Tokhtaeva Elmira, Capri Joe, Marcus Elizabeth A, Whitelegge Julian P, Khuzakhmetova Venera, Bukharaeva Ellya, Deiss-Yehiely Nimrod, Dada Laura A, Sachs George, Fernandez-Salas Ester, Vagin Olga
From the Departments of Physiology and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073.
The Neuropsychiatric Institute-Semel Institute, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, California 90024.
J Biol Chem. 2015 Feb 27;290(9):5280-97. doi: 10.1074/jbc.M114.616201. Epub 2015 Jan 9.
Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.
Septin蛋白是一个由14种细胞骨架蛋白组成的家族，它们动态形成异源寡聚体，并为蛋白质复合物组织膜微区。先前报道的与SNARE蛋白的相互作用表明Septin蛋白参与胞吐作用。然而，在各种细胞和小鼠模型中Septin-5上调或下调的矛盾结果，以及小鼠中Septin-4的矛盾结果，表明这些Septin蛋白在胞吐作用中具有抑制或刺激作用。迄今为止，尚未探索普遍表达的Septin-2或一般的Septin聚合在胞吐作用中的参与情况。在这里，通过对小鼠脑内Septin-2相互作用组进行纳升级液相色谱串联质谱分析和免疫印迹分析，我们不仅鉴定出了SNARE蛋白，还鉴定出了Munc-18-1（稳定组装好的SNARE复合物）、N-乙基马来酰亚胺敏感因子（NSF）（在每次膜融合事件后拆解SNARE复合物）以及伴侣蛋白Hsc70和突触核蛋白（在复合物拆解后维持SNARE蛋白的功能构象）。重要的是，介导NSF与SNARE复合物结合的衔接蛋白α-可溶性NSF附着蛋白（SNAP）不与Septin-2相互作用，这表明Septin蛋白在每个胞吐循环中都会发生重组。通过小干扰RNA（siRNA）部分耗尽Septin-2或用氯吡脲损害Septin动力学，会抑制各种细胞类型中分泌蛋白和跨膜蛋白的组成型和刺激性胞吐作用。氯吡脲损害了SNAP-25与其伴侣蛋白Hsc70之间的相互作用，降低了培养的神经内分泌细胞中SNAP-25的水平，并抑制了小鼠运动神经元中自发和刺激性乙酰胆碱的分泌。结果表明Septin-2在胞吐作用中具有刺激作用，并且Septin寡聚体在胞吐作用中会发生动态重组。
