Analysis of the mechanisms by which chloroplast HSP70 chaperone activity is regulated

通过其折叠蛋白质底物的能力，HSP70家族的分子伴侣参与了过多的必要的细胞过程。叶绿体中的Hsp70也是如此，这从基因敲除突变体的致命性中得到了反映。尽管它们对植物细胞很重要，但人们对叶绿体Hsp70的生化知之甚少。这主要是由于在大肠杆菌中重组表达的叶绿体HSP70是无功能的。我们以前可以通过将莱茵衣藻的叶绿体HSP70B与其护航蛋白HEP2在大肠杆菌中共表达来解决这个问题。这一发现最终为叶绿体HSP70B的生化特性铺平了道路，这是该项目的目标。为此，我们想解决三个主要问题：(I)我们之前已经证明，HSP70B的CGE1辅助伴侣具有暗示叶绿体系统特定适应的特征。此外，我们还发现叶绿体HSP70B和HSP90C构成了一个多分子伴侣复合体。因此，我们想要确定，CGE1是如何调节HSP70B的伴侣活性的，以及是否通过与HSP90C的合作来改善它。(Ii)HSP70B被发现变成谷胱甘肽，并且是硫氧还蛋白底物。这表明叶绿体的氧化还原状态对HSP70B的伴侣活性的调节，我们想要详细研究这一点。(Iii)线粒体Hsp70还需要HEP同源物才能发挥功能，而细菌Hsp70则不需要。因此，我们想要阐明为什么只有细胞器Hsp70需要HEPs来实现功能，以及这是通过什么机制实现的。

By their ability to (un)fold protein substrates, molecular chaperones of the HSP70 family are involved in a plethora of essential cellular processes. This is true also for HSP70s in the chloroplast, as reflected by the lethality of knockout mutants. Despite of their importance for the plant cell, only little is known on the biochemistry of chloroplast HSP70s. This is mainly due to the fact that chloroplast HSP70 recombinantly expressed in E. coli was non-functional. We could previously solve this problem by co-expressing chloroplast HSP70B from Chlamydomonas reinhardtii with its escort protein HEP2 in E.coli. This finding finally paves the path for a biochemical characterization of chloroplast HSP70B, which is the goal of this project. For this we would like to address three main questions: (i) we have previously shown that the CGE1 co-chaperone of HSP70B harbours traits that suggest specific adaptations of the chloroplast system. Moreover, we have found that chloroplast HSP70B and HSP90C constitutively form a multi-chaperone complex. Hence, we want to determine, how HSP70B’s chaperone activity is regulated by CGE1 and whether it is improved by collaboration with HSP90C. (ii) HSP70B was found to become glutathionylated and to be a thioredoxin substrate. This suggests regulation of HSP70B’s chaperone activity by the chloroplast’s redox state, which we want to study in detail. (iii) Also mitochondrial HSP70s require a HEP homolog for becoming functional, whereas bacterial HSP70s do not. Hence, we want to elucidate why only organellar HSP70s require HEPs for functionality and by which mechanism this is achieved.