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.

中文翻译：

---

ClpB3 的生化特征，一种来自拟南芥的叶绿体解聚酶。

关键信息

首次报道了叶绿体解聚酶的生化特征（拟南芥ClpB3）。ClpB3 寡聚成活性六聚体，使用 ATP 重新溶解聚集的底物，无需合作伙伴的帮助。

抽象的

Hsp100/Clp 家族的解聚酶是一种参与分解蛋白质聚集体的分子伴侣。植物细胞在胞质溶胶、线粒体和叶绿体中具有独特的 ClpB 蛋白。叶绿体 ClpB 蛋白与未折叠蛋白反应等关键过程有关。但是，尚未对其进行详细研究。在这项研究中，我们探索了叶绿体 ClpB 解聚酶的生化特性，特别是来自A 的ClpB3 。拟南芥_ ClpB3 在大肠杆菌中重组产生和亲和纯化到接近同质性。ClpB3 在 MgATP 存在下形成六聚体复合物并显示出内在的 ATPase 活性。我们证明 ClpB3 在广泛的 pH 值和温度值范围内具有 ATP 酶活性，并且特别耐热。ClpB3 专门针对非结构化多肽并在没有 Hsp70 系统帮助的情况下介导热变性模型底物的再激活。总体而言，这项工作代表了对来自植物的 ClpB 蛋白的首次深入生化描述，并强烈支持其在叶绿体中作为假定的解聚酶伴侣的作用。