Department of Molecular Physiology and Biological Physics, Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22904.
Proc Natl Acad Sci U S A. 2023 Mar 7;120(10):e2207461120. doi: 10.1073/pnas.2207461120. Epub 2023 Feb 27.
The composition of the plasma membrane (PM) must be tightly controlled despite constant, rapid endocytosis, which requires active, selective recycling of endocytosed membrane components. For many proteins, the mechanisms, pathways, and determinants of this PM recycling remain unknown. We report that association with ordered, lipid-driven membrane microdomains (known as rafts) is sufficient for PM localization of a subset of transmembrane proteins and that abrogation of raft association disrupts their trafficking and leads to degradation in lysosomes. Using orthogonal, genetically encoded probes with tunable raft partitioning, we screened for the trafficking machinery required for efficient recycling of engineered microdomain-associated cargo from endosomes to the PM. Using this screen, we identified the Rab3 family as an important mediator of PM localization of microdomain-associated proteins. Disruption of Rab3 reduced PM localization of raft probes and led to their accumulation in Rab7-positive endosomes, suggesting inefficient recycling. Abrogation of Rab3 function also mislocalized the endogenous raft-associated protein Linker for Activation of T cells (LAT), leading to its intracellular accumulation and reduced T cell activation. These findings reveal a key role for lipid-driven microdomains in endocytic traffic and suggest Rab3 as a mediator of microdomain recycling and PM composition.
尽管不断发生快速的胞吞作用，但质膜（PM）的组成必须受到严格控制，这需要对内质网中摄取的膜成分进行主动、选择性的回收。对于许多蛋白质来说，PM 回收的机制、途径和决定因素仍然未知。我们报告称，与有序的、受脂质驱动的膜微区（称为脂筏）的结合对于跨膜蛋白的亚群的 PM 定位是足够的，并且破坏脂筏的结合会破坏它们的运输，并导致它们在溶酶体中降解。我们使用具有可调脂质筏分配的正交、基因编码探针进行筛选，以寻找从内体有效回收工程化微区相关货物所需的运输机制。使用该筛选，我们确定 Rab3 家族是微区相关蛋白 PM 定位的重要介质。Rab3 的破坏减少了脂筏探针的 PM 定位，并导致它们在内体 Rab7 阳性区积累，表明回收效率低下。Rab3 功能的破坏也使内源性脂筏相关蛋白 Linker for Activation of T cells（LAT）定位错误，导致其在细胞内积累和 T 细胞激活减少。这些发现揭示了脂质驱动的微区在胞吞作用中的关键作用，并表明 Rab3 是微区回收和 PM 组成的介质。
