DEGRADATION OF CONNEXIN43 GAP-JUNCTIONS INVOLVES BOTH THE PROTEASOME AND THE LYSOSOME

Intercellular communication may be modulated by the rather rapid turno ver and degradation of gap junction proteins, since many connexins hav e half-lives of 1-3 h. While several morphological studies have sugges ted that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-pr oteasome pathway in proteolysis of the connexin43 polypeptide. The pre sent study was designed to reconcile these observations by examining t he degradation of connexin43-containing gap junctions in rat heart-der ived BWEM cells. After treatment of BWEM cells with Brefeldin A to pre vent transport of newly synthesized connexin43 polypeptides to the pla sma membrane, quantitative confocal microscopy showed the disappearanc e of immunoreactive connexin43 from the cell surface with a half-life of similar to 1 h. This loss of connexin43 immunoreactivity was inhibi ted by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactac ystin) or lysosomal inhibitors (leupeptin or E-64). Similar results we re seen when connexin43 export was blocked with monensin. After treatm ent of BWEM cells with either proteasomal or lysosomal inhibitors alon e, immunoblots showed accumulation of connexin43 in both whole cell ly sates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lact acystin-treated cells, but in vesicles in BWEM cells treated with lyso somal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions. (C ) 1997 Academic Press.