Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
Nat Cell Biol. 2024 Oct;26(10):1804-1816. doi: 10.1038/s41556-024-01495-8. Epub 2024 Aug 29.
Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled anchor-away/recruiter system comprises an engineered, 15 kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP-GFP nanobody interaction. Applications of the mitosis-enabled anchor-away/recruiter system include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.
用于急性蛋白质定位操作的工具可以阐明时空定义的功能，但它们对外源触发的依赖可能会干扰细胞生理学。这一限制在研究有丝分裂时尤为明显，有丝分裂的高度协调事件对干扰很敏感。在这里，我们利用支架蛋白 PLEKHA5 的磷酸化控制的、细胞周期依赖性定位变化的偶然发现，开发了一种用于有丝分裂特异性蛋白质募集到质膜的系统，该系统不需要外源刺激。有丝分裂使能的锚定/招募系统由源自 PLEKHA5 的工程化的 15 kDa 模块组成，在有丝分裂期间，该模块能够通过直接融合或 GFP-GFP 纳米抗体相互作用将功能性蛋白质货物募集到质膜。有丝分裂使能的锚定/招募系统的应用包括快速敲除侧向，以便在有丝分裂期间从其天然定位中提取蛋白质，以及条件性招募脂质代谢酶，用于有丝分裂选择性编辑质膜脂质含量，而无需外源触发或干扰性同步方法。
