We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane-specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CARriers of the TGN to the cell Surface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)-G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.

Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process.DOI: http://dx.doi.org/10.7554/eLife.10982.001

The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans-Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4-phosphate (PI(4)P)-containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD-dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.

Sphingomyelin-mediated organization of resident transmembrane proteins into specific membrane domains at the trans-Golgi network is necessary for normal enzymatic activity.

The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis.

The question of whether the Golgi complex is a stable compartment or is constantly regenerated from the endoplasmic reticulum (ER) is an important issue under debate. Using an ER trapping procedure and Golgi-specific O-linked glycosylation of a resident ER protein, this study demonstrates that Golgi enzymes do not cycle through the ER during secretion and mitosis.

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.

Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process.