Ubiquitin Conjugation Regulates Hypotonic Stress‐Induced Trafficking of Short ClC‐3 Channel

Abstract

Short ClC‐3 channel (sClC‐3) functions include lysosomal acidification and cell volume regulation, requiring lysosomal and surface membrane localization, respectively. As sClC‐3 is primarily a cytosolic protein how ClC‐3‐dependent volume regulation occurs remains undefined. Immunofluorescent labeling of COS‐7 cells revealed both native and expressed ClC‐3 are associated with lysosome‐like vesicles, and less than 10% resides at the cell surface. Hypotonic stress induced export trafficking of sClC‐3 to the cell surface membrane. The surface membrane exposure of sClC‐3 was transient and was followed by spontaneous or hypertonic stress‐induced internalization. Membrane‐associated sClC‐3 underwent ubiquitin conjugation followed by endocytosis of membrane sClC‐3. Inhibition of proteolysis with MG132 and Bafilomycin A potentiated both the surface retention of Flag‐sClC‐3A and the hypotonic stress‐induced Cl− current. Ubiquitin‐specific protease (USP) 7 and much less USP2 or ubiquitin C‐terminal hydrolyze, L1, mediated de‐ubiquitination of sClC‐3 in vitro. These results demonstrate that sClC‐3 can shuttle between the cytosol and cell surface membrane and the latter could contribute to cell volume regulation. Ubiquitination and de‐ubiquitination of sClC‐3 may play a role in the fine regulation of the cell volume during hypotonic stress. This study was supported by NCRR grant P20RR 15581.