title: Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.
abstract: The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.
authors.affiliation: From the Department of Molecular and Experimental Medicine and.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Johnson
authors.affiliation: From the Department of Molecular and Experimental Medicine and.
authors.forename: Jing
authors.initials: J
authors.lastname: He
authors.affiliation: From the Department of Molecular and Experimental Medicine and.
authors.forename: Mahalakshmi
authors.initials: M
authors.lastname: Ramadass
authors.affiliation: Cancer Center Microscopy Shared Resource, University of California San Diego, La Jolla, California 92093.
authors.forename: Kersi
authors.initials: K
authors.lastname: Pestonjamasp
authors.affiliation: Light Microscopy Core Facility, The Scripps Research Institute, La Jolla, California 92037 and.
authors.forename: William B
authors.initials: WB
authors.lastname: Kiosses
authors.affiliation: From the Department of Molecular and Experimental Medicine and.
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the Department of Molecular and Experimental Medicine and scatz@scripps.edu.
authors.forename: Sergio D
authors.initials: SD
authors.lastname: Catz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005089:Exocytosis; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013047:Specific Pathogen-Free Organisms; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C507969:UNC13D protein, human; C520225:Unc13d protein, mouse
keywords: Munc13-4; NADPH oxidase; Rab; Rab11; Rab27; docking; intracellular trafficking; neutrophil; recycling endosome; super-resolution microscopy