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 int egral membrane proteins. The molecular basis for their cellular localizatio n and ion channel activity remains unclear. To help in addressing these pro blems, we identified novel rat brain CLIC4 (p64H1) binding partners by affi nity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha -tubulin, beta -actin, creatine kinase and tw o 14-3-3 isoforms: the interactions are confirmed in vivo by immunoprecipit ation. Gel overlay and reverse pull-down assays indicate that the binding o f CLIC4 to dynamin I and 14-3-3 zeta is direct. In HEK-293 cells, biochemic al and immunofluorescence analyses show partial co-localization of recombin ant CLIC4 xith caveolin and with functional caveolae, which is consistent w ith a dynamin-associated role for CLIC4 in caveolar endocytosis. We specula te that brain CLIC4 might be involved in the dynamics of neuronal plasma me mbrane microdomains (micropatches) containing caveolin-like proteins and mi ght also have other cellular roles related to membrane trafficking. Our res ults provide the basis for new hypotheses concerning novel ways in which CL IC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity.