇不同。

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.