Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging.
Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging.
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DOI：
10.1091/mbc.e18-07-0424
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发表时间：
2018-11-01
影响因子：
3.3
通讯作者：
Thorner J
中科院分区：
文献类型：
--
作者：
Emmerstorfer-Augustin A;Augustin CM;Shams S;Thorner J
为了通过活细胞中的荧光显微镜实时观察酵母信息素受体 Ste2 的内化，我们通过在该受体的 N 末端用细胞外荧光激活蛋白 (FAP) 标记该受体，仅可视化激动剂接合时存在于细胞表面的那些分子（而不是整个细胞池）。 FAP 是一种单链抗体，经过设计可紧密结合非荧光、细胞不可渗透的染料（荧光剂），从而产生荧光复合物。 FAP 标记在研究具有细胞壁的酵母中整合膜蛋白的运输方面的用途此前尚未被研究过。探索了一组不同的信号肽和前肽序列以最大化表达。维持最佳 FAP-Ste2 嵌合体完整需要删除两个旁系同源的糖基磷脂酰肌醇 (GPI) 锚定的细胞外天冬氨酰蛋白酶（Yps1 和 Mkc7）。 FAP-Ste2 表现出比 Ste2-GFP 或 Ste2-mCherry 更亮、更清晰的质膜信号，但表现却非常相似。使用 FAP-Ste2，获得了有关其内化机制的新信息，包括有关货物选择性内吞接头 Ldb19/Art1、Rod1/Art4 和 Rog3/Art7 作用的新见解。
To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.