Perturbation of a very late step of regulated exocytosis by a secretory carrier membrane protein (SCAMP2)-derived peptide.
Secretory carrier membrane proteins (SCAMPs) are conserved four transmembrane-spanning proteins associated with recycling vesicular carriers. In mast cells, as in other cell types, SCAMPs 1 and 2 are present in secretory granule membranes and other intracellular membranes. We now demonstrate a population of these SCAMPs in plasma membranes. Although small, this population partially colocalizes with SNARE proteins SNAP-23 and syntaxin 4. A fraction of SCAMPs 1 and 2 also coimmunoprecipitates with SNAP-23. An oligopeptide, E peptide, within the cytoplasmic segment linking the second and third transmembrane spans, particularly of SCAMP2, potently inhibits exocytosis in streptolysin O-permeabilized mast cells. The E peptide is unique to SCAMPs and highly conserved among SCAMP isoforms, and minor changes in its sequence abrogate inhibition. It blocks fusion beyond the putative docking step where granules contact the cell surface and each other during compound exocytosis. Blockade is also beyond Ca(2+)/ATP-dependent relocation of SNAP-23, which regulates compound exocytosis, and beyond ATP-dependent priming of fusion. Kinetic ordering of exocytotic inhibitors has shown that E peptide acts later than other perturbants at a stage closely associated with membrane fusion. These findings identify a new reagent for analyzing the final stage of exocytosis and point to the likely action of SCAMP2 in this process.
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Zhenheng
Z
Guo
Lixia
L
Liu
David
D
Cafiso
David
D
Castle
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008297:Male; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000502:physiology*; D011233:Precipitin Tests; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C466776:SCAMP2 protein, human