Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, MD 20892.
Advanced Imaging and Microscopy Resource, NIH, Bethesda, MD 20892;
Proc Natl Acad Sci U S A. 2019 Oct 22;116(43):21789-21799. doi: 10.1073/pnas.1907397116. Epub 2019 Oct 9.
Bacterial spores are dormant cells that are encased in a thick protein shell, the "coat," which participates in protecting the organism's DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In , 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM's localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.
细菌孢子是休眠细胞，被包裹在厚厚的蛋白质外壳（“外壳”）中，参与保护生物体的 DNA 免受环境侵害。外壳由数十种蛋白质组成，在孢子形成过程中以协调的方式组装。在 中，2 种蛋白质启动外壳组装：SpoVM，优先结合到微米级的凸面膜上，并将发育中的孢子表面标记为外壳组装的部位；和 SpoIVA，一种被 SpoVM 募集的结构蛋白，利用 ATP 水解在发育中的孢子周围不可逆地聚合。在这里，我们描述了 SpoVM 和 SpoIVA 启动外壳组装的过程。我们使用体内单分子荧光显微镜在孢子形成细胞中和体外合成的孢子上进行实验，报告说 SpoVM 的定位主要是由在没有其他外壳蛋白的情况下优先曲率的膜上较低的离解率驱动的。SpoIVA 的募集和聚合导致 SpoVM 被捕获在前孢子表面上。使用实验得出的反应参数，我们表明，二维棘轮模型可以描述 SpoVM 和 SpoIVA 的相互依赖的定位动力学，其中 SpoVM 在前孢子表面上具有更长的停留时间，这有利于 SpoIVA 募集到该位置。局部化的 SpoIVA 聚合反过来又阻止了预先局部化的 SpoVM 分子进一步采样其他膜。因此，我们的模型描述了结构蛋白在细胞内正确的位置和时间定位和组装形成超分子复合物的动力学。
