title: SNAP-23 regulates phagosome formation and maturation in macrophages.
abstract: Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus-tagged SNAP-23 was established. These cells showed enhanced Fc receptor-mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H(+)-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.
authors.affiliation: Department of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan.
authors.forename: Chiye
authors.initials: C
authors.lastname: Sakurai
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hashimoto
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Seisuke
authors.initials: S
authors.lastname: Arai
authors.forename: Yoh
authors.initials: Y
authors.lastname: Wada
authors.forename: Ge-Hong
authors.initials: GH
authors.lastname: Sun-Wada
authors.forename: Ikuo
authors.initials: I
authors.lastname: Wada
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Hatsuzawa
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050766:Qb-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D017382:Reactive Oxygen Species; C100493:SNAP23 protein, human; D019255:NADPH Oxidases; D025262:Vacuolar Proton-Translocating ATPases