TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Many questions in cell biology can be solved by state-of-the-art technology of live cell imaging. One good example is the mechanism of membrane traffic, in which small membrane carriers are rapidly moving around in the cytoplasm to deliver cargo proteins between organelles. For directly visualizing the events in membrane trafficking system, researchers have long awaited the technology that enables simultaneous multi-color and four-dimensional observation at high space and time resolution. Super-resolution microscopy methods, for example STED, PALM/STORM, and SIM, provide greater spatial resolution, however, these methods are not enough in temporal resolution. The super-resolution confocal live imaging microscopy (SCLIM) that we developed has now achieved the performance required. By using SCLIM, we have conducted high spatiotemporal visualization of secretory cargo together with early and late Golgi resident proteins tagged with three different fluorescence proteins. We have demonstrated that secretory cargo is indeed delivered within the Golgi by cisternal maturation. In addition, we have visualized details of secretory cargo trafficking in the Golgi, including formation of zones within a maturing cisterna, in which Golgi resident proteins are segregated, and movement of cargo between these zones. This protocol can be used for simultaneous three-color and four-dimensional observation of various phenomena in living cells, from yeast to higher plants and animals, at high spatiotemporal resolution.

Specialization of many cells, including the acinar cells of the salivary glands and pancreas, milk-producing cells of mammary glands, mucus-secreting goblet cells, antibody-producing plasma cells, and cells that generate the dense extracellular matrices of bone and cartilage, requires scaling up both secretory machinery and cell-type specific secretory cargo. Using tissue-specific genome-scale analyses, we determine how increases in secretory capacity are coordinated with increases in secretory load in the Drosophila salivary gland (SG), an ideal model for gaining mechanistic insight into the functional specialization of secretory organs. Our findings show that CrebA, a bZIP transcription factor, directly binds genes encoding the core secretory machinery, including protein components of the signal recognition particle and receptor, ER cargo translocators, Cop I and Cop II vesicles, as well as the structural proteins and enzymes of these organelles. CrebA directly binds a subset of SG cargo genes and CrebA binds and boosts expression of Sage, a SG-specific transcription factor essential for cargo expression. To further enhance secretory output, CrebA binds and activates Xbp1 and Tudor-SN. Thus, CrebA directly upregulates the machinery of secretion and additional factors to increase overall secretory capacity in professional secretory cells; concomitant increases in cargo are achieved both directly and indirectly.

The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.

Sorting of proteins for secretion from cells is crucial for normal physiology and the regulation of key cellular events. Although the sorting of lysosomal hydrolases at the trans-Golgi network (TGN) for delivery to pre-lysosomes is well characterized, the corresponding mechanism by which secreted proteins are sorted for plasma-membrane delivery remains poorly understood. Recent discoveries have revealed a novel sorting mechanism that requires the linkage between the cytoplasmic actin cytoskeleton to the membrane-anchored Ca(2+) ATPase, SPCA1 (secretory pathway calcium ATPase 1), and the luminal 45 kDa Ca(2+)-binding protein, Cab45, for successful sorting of a subset of proteins at the TGN. We review progress in understanding these processes.