Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

可以通过对货物蛋白通过分泌途径时进行成像来研究膜运输。用于此目的的最佳工具首先阻止内质网 (ER) 中分泌货物的输出，然后释放该阻碍以产生货物波。然而，先前开发的可调节分泌货物通常难以使​​用或特定于单一模型生物。为了克服芽殖酵母的这些障碍，我们最近优化了一种人工荧光分泌蛋白，该蛋白借助与哺乳动物 Surf4 同源的 Erv29 货物受体离开内质网。荧光分泌蛋白在内质网腔中形成聚集体，并且可以通过添加配体快速解聚以产生几乎同步的货物波。在这里，我们将这种可调节分泌蛋白命名为 ESCargo（ E rv29/Surf4 依赖性分泌货物），并证明其不仅在酵母细胞中有用，而且在培养的哺乳动物细胞、果蝇细胞和纤毛虫嗜热四膜虫中也有用。动力学研究表明，从 ER 快速输出需要被 Erv29/Surf4 识别。通过选择合适的 ER 信号序列和表达载体，这种简单的技术可能适用于许多模型生物。