The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.

中文翻译：

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TTYH 家族成员在细胞膜内形成四聚体复合物

保守的 Tweety 同源 (TTYH) 家族由脊椎动物中的三个旁系同源物组成，显示出无处不在的表达模式。尽管近二十年来一直被认为是离子通道，但最近的结构和功能分析驳斥了这种作用。有趣的是，虽然在洗涤剂溶解后研究的所有旁系同源物共享二聚化学计量，但它们的空间组织不同。在这里，我们确定了细胞中完整小鼠 TTYH (mTTYH) 复合物的化学计量。使用交联和单分子荧光显微镜，我们证明 mTTYH1 和 mTTYH3 在质膜上形成四聚体。蓝色天然 PAGE 和荧光检测大小排阻色谱分析表明，洗涤剂增溶导致四聚体溶解成二聚体，表明二聚体的二聚体组装模式。由于可溶性胞外域的交联分析也显示出四聚体的化学计量，我们探索了膜增溶和二硫键完整性的影响，并确定了它们对四聚体稳定性的贡献。对此处表征的天然四聚体 TTYH 的未来研究可能会阐明其长期寻求的细胞功能。