Rossi Guendalina, Lepore Dante, Kenner Lillian, Czuchra Alexander B, Plooster Melissa, Frost Adam, Munson Mary, Brennwald Patrick
Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA.
J Cell Biol. 2020 Feb 3;219(2). doi: 10.1083/jcb.201904161.
The exocyst complex plays a critical role in determining both temporal and spatial dynamics of exocytic vesicle tethering and fusion with the plasma membrane. However, the mechanism by which the exocyst functions and how it is regulated remain poorly understood. Here we describe a novel biochemical assay for the examination of exocyst function in vesicle tethering. Importantly, the assay is stimulated by gain-of-function mutations in the Exo70 component of the exocyst, selected for their ability to bypass Rho/Cdc42 activation in vivo. Single-particle electron microscopy and 3D reconstructions of negatively stained exocyst complexes reveal a structural change in the mutant exocyst that exposes a binding site for the v-SNARE. We demonstrate a v-SNARE requirement in our tethering assay and increased v-SNARE binding to exocyst gain-of-function complexes. Together, these data suggest an allosteric mechanism for activation involving a conformational change in one subunit of the complex, which is relayed through the complex to regulate its biochemical activity in vitro, as well as overall function in vivo.
外排体复合物在决定胞吐囊泡与质膜拴系和融合的时间和空间动态方面起着关键作用。然而，外排体发挥功能的机制及其调控方式仍知之甚少。在此，我们描述了一种用于检测外排体在囊泡拴系中功能的新型生化分析方法。重要的是，该分析受到外排体Exo70组分功能获得性突变的刺激，这些突变因其能够在体内绕过Rho/Cdc42激活而被选择。单颗粒电子显微镜和负染外排体复合物的三维重建揭示了突变外排体的结构变化，该变化暴露了v-SNARE的结合位点。我们在拴系分析中证明了对v-SNARE的需求，并增加了v-SNARE与外排体功能获得性复合物的结合。总之，这些数据表明了一种变构激活机制，涉及复合物一个亚基的构象变化，该变化通过复合物传递以在体外调节其生化活性以及在体内调节其整体功能。
