RRC ID 68665
Author Sakurai C, Hashimoto H, Nakanishi H, Arai S, Wada Y, Sun-Wada GH, Wada I, Hatsuzawa K.
Title SNAP-23 regulates phagosome formation and maturation in macrophages.
Journal Mol Biol Cell
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.
Volume 23(24)
Pages 4849-63
Published 2012-12-1
DOI 10.1091/mbc.E12-01-0069
PII mbc.E12-01-0069
PMID 23087210
PMC PMC3521691
MeSH Animals Blotting, Western Cell Line Fluorescence Resonance Energy Transfer Humans Luminescent Proteins / genetics Luminescent Proteins / metabolism Lysosomes / metabolism Macrophages / cytology Macrophages / metabolism* Mice Microscopy, Confocal NADPH Oxidases / metabolism Phagocytosis Phagosomes / metabolism* Protein Binding Qa-SNARE Proteins / metabolism Qb-SNARE Proteins / genetics Qb-SNARE Proteins / metabolism* Qc-SNARE Proteins / genetics Qc-SNARE Proteins / metabolism* R-SNARE Proteins / metabolism RNA Interference Reactive Oxygen Species / metabolism Vacuolar Proton-Translocating ATPases / metabolism
IF 3.791
Human and Animal Cells J774.1