The Cpn10(1) Co-Chaperonin of A. thaliana Functions Only as a Hetero-Oligomer with Cpn20

Abstract

The A. thaliana genome encodes five co-chaperonin homologs, three of which are destined to the chloroplast. Two of the proteins, Cpn10(2) and Cpn20, form functional homo-oligomers in vitro. In the current work, we present data on the structure and function of the third A. thaliana co-chaperonin, which exhibits unique properties. We found that purified recombinant Cpn10(1) forms inactive dimers in solution, in contrast to the active heptamers that are formed by canonical Cpn10s. Additionally, our data demonstrate that Cpn10(1) is capable of assembling into active hetero-oligomers together with Cpn20. This finding was reinforced by the formation of active co-chaperonin species upon mixing an inactive Cpn20 mutant with the inactive Cpn10(1). The present study constitutes the first report of a higher plant Cpn10 subunit that is able to function only upon formation of hetero-oligomers with other co-chaperonins.

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

“…Previous studies have shown that hetero‐oligomers consisting of Cpn10 and Cpn20 occur frequently in vitro , which raised the question whether such hetero‐oligomers also represent functional co‐chaperonins in vivo (Tsai et al ., ; Vitlin Gruber et al ., ). To confirm the existence of chloroplast co‐chaperonin hetero‐oligomers in vivo , we carried out co‐immunoprecipitation assays from Chlamydomonas chloroplast stroma and total protein using an antiserum against CPN20.…”

“…Chloroplast co‐chaperonin subunits are also divided into two subtypes, the conventional ~10‐kDa GroES‐like Cpn10 and the ~20‐kDa Cpn20, which consists of two tandem Cpn10 domains joined head to tail (Musgrove et al ., ; Bertsch et al ., ; Baneyx et al ., ). Both chloroplast chaperonin and co‐chaperonin complexes are liable to exist as intricate hetero‐oligomers, although Cpn60β and Cpn20 homo‐oligomers have been shown to be functional in vitro (Viitanen et al ., ; Dickson et al ., ; Tsai et al ., ; Vitlin Gruber et al ., ; Bai et al ., ). Another unique aspect of the chloroplast chaperonin system compared with its bacterial and mitochondrial counterparts is the capability to assist in the folding and assembly of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco), which accounts for ~30–50% of soluble proteins in chloroplasts (Barraclough and Ellis, ; Dhingra et al ., ).…”

“…Regarding chloroplast co‐chaperonins, it is not clear how the six‐ or eightfold symmetry realized in functional Cpn20 homo‐oligomers matches with the heptameric chaperonin cylinder, although a recent biochemical study has claimed that a perfect match with the chaperonin was no absolute prerequisite for a functional interaction (Baneyx et al ., ; Koumoto et al ., ; Guo et al ., ). Several in vitro studies have suggested that, similar to Cpn60s, the co‐chaperonins might also form Cpn20–Cpn10 hetero‐oligomers in vivo (Tsai et al ., ; Vitlin Gruber et al ., ). It appears that hetero‐oligomerization of different subunits might not only be a smart way to solve the symmetry mismatch problem, but might also increase the flexibility of the chloroplast chaperonin system to provide folding environments adapted for specific substrates.…”

Asymmetric functional interaction between chaperonin and its plastidic cofactors

“… reported that none of the three individual cochaperonin (CrCPN11, CrCPN20 and CrCPN23) of Chlamydomonas was functionally active, although hetero‐oligomers consisting of seven 10‐kDa domains cooperated with chaperonin in refolding model substrates. By contrast, in Arabidopsis , both homo‐oligomers of Cpn10(2) and Cpn20 were functional as cochaperonins , whereas Cpn10(1) was incorporated into Cpn20 oligomers to cooperate with chaperonin . In the present study, we investigated the cooperation of various cochaperonins with the homo‐oligomeric GroEL and hetero‐oligomeric CrCPN60 chaperonins.…”

“…Recently, Vitlin Gruber et al . found that, in contrast to AtCpn10(2), which forms heptamers interacting with chaperonin as cofactors, Arabidopsis AtCpn10(1) was not functional unless in the hetero‐oligomeric form with AtCpn20. Moreover, AtCpn20 is postulated to play multiple functions in chloroplasts independent of its co‐chaperonin role in Arabidopsis (e.g.…”