We investigated the degradation of the gap junction protein connexin43 in E36 Chinese hamster ovary cells and rat cardiomyocyte-derived BWEM cells. Treatment of E36 cells with the lysosomotropic amine, primaquine, for 16 h doubled the amount of connexin43 detected by immunoblotting and modestly increased the half-life of connexin43 in pulse-chase studies, suggesting that the lysosome played a minor role in connexin43 proteolysis. In contrast, treatment with the proteasomal inhibitor N-acetyl-L-leucyl-L-leucinyl-norleucinal led to a 6-fold accumulation of connexin43 and increased the half-life of connexin43 to 9 h. The role of ubiquitin in connexin43 degradation was examined in an E36-derived mutant, ts20, which contains a thermolabile ubiquitin-activating enzyme, E1. E36 and ts20 cells grown at the permissive temperature contained similar amounts of connexin43 detectable by immunoblotting. Heat treatment dramatically reduced the amount of connexin43 detected in E36 cells, while connexin43 levels in heat-treated ts20 cells did not change. E36 cells that were heat-treated in the presence of N-acetyl-L-leucyl-L-leucinyl-norleucinal did not lose their connexin43. Pulse-chase experiments showed the reversibility of the block to connexin43 degradation in ts20 cells that were returned to the permissive temperature. Finally, sequential immunoprecipitation using anti-connexin43 and anti-ubiquitin antibodies demonstrated polyubiquitination of connexin43. These results indicate that ubiquitin-mediated proteasomal proteolysis may be the major mechanism of degradation of connexin43.

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