Molecular organization of tubular post-Golgi intermediates forming at the trans -Golgi network exit sites

The ability of cells to export proteins is essential for numerous cellular functions. Proteins for export are synthesised in the endoplasmic reticulum (ER) and move through the Golgi complex to the trans-Golgi network (TGN), from where they are shipped to various final destinations, including the plasma membrane (PM) (apical or basolateral, in polarized cells), early and late endosomes, and secretory granules. The membranous carriers involved in the trafficking step from the TGN directly to the PM are much larger than vesicles, and are pleiomorphic rather than vesicular. They also do not possess an external coat, as has been seen with other types of carriers (e.g. clathrin, COPI and COPII vesicles). At present, the molecular mechanisms of the formation of these large pleiomorphic carriers are completely unknown. I built up a thematic interactome to look at interactions between the many players involved in TGN-to-PM trafficking, and I have added some newly discovered interesting interactions through use of a two-hybrid matrix system. I thus focused my attention on some key elements such as the Arf1 and Arl1 interacting protein Arfaptin, the TRA nsport P rotein P article (TRAPP) complex, protein kinase D (PKD) and C-terminal binding protein/BFA-ribosylated substrate (Ctbp1-S/BARS) since these were revealed as being important nodes into this thematic interactome. Arfaptin is a protein specifically recruited to the TGN through a BAR domain which can interact with membranes, possibly bending them; thus, Arfaptin might be relevant in the initial formation of carriers. We have discovered that PKD (a protein involved in fission) phosphorylates and displaces Arfaptin from the Golgi complex, regulating its action in time, since the fission-mediated action of PKD occurs later. The fission activity of PKD can occur via Ctbp1-S/BARS, which is already known to be involved in membrane fission. Moreover, we have identified Ctbp1-S/BARS as a PKD phosphorylation substrate. A possible role for the mammalian TRAPP complex in the exit of VSVG from the Golgi to the PM (TGN-to-PM), comes from the study of interaction networks after our two-hybrid screening. This has shown some links between TRAPP and other relevant players, such as PKD and 14-3-3γ. In yeast, TRAPPII has been characterized as being relevant for post-Golgi exocytic trafficking. The TRAPPII complex comprises TRAPPI (Bet5p, Trs20p, Bet3p, Trs23p, Trs31p, Trs33p, Trs85p) plus three TRAPPII-specific components (Trs65p, Trs120p, Trs130p). In yeast, as in mammals, Bet3 is an essential component, since it can anchor the rest of the complex to Golgi membranes. The removal of Bet3 both chronically, with specific siRNA and acutely, with microinjection of specific antibodies, shows for the first time a role for TRAPP in TGN-to-PM trafficking: Bet3 removal strongly delays the transport of VSVG specifically from the TGN to the PM, without affecting its ER-to-Golgi trafficking.