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Compartmentalization of ClC‐3 and TNF‐induced Superoxide Production
Author(s) -
Bacon Matthew,
Nguyen HongNgan,
Choi Hyehun,
Ettinger Nicholas,
Lamb Fred
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1046.7
Subject(s) - endosome , rab , microbiology and biotechnology , small gtpase , vesicle , chemistry , green fluorescent protein , gtpase , mcherry , compartmentalization (fire protection) , signal transduction , biology , biochemistry , intracellular , membrane , gene , enzyme
ClC‐3 is required for NADPH Oxidase (Nox)‐dependent endosomal superoxide (O 2 ‐ ) production and NF‐kB activation in response to cytokines. We co‐localized ClC‐3, Rab‐GTPases and O 2 ‐ following TNFa treatment of HEK293 cells. ClC‐3, Rab5(A, B, C), Rab7 (GFP, mCherry, RFP tags) and O 2 ‐ (ClC‐3‐HyPer fusion protein) were visualized by confocal microscopy. Expressed ClC‐3‐GFP was precipitated using bead‐immobilized GFP‐binding protein under detergent‐free conditions and Western analysis assessed ClC‐3‐Rab associations. An NF‐kB luciferase reporter quantified activation. Alone, ClC‐3 localizes to enlarged vesicles. Tagged ClC‐3 associates with Rab5A, B, and C in early endosomes and vesicles expressing both proteins were small. ClC‐3‐GFP pulled down primarily endogenous Rab5A. Dominant negative (S34N) and constitutively active (Q79L) Rab5A altered the distribution of ClC‐3 and drastically reduced NF‐kB activation by TNFa. The S34N mutant induced discrete small vesicles while Q79L yielded tight multi‐vesicle clusters. ClC‐3 and Rab7 co‐expression produced larger late endosomal vesicles and the overlap of ClC‐3 with Rab7 was more extensive than with Rab5. Under resting conditions ClC‐3‐HyPer co‐localized best with Rab7 vesicles and this signal was enhanced by TNFa and quenched by N‐acetylcysteine. Localization of O 2 ‐ with Rab5 is ongoing. These data demonstrate that ClC‐3 is both an early and late endsosomal protein. In early endosomes it is trafficked by Rab5A and altered function of this GTPase disrupts both localization and TNFa signaling. In resting cells, basal O 2 ‐ production occurs primarily in late endosomes (Rab7). Future work aims to determine if O 2 ‐ is produced in endocytic vesicles or early endosomes following activation of TNFa receptors.