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

Abstract

Summary The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60β1 and CPN60β2 and the co‐chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo‐oligomers and all possible other chloroplast co‐chaperonin hetero‐oligomers are functional, but only that consisting of CPN11/20/23‐CPN60αβ1β2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60β1:CPN60β2. The cryo‐EM structures of endogenous CPN60αβ1β2/ADP and CPN60αβ1β2/co‐chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero‐oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero‐oligomerization, the chloroplast co‐chaperonin CPN11/20/23 forms seven GroES‐like domains, which symmetrically interact with CPN60αβ1β2. Our structure also reveals an uneven distribution of roof‐forming domains in the dome‐shaped CPN11/20/23 co‐chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αβ1β2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

Engineering of the cytosolic form of phosphoglucose isomerase into chloroplasts improves plant photosynthesis and biomass

“…Intact chloroplasts were isolated from 4‐wk‐old Arabidopsis thaliana plants as described in prior reports (Aronsson & Jarvis, 2002; Zhao et al ., 2019). Approximately 5 g of leaf material were mashed up in 50 ml ice‐cold isolation buffer (0.33 M sorbitol, 5 mM MgCl 2 , 5 mM EGTA, 5 mM EDTA, 20 mM HEPES/KOH pH 8.0, 10 mM NaHCO 3 , 0.25% w/v BSA, 4.5 mM d ‐erythorbic acid).…”

OsCpn60β1 is Essential for Chloroplast Development in Rice (Oryza sativa L.)

“…It has been proposed that Cpn60 hetero-oligomers contain seven α subunits and seven β subunits (Cpn60α7β7) [16,18,26]. However, a recent study found that in Chlamydomonas reinhardtii, Cpn60α:Cpn60β1:Cpn60β2 was determined in a 6:2:6 ratio [27], which indicated that the structures of Cpn60 vary significantly among species and need to be further studied.…”

The cryo-EM structure of the chloroplast ClpP complex reveals an interaction with the co-chaperonin complex that inhibits ClpP proteolytic activity

“…1). Previous work from us and others showed that the authentic co-chaperonin complex in vivo consists of two Cpn20 subunits, one Cpn23 subunit, and one Cpn11 subunit 37, 38 . Compared to Clp-S1, the ClpP-S2c map showed a dome-like density located on the top of the ClpP core complexes (Fig.…”

“…The forward and reverse primers were 5’-GCGGATCCATGTCATACAGCAGCGGCGAACG-3’ and 5’-CCCAAGCTTTCAATTACGATGGGTCAGAATCGAATCGACCAG-3’ containing BamHI and HindIII restriction sites, respectively. The construction of the co-chaperonin expression plasmids, GroES-pET11a, CrCPN20-pQlinkT and CrCPN11/ 20/23-pQlinkT, were described earlier 37 .…”