We also report the specific requirement of arfaptin1 in the trafficking of chromogranin A (Cg A) the description of our findings follows. Moreover, the binding of arfaptin1 to PI(4)P is regulated by PKD whereas the binding of arfaptin2 to PI(4)P is insensitive to PKD activity. We have made a surprising finding that BAR domain-containing proteins arfaptin1 and arfaptin2 are recruited to the TGN by a PI(4)P-dependent reaction. Strangely, thus far there is no evidence of a coat (like clathrin, COPI or COPII) or a BAR (Bin/Amphiphysin/Rvs) domain-containing protein in the PKD-dependent biogenesis of transport carriers. More recently, it has been shown that PKD and CtBP1-S/BARS are linked by 14-3-3γ to recruit and control a number of other proteins, which together regulate membrane fission ( Valente et al, 2012). Based on these findings, it has been proposed that the PKD-dependent PI(4)P production, CERT and OSBP recruitment, and control of the binding of those proteins are essential for events leading to membrane fission to generate specific transport carriers ( Graham and Burd, 2011 Bankaitis et al, 2012 Campelo and Malhotra, 2012). PKD phosphorylates CERT and OSBP and they detach from the TGN ( Fugmann et al, 2007 Nhek et al, 2010). As a result of the PI(4)P synthesis, the ceramide transfer protein (CERT), oxysterol binding protein (OSBP), and FAPP1 and FAPP2 are recruited to the TGN by virtue of their Pleckstrin homology (PH) domains ( Levine and Munro, 1998, 2002 Godi et al, 2004). DAG and Arf1 recruit PKD to the TGN, PKD is then activated by PKCη and the trimeric G protein subunits Gβγ, and promotes the production of PI(4)P by activating phosphatidylinositol 4-kinase IIIβ (PI(4)KIIIβ) ( Baron and Malhotra, 2002 Diaz Anel and Malhotra, 2005 Hausser et al, 2005 Pusapati et al, 2010). Two lipids diacylglycerol (DAG) and PI(4)P are essential for the PKD-dependent transport carrier biogenesis. We are interested in membrane fission regulated by PKD, which is necessary for the generation of a specific class of transport carriers at the TGN. For example, does increase in secretory cargo affect the biogenesis of clathrin-coated or COPI vesicles and favour the formation of carriers destined for the cell surface? How is this compartmentation or spatial segregation of transport machinery achieved at the TGN? It is also not known whether different carriers, mentioned above, form continuously or if there is a competition among different classes depending on the cargo. In other words, there are components shared by processes that generate carriers destined for different membranes of the cell.

How is the biogenesis of these different classes of transport carriers regulated at the TGN? For example, phosphatidylinositol 4-phosphate (PI(4)P) and the small GTPase Arf1 are required for the formation of clathrin-coated vesicles and CARTS but protein kinase D (PKD), which promotes the production of PI(4)P at the TGN, is required only for the biogenesis of the CARTS. The TGN is also the source of secretory storage granules in secretory cells and for the production of apical- and basolateral-specific carriers in polarized cells ( Mellman and Nelson, 2008). A large number of carriers form at the trans-Golgi network (TGN) that include clathrin-coated vesicles for trafficking to the endosomes, COPI for retrograde transport to the preceding Golgi cisternae and to the endoplasmic reticulum (ER), and carriers called CARTS for the trafficking of specific cargoes to the cell surface ( De Matteis and Luini, 2008 Emr et al, 2009 Campelo and Malhotra, 2012 Valente et al, 2012 Wakana et al, 2012).

Snapgene confocal microscopy