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 (YXXempty set), 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 YXXempty set signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXempty set motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biatinylation 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.