Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic β cell-specific cholesterol homeostasis in insulin secretion as well as in β cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in β cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in β cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse β cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.

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...

The epidermal growth factor receptor (EGFR) is targeted for lysosomal degradation by ubiquitin-mediated interactions with the ESCRTs (endosomal-sorting complexes required for transport) in multivesicular bodies (MVBs). We show that secretory carrier membrane protein, SCAMP3, localizes in part to early endosomes and negatively regulates EGFR degradation through processes that involve its ubiquitylation and interactions with ESCRTs. SCAMP3 is multimonoubiquitylated and is able to associate with Nedd4 HECT ubiquitin ligases and the ESCRT-I subunit Tsg101 via its PY and PSAP motifs, respectively. SCAMP3 also associates with the ESCRT-0 subunit Hrs. Depletion of SCAMP3 in HeLa cells by inhibitory RNA accelerated degradation of EGFR and EGF while inhibiting recycling. Conversely, overexpression enhanced EGFR recycling unless ubiquitylatable lysines, PY or PSAP motifs in SCAMP3 were mutated. Notably, dual depletions of SCAMP3 and ESCRT subunits suggest that SCAMP3 has a distinct function in parallel with the ESCRTs that regulates receptor degradation. This function may affect trafficking of receptors from prelysosomal compartments as SCAMP3 depletion appeared to sustain the incidence of EGFR-containing MVBs detected by immunoelectron microscopy. Together, our results suggest that SCAMP3, its modification with ubiquitin, and its interactions with ESCRTs coordinately regulate endosomal pathways and affect the efficiency of receptor down-regulation. INTRODUCTIONThe internalization of cell surface receptors and transporters coupled to degradation or recycling is critical for nutrient uptake and regulating cell signaling. The epidermal growth factor receptor (EGFR) has been studied extensively as a prototypical receptor that is targeted for lysosomal degradation after ligand-stimulated internalization. Disrupted degradation of the EGFR and other receptors has been linked to the pathogenesis of many diseases including several cancers. An essential element in the down-regulation process is ubiquitin conjugation to EGFR by Cbl, an E3 ubiquitin ligase (reviewed in Marmor and Yarden, 2004). It is thought that the ubiquitin enables successive passage of the receptor to the endosomal-sorting complexes required for transport: ESCRT-0, I, II, and III. These complexes are evolutionarily conserved and specialize in targeting and packaging of membrane proteins into intraluminal vesicles (ILVs) upstream of lysosomal degradation. The ESCRT-0 complex is composed of Hrs, STAM, and Eps15b and is enriched in flat clathrin patches on early endosomes via interactions involving Hrs' FYVE and coiled-coiled domains (Raiborg et al., 2001a(Raiborg et al., , 2002Bache et al., 2003b;Roxrud et al., 2008). Ubiquitin-interacting motifs (UIMs) in ESCRT-0 subunits bind ubiquitylated ligands such as EGFR; interaction of Hrs with the UEV domain of ESCRT-I protein Tsg101 is thought to facilitate passage of the receptor to ESCRT-I (Polo et al., 2002;Bache et al., 2003a;Lu et al., 2003). ESCRT-I in turn recruits ESCRT-II, which may function in sequential liga...

Intracellular transport and secretion of salivary proteins are major activities of salivary acinar cells. While the major intracellular pathway followed by salivary proteins following their synthesis has been described previously, there is only limited understanding of how this process is regulated at the molecular level. Studies of salivary proteins, especially proline-rich proteins, expressed in an endocrine cell line have begun to provide insight regarding intermolecular interactions during transport and the role played by structural signals during intracellular sorting. Analysis of the secretion of newly synthesized salivary proteins in parotid tissue has shown that there are multiple pathways of discharge from acinar cells. While granule exocytosis is the major pathway, at least two other pathways that export salivary proteins have been found to originate from maturing secretion granules. These pathways may contribute to other acinar cell functions, including secretion of proteins in the absence of acute stimulation and support of the secretory process for fluid and electrolytes.

In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of (35)S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide-containing domain.