Donovan Kirk W, Bretscher Anthony
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853
J Cell Biol. 2015 Jul 20;210(2):181-9. doi: 10.1083/jcb.201501118. Epub 2015 Jul 13.
Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.
高尔基体后分泌囊泡运输是一个协调的过程，具有确保适当胞吐作用的运输和调节机制。虽然许多单个调节蛋白对这一过程的贡献已得到充分研究，但事件的时间安排和依赖性尚未明确。在这里，我们在体内追踪出芽酵母中系留和融合过程中的单个分泌囊泡及相关蛋白。分泌囊泡与质膜的系留非常可重复，持续约18秒，在膜融合缺陷的细胞中会延长，当胞吐Rab的GTP水解延迟时，系留时间会显著延长且更具变异性。此外，肌球蛋白-V Myo2p通过一种与其与外排体组分Sec15p相互作用无关的机制调节系留时间。对系留囊泡与Myo2p、鸟苷酸交换因子Sec2p以及几个外排体组分进行双色成像，使我们能够记录酵母胞吐作用的时间线，其中Myo2p在融合前4秒离开, 而Sec2p和外排体的所有组分在融合时同时分散。
