SCAMP2 Interacts with Arf6 and Phospholipase D1 and Links Their Function to Exocytotic Fusion Pore Formation in PC12 Cells

Abstract

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores. INTRODUCTIONNeuroendocrine cells such as PC12 cells have been used widely to define the molecular machinery controlling exocytosis. Their secretory granules, dense core vesicles (DCVs), undergo rapid fusion with the plasma membrane in response to stimulation. This process involves several steps, including docking/tethering DCVs to the plasma membrane, priming the secretory machinery, assembling trans-SNARE complexes, and triggering fusion (Chapman, 2002;Jahn et al., 2003;Martin, 2003). Furthermore, experiments using PC12 cells have provided considerable insight concerning the molecular organization of DCV fusion sites (Lang et al., 2001;Holroyd et al., 2002;Liu et al., 2002;Han et al., 2004) and interactions between SNARE proteins and synaptotagmins, candidate calcium sensors and effectors of membrane fusion (Zhang et al., 2002;Tucker et al., 2003;Bai et al., 2004b).Several studies have been instrumental in showing that phosphatidic acid (PA) and inositol phospholipids, especially phosphatidylinositol 4,5-bisphosphate (PIP 2 ), make critical contributions in promoting DCV exocytosis (Hay et al., 1995;Holz et al., 2000;Vitale et al., 2001Vitale et al., , 2002Tucker et al., 2003;Bai et al., 2004a). PIP 2 synthesis at the cell surface is supported by type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K-1␥), which is activated by Arf6 and by PA (Honda et al., 1999;Aikawa and Martin, 2003). PA is produced by phospholipase D (PLD), which is also activated by Arf6 and by PIP 2 (Sciorra et al., 1999;Dana et al., 2000), and like PIP5K-1␥ is concentrated at the PC12 cell's plasma membrane (Vitale et al., 2001(Vitale et al., , 2002. Thus, Arf...

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

“…Alternatively, because SERT/SCAMP2-positive compartments, in particular in RN46A cells, appear to be located very close to the plasma membrane, their co-localization with syntaxin 1A could represent SNARE complex-dependent attachment sites for recycling vesicles. This possibility is supported by a previous study showing that in PC12 cells SCAMP2 associates with dense core vesicles at putative vesicle fusion sites, where it co-localizes with syntaxin 1A in the plasma membrane (25,37).…”

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…As noted by Liu et al (2005), a characteristic feature of these proteins is that they are concentrated on intracellular membranes but exercise a function at the PM. This seems to be borne out in the case of SCAMP1 to SCAMP4, which are found predominantly on the membranes of exocrine and endocrine storage granules and post-Golgi recycling carriers (Guo et al, 2002).…”

“…Only in the case of SCAMP2 is a higher proportion detectable at the PM (;25% of total cell SCAMPs). In PC12 cells, a neuroendocrine cell line, SCAMP2 proteins are localized to specific foci randomly distributed throughout the cell surface, and these represent part of a fusion pore complex for dense core vesicles (Liu et al, 2005). This function of SCAMP2 involves the E peptide, a highly conserved segment lying at the cytosolic surface of the molecule and linking the second and third transmembrane domains.…”

“…SCAMPS are found in both the TGN and the endosomal recycling compartment in NRK cells, and they appear to be concentrated within the motile population of early and recycling endosomes . Thus, SCAMPs appear to be reliable indicators for postGolgi endocytic and exocytic trafficking in animal cells (FernandezChacon and Sudhof, 2000;Castle and Castle, 2005;Liu et al, 2005).…”

Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans

“…This notion is supported by the multiple interactions of TVPs with lipids, notably cholesterol (8) or various components of the recycling machinery including soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) (9)(10)(11), dynamin (12,13), adaptor proteins (14), and eps15 homology (EH)-domain proteins (15). In addition, it has been postulated that TVPs are directly responsible for microvesicle formation (8,16,17) and are involved in fusion pore formation (18)(19)(20)(21).…”