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

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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.