Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal

Gap junctions are specialized cell–cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present at amino acids 286–289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXXØ signal. Indeed, expression of a Cx43–Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXØ motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43–Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the protein. Furthermore, the internalization of Cx43 induced by Cx43–Ub was shown to depend on its interaction with epidermal growth factor receptor substrate 15.