In vitrocharacterization of bacterial and chloroplast Hsp70 systems reveals an evolutionary optimization of the co-chaperones for their Hsp70 partner

Abstract

The chloroplast Hsp70 (heat-shock protein of 70 kDa) system involved in protein folding in Chlamydomonas reinhardtii consists of HSP70B, the DnaJ homologue CDJ1 and the GrpE-type nucleotide-exchange factor CGE1. The finding that HSP70B needs to be co-expressed with HEP2 (Hsp70 escort protein 2) to become functional allowed the reconstitution of the chloroplast Hsp70 system in vitro and comparison with the homologous Escherichia coli system. Both systems support luciferase refolding and display ATPase and holdase activities. Steady-state activities are low and strongly stimulated by the co-chaperones, whose concentrations need to be balanced to optimally support luciferase refolding. Although the co-chaperones of either system generally stimulate ATPase and folding-assistance activities of the other, luciferase refolding is reduced ~10-fold and <2-fold if either Hsp70 is supplemented with the foreign DnaJ and GrpE protein respectively, suggesting an evolutionary specialization of the co-chaperones for their Hsp70 partner. Distinct features are that HSP70B's steady-state ATPase exhibits ~20-fold higher values for Vmax and Km and that the HSP70B system displays a ~6-fold higher folding assistance on denatured luciferase. Although truncating up to 16 N-terminal amino acids of CGE1 does not affect HSP70B's general ATPase and folding-assistance activities in the physiological temperature range, further deletions hampering dimerization of CGE1 via its N-terminal coiled coil do.

HSP33 in eukaryotes – an evolutionary tale of a chaperone adapted to photosynthetic organisms

“…Protection from damages inflicted by oxidative stress in land plants is provided by alternative mechanisms. Indeed, the chloroplast ortholog of DnaK, HSP70B, exhibits increased expression levels under oxidizing conditions (Drzymalla et al ., ; Veyel et al ., ).…”

“…In photosynthetic organisms, which experience light‐induced oxidative stress on a regular basis (Foyer and Noctor, ; Couturier et al ., ), the chloroplast proteome contains a variety of chaperones, including members of the DnaK/J/GrpE families, along with other chaperones (Liu et al ., , ; Merchant et al ., ; Nordhues et al ., ; Muhlhaus et al ., ; Veyel et al ., ). However, as the ability of these chaperones to function during oxidative stress is inhibited, it is possible that HSP33 could take over this missing function in photosynthetic organisms, as shown for bacteria (Hoffmann et al ., ; Winter et al ., ).…”

Hetero‐oligomeric CPN60 resembles highly symmetric group‐I chaperonin structure revealed by Cryo‐EM

“…Several biochemical assays (Figures , S2 and S3), including co‐migration in gel filtration, ATPase activity inhibition and in vitro refolding of RrRubisco, indicated that different chloroplast co‐chaperonin compositions have different bioactivities, which may correlate with different functions in vivo . In all our biochemical assays, the co‐chaperonins (GroES or CPN20 combinations) were functional with both bacterial and green algal chaperonins, but the homologous CPN11/20/23‐CPN60αβ1β2 complex performed best (Figure a), similar to what has been reported for chloroplast and bacterial Hsp70 systems (Veyel et al ., ). These results indicate a strong functional conservation between the bacterial and green algal chaperonin systems, despite substantial subunit differentiation.…”

Systems-Wide Analysis of Acclimation Responses to Long-Term Heat Stress and Recovery in the Photosynthetic Model OrganismChlamydomonas reinhardtii

“…During long-term HS, levels of HSP70B increased 2.6-fold, while those of CGE1 only by 1.6-fold (Supplemental Data Set 2). As CGE1 accelerates nucleotide exchange, the increased HSP70B to CGE1 ratio during HS may increasingly shift HSP70B toward the ADP-bound state and, thus, from a folding-supportive to a holdase activity (Veyel et al, 2014b). Hence, decreasing HSP70B and increasing CGE1 levels during recovery may rapidly shift the system back to a folding-supportive function.…”