Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.
Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity.
Suginta
W
W
Department of Biomedical Sciences, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK.
Karoulias
N
N
Aitken
A
A
Ashley
R H
RH
D048948:14-3-3 Proteins; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D034284:Dynamin I / Q000378:metabolism*; D004705:Endocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000502:physiology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D000199:Actins; C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D018118:Chloride Channels; C467578:Clic4 protein, rat; D017931:DNA Primers; D007473:Ion Channels; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; D034284:Dynamin I