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

HSP70家族的分子伴侣通过其折叠蛋白质底物的能力参与了大量的基本细胞过程。叶绿体中的HSP 70也是如此，如敲除突变体的致死性所反映的。尽管它们对植物细胞的重要性，但对叶绿体HSP 70的生物化学知之甚少。这主要是由于叶绿体HSP70在E.大肠杆菌是无功能的。我们以前可以通过在大肠杆菌中共表达莱茵衣藻叶绿体HSP 70 B及其护送蛋白HEP 2来解决这个问题。这一发现最终为叶绿体HSP 70 B的生化表征铺平了道路，这是本项目的目标。为此，我们想解决三个主要问题：（i）我们以前已经表明，热休克蛋白70 B的CGE1共伴侣窝藏性状，表明叶绿体系统的具体适应。此外，我们还发现叶绿体HSP70 B和HSP90 C组成型地形成多分子伴侣复合物。因此，我们想确定，如何热休克蛋白70 B的伴侣活性是由CGE1调节，以及是否与热休克蛋白90 C的合作得到改善。(ii)HSP70 B被发现成为谷胱甘肽化，并成为硫氧还蛋白底物。这表明叶绿体的氧化还原状态对HSP70 B的伴侣活性的调节，我们想详细研究。(iii)此外，线粒体HSP70需要HEP同系物才能发挥功能，而细菌HSP70则不需要。因此，我们想阐明为什么只有细胞器HSP70需要HEPs的功能，以及通过何种机制实现这一点。

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.