SCAMP2 interacts with Arf6 and phospholipase D1 and links their function to exocytotic fusion pore formation in PC12 cells.
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.
Liu
Lixia
L
Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA.
Liao
Haini
H
Castle
Anna
A
Zhang
Jie
J
Casanova
James
J
Szabo
Gabor
G
Castle
David
D
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D005089:Exocytosis / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010446:Peptide Fragments / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D000089982:ADP-Ribosylation Factor 6; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D010446:Peptide Fragments; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; C106801:phospholipase D1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat; D009638:Norepinephrine