Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis

Gap junction (GJ) channels that electrically and chemically couple neighboring cells are formed when two hemichannels (connexons) of apposed cells dock head-on in the extracellular space. Remarkably, docked connexons are inseparable under physiological conditions, and we and others have shown that GJs are internalized in whole, utilizing the endocytic clathrin machinery. Endocytosis generates double-membrane vesicles (annular GJs or connexosomes) in the cytoplasm of one of the apposed cells that are degraded by autophagosomal and, potentially, endo/lysosomal pathways. In this study, we investigated the structural motifs that mediate Cx43 GJ endocytosis. We identified three canonical tyrosine-based sorting signals of the type "YXXΦ" in the Cx43 C-terminus, two of which function cooperatively as AP-2 binding sites. We generated a set of green fluorescent protein-tagged and untagged Cx43 mutants that targeted these two sites either individually or together. Mutating both sites completely abolished Cx43-AP-2/Dab2/clathrin interaction and resulted in increased GJ plaque size, longer Cx43 protein half-lives, and impaired GJ internalization. Interestingly, Dab2, an accessory clathrin adaptor found earlier to be important for GJ endocytosis, interacts indirectly with Cx43 via AP-2, permitting the recruitment of up to four clathrin complexes per Cx43 protein. Our analyses provide a mechanistic model for clathrin's efficient internalization of large plasma membrane structures, such as GJs.