AP-1 and GGAs: Two Adaptors for TGN to Endosome Trafficking

Abstract

We have recently identified a novel family of proteins, the GGAs. The GGAs consist of four distinct domains and appear to be monomeric. Studies making use of both yeast and mammalian cells indicate that both the GGAs and the AP-1 adaptor complex facilitate trafficking from the T G N to an endosomal compartment. Our current model is that the GGAs are monomeric adaptors, with the N-terminal VHS domain involved in cargo selection, the GAT domain serving to target the GGAs to the TGN, the variable hinge-like domain interacting with clathrin, and the C-terminal gamma-adaptin ear-like domain recruiting accessory proteins onto the membrane. Using GST pulldowns, we have identified two novel potential accessory proteins, p200 and p56, which bind to both GGA ears and gamma ears. p200 is a highly conserved protein, with homologues in Drosophila, C. elegans, and S. cerevisiae. p56 is a protein predicted to have a large amount of coiled coil structure. Antibodies are currently being raised against both proteins for further characterisation.Assembly of SNARE proteins between opposing membranes is thought to be a key event in intracellular membrane fusion. In vitro, complex forming domains of the individual SNARE proteins are largely unstructured. Complex formation is associated with major conformational rearrangements and increased stability. The central domain of the ternary SNARE complex consists of a four-helix bundle, to which synaptobrevin and syntaxin each contribute a single and SNAP-25 two helices. Syntaxin and SNAP-25 can form a ibinaryi complex consisting of two syntaxins and one SNAP-25. This binary complex consists of a four-helix bundle similar to the ternary complex. To investigate the events of the assembly reaction in detail, thermodynamic and kinetic studies were carried out. The ternary SNARE complex unfolds at temperatures above SOT, whereas refolding occurs only below 65°C. A similar hysteresis between ternary complex formation and unfolding was observed in the presence of denaturant. Hence, it is likely that the pathways for ternary SNARE complex formation and unfolding are different. The binary complex is less stable and reversibly unfolds at temperatures around 45°C. Kinetic data imply that successful interaction of syntaxin and SNAP-25 must precede to allow for ternary complex formation. Together, these data suggest that the binary complex is an intermediate for ternary complex formation.Phospholipase D (PLD) activation has been implicated in the regulation of a number of cellular events including cytoskeletal change and regulated secretion. Both PLDl and 2 are regulated by PtdIns(4,5)P2 through ligation of a PtdIns(4,5)P2 specific P H domain. This domain is important in regulating both membrane association and enzyme activity. PLDl is activated by a Rho family protein, an ARF family protein and protein kinase C. BIAcore studies show the regulation to be mediated by direct, independent molecular interaction. In the RBL2H3 mast cell line antigen stimulates the activation of PLD-dependent secretion. In ce...