Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000, Rosario, Argentina.
Plant Mol Biol. 2020 Nov;104(4-5):451-465. doi: 10.1007/s11103-020-01050-7. Epub 2020 Aug 16.
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. 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.
首次对叶绿体解聚酶进行了生化特性分析（拟南芥 ClpB3）。ClpB3 寡聚形成有活性的六聚体，可在没有伴侣协助的情况下使用 ATP 重新溶解聚集的底物。Hsp100/Clp 家族的解聚酶是一种参与解聚蛋白聚集体的分子伴侣。植物细胞在细胞质、线粒体和叶绿体中特有的 ClpB 蛋白。叶绿体 ClpB 蛋白已被牵连到关键过程，如未折叠蛋白反应；然而，它们尚未被详细研究。在这项研究中，我们探索了一种叶绿体 ClpB 解聚酶的生化特性，特别是来自拟南芥的 ClpB3。ClpB3 在大肠杆菌中通过重组产生并通过亲和纯化至近乎均一。ClpB3 在存在 MgATP 的情况下形成六聚体复合物，并显示出内在的 ATP 酶活性。我们证明 ClpB3 在广泛的 pH 和温度范围内具有 ATP 酶活性，并且特别耐热。ClpB3 特异性靶向无结构多肽，并介导热变性模型底物的复活，而无需 Hsp70 系统的帮助。总的来说，这项工作代表了对植物 ClpB 蛋白的首次深入生化描述，并强烈支持其作为叶绿体中假定的解聚酶伴侣的作用。
