Core Center for Molecular Morphology, Section for Stereology and Microscopy, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
Department of Pathology, Aarhus University Hospital, Aarhus, Denmark.
Mol Biotechnol. 2022 Dec;64(12):1319-1327. doi: 10.1007/s12033-022-00504-1. Epub 2022 May 24.
The tripartite partition defect (PAR) polarity complex, which includes the proteins PAR3, atypical protein kinase C (aPKC), and PAR6, is a major regulator of cellular polarity. It is highly conserved and expressed in various tissues. Its largest component, PAR3, controls protein-protein interactions of the PAR complex with a variety of interaction partners, and PAR3 self-association is critical for the formation of filament-like structures. However, little is known about the structure of the PAR complex. Here, we purified non-filamentous PAR3 and the aPKC-PAR6 complex and characterized them by single-particle electron microscopy (EM). We expressed and purified an oligomerization-deficient form of PAR3, PAR3, and the active aPKC-PAR6 dimer. For PAR3, engineering at two positions is sufficient to form stable single particles with a maximum dimension of 20 nm. aPKC-PAR6 forms a complex with a maximum dimension of 13.5 nm that contains single copies of aPKC. Thus, the data present a basis for further high-resolution studies of PAR proteins and PAR complex formation.
三联体分裂缺陷 (PAR) 极性复合物，包括蛋白 PAR3、非典型蛋白激酶 C (aPKC) 和 PAR6，是细胞极性的主要调节因子。它高度保守，在各种组织中表达。其最大的组成部分 PAR3 控制 PAR 复合物与各种相互作用伙伴的蛋白-蛋白相互作用，并且 PAR3 自身缔合对于形成丝状结构至关重要。然而，关于 PAR 复合物的结构知之甚少。在这里，我们纯化了非丝状的 PAR3 和 aPKC-PAR6 复合物，并通过单颗粒电子显微镜 (EM) 对其进行了表征。我们表达和纯化了 PAR3 的寡聚缺陷形式和活性 aPKC-PAR6 二聚体。对于 PAR3，在两个位置进行工程改造足以形成最大尺寸为 20nm 的稳定单颗粒。aPKC-PAR6 形成一个最大尺寸为 13.5nm 的复合物，其中包含单个 aPKC 拷贝。因此，这些数据为进一步研究 PAR 蛋白和 PAR 复合物形成提供了基础。
