An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.</p

Experimental investigation on the influence of portal-blocking speed on fire behaviors in tunnel structure

This work provides a series of fire tests in a 1/7 model tunnel to explore the influence of portal-blocking speed on tunnel fire behaviors. Burning rate, ceiling temperature, temperature distribution inside tunnel and thermal characteristic near tunnel portal are obtained and analyzed. Results shown that the comprehensive effect of portal-blocking speed on fire behavior is related to fuel area of fire source. For the fire scenario with small fuel area (Sfuel≤0.18 m2), the effect of blocking speed is not significant. While for the fire scenario with large fuel area (Sfuel≥0.54 m2), burning rate decreases rapidly, due to the blocking of tunnel portals. A critical blocking ratio existed due to the competitive between two action mechanisms of the reduction of air supplement and heat loss. The larger the fuel area is, the smaller the critical blocking ratio is. There is a delay effect on the burning suppression of fire source. For the fire scenario with large fuel area (Sfuel≥0.54 m2), the ceiling temperature distribution was more “uniform”, compared to that of the fire scenario with small fuel area (Sfuel≤0.18 m2). The faster blocking speed can make the ceiling temperature decay to a relatively low level earlier. The larger the fuel area is, the better the suppression effect of blocking strategy is. Earlier and faster blocking operation can effectively suppress the high temperature region, which is beneficial to the protection of tunnel lining

A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations

The plant unique ESCRT component FREE1 regulates autophagosome closure

Nutrient sensing impinges on the autophagosome biogenesis. Here, the authors present evidence that plant energy sensing regulates the autophagosome closure by modulating the phosphorylation status and localization of the ESCRT machinery