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PMID:21737456

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Citation

Rosano, GL, Bruch, EM and Ceccarelli, EA (2011) Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 286:29671-80

Abstract

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ∼19.80 mm, V(max) ∼0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (∼40% for AtClpC2 and ∼20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.

Links

PubMed PMC3191008 Online version:10.1074/jbc.M110.211946

Keywords

Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/genetics; Adenosine Triphosphate/metabolism; Arabidopsis/enzymology; Arabidopsis/genetics; Arabidopsis Proteins/genetics; Arabidopsis Proteins/metabolism; Chloroplasts/enzymology; Chloroplasts/genetics; Heat-Shock Proteins/genetics; Heat-Shock Proteins/metabolism; Protein Multimerization/physiology; Protein Transport/physiology

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