Groupe d'Étude des Protéines Membranaires, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
J Biol Chem. 2012 Oct 19;287(43):35912-21. doi: 10.1074/jbc.M112.402057. Epub 2012 Aug 28.
Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.
阐明神经递质受体的亚基数量最好在哺乳动物表达系统中进行，因为在该系统中严格遵循天然蛋白质组装的规则。虽然在非洲爪蟾卵母细胞中取得了成功，但由于背景荧光过高、表达控制不佳以及 GFP 成熟效率低，手动计数 GFP 标记的亚基的光漂白步骤在哺乳动物细胞中受到阻碍。在这里，我们提出了一种全自动的单分子荧光计数方法，可将质膜上的标记蛋白与细胞质或内质网中的背景荧光和杂质蛋白区分开来，并确定蛋白的数量。使用单体形式的超折叠 GFP，部分抵消了在 37°C 下培养的细胞中观察到的 GFP 成熟率较低的问题。我们能够正确识别 GluK2 和 α1 甘氨酸受体的数量。我们的方法允许使用任何市售的 TIRF 显微镜在哺乳动物细胞中阐明各种质膜蛋白的数量。
