Abstract
The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.
Highlights
The chloroplast chaperonin system is encoded by multiple genes
Formation of heptamer Cpn10/Cpn20 hetero-oligomers was observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60␣77
Structural Features of Bacterial and Eukaryotic Chaperonin Cofactors—A sequence alignment of the ϳ10-kDa domains of the chaperonin cofactors of E. coli (GroES), T4 phage, human mitochondria (Hsp10), A. thaliana (AtCpn10 and AtCpn20), and C. reinhardtii (CrCpn10, CrCpn20, and CrCpn23) reveals that AtCpn10 and the N-terminal ϳ10-kDa domain of CrCpn20 (CrCpn20N) are lacking the complete sequence corresponding to a -hairpin that forms the roof of the dome-shaped oligomer (Fig. 1) [4, 36, 37]
Summary
Results: The chaperonin cofactors of the green alga C. reinhardtii and the plant A. thaliana form hetero-oligomeric ring complexes containing seven ϳ10-kDa modules. We characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ϳ10-kDa subunits. Formation of heptamer Cpn10/Cpn hetero-oligomers was observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60␣77. Hetero-oligomeric Chaperonin Cofactors of Chloroplasts (AtCpn10) has low functional activity in vitro [19], AtCpn has been reported to form multiple oligomeric states, including tetramers [20, 21], which cooperate with E. coli GroEL in protein refolding (19 –22). The C. reinhardtii Cpn cofactors must form hetero-oligomers with Cpn, AtCpn can function as a homotetramer with one ϳ10-kDa domain being excluded from interacting with chaperonin. Different subunit configurations of the cofactors may correlate with a differential ability to assist the folding of specific client proteins by modifying the chemical environment of the chaperonin folding cage
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