Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana
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Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana Ivana L. Parcerisa1 · Germán L. Rosano1 · Eduardo A. Ceccarelli1 Received: 7 June 2020 / Accepted: 11 August 2020 © Springer Nature B.V. 2020
Abstract Key message The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize aggregated substrates using ATP and without the aid of partners. Abstract Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and affinity-purified to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifically targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the first in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts. Keywords ClpB · Disaggregase · Chloroplasts · Arabidopsis thaliana · ATPase · Chaperone
Introduction At the molecular level, different types of stress, such as high temperature or alterations in the intracellular redox status, can alter the correct folding and structure of proteins. If these conditions are severe or long-lasting, unfolded proteins accumulate as cytotoxic aggregates that threaten cell survival. To counter this imbalance in protein homeostasis, a quality control network of chaperones and proteases operates within the cell to restore the proteome (Bukau et al. 2006; Chen et al. 2011; Kim et al. 2013). Different classes of Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11103-020-01050-7) contains supplementary material, which is available to authorized users. * Germán L. Rosano rosano@ibr‑conicet.gov.ar 1
Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina
chaperones work in synergy to restore protein homeostasis, and they can be classified based on their mode of action into (un)foldases, holdases, and disaggregases. Foldases such as the chaperonin system Hsp60, Hsp70, Trigger factor, and Hsp90 provide active forces to assist in the folding of their substrates in an ATP-dependen
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