Streptavidin is fused to a resident ER protein, and it traps a secretory cargo that is tagged with the streptavidin-binding peptide (SBP). Perhaps the most popular of the recently developed methods is retention using streptavidin “hooks” (RUSH) ( Boncompain et al., 2012 Boncompain and Perez, 2013 Chen et al., 2017). A newer approach is to fuse multiple copies of the dimeric plant photoreceptor protein UVR8 to a secretory cargo to generate ER-localized aggregates, which can be dissolved by a pulse of UV light to trigger ER export ( Chen et al., 2013). These regulatable secretory cargoes are unlikely to be suitable for other model organisms. Similarly, procollagen accumulates in the ER at 40☌ and can be released for ER export by reducing the temperature and adding ascorbic acid ( Mironov et al., 2001). Tagged versions of the thermosensitive tsO45 mutant of the viral glycoprotein VSV-G accumulate in the ER at 40☌ and can be released for ER export by dropping the temperature to 32☌ ( Presley et al., 1997 Scales et al., 1997).
Several regulatable secretory cargoes have been generated for mammalian cells. Second, when the accumulated cargo is released to allow ER export, the resulting wave of transport illuminates the stages of cargo movement through the secretory pathway ( Trucco et al., 2004 Boncompain and Perez, 2013). First, a residence period in the ER gives the fluorescent protein portion of the cargo time to acquire a mature chromophore. The optimal approach is to trap the secretory cargo initially in the endoplasmic reticulum (ER), for two reasons. A natural or artificial secretory cargo is typically labeled with a fluorescent protein. In this regard, a powerful technique is the tracking of secretory cargoes in live cells using fluorescence microscopy ( Lippincott-Schwartz et al., 2000). Yet fundamental questions remain about how these components work together to drive membrane traffic. Most of the key players have been identified, and they are being characterized at the biochemical and structural levels. Our knowledge of the secretory pathway has progressively extended beyond morphological observations to studies of the underlying molecular machinery. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. 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. 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. 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. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. 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. Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway.