FIGURE 4:

Sorting-motif mutant YA/VD-Cx43 restores defective gap junction assembly by incorporating into gap junctions and inhibiting clathrin-mediated endocytosis. (A) BxPC3 and Capan-1 cells expressing endogenous Cx43 were transiently transfected with YA/VD-Cx43-Myc (YA/VD-Myc) and immunostained for Myc and Cx43. Note the robust colocalization of YA/VD-Cx43-Myc with total Cx43 (WT) and the formation of gap junctions. (B) Coimmunoprecipitation of WT-Cx43 and YA/VD-Cx43. HeLa cells were transfected with YA/VD-Cx43EGFP (YA/VD-GFP), WT-Cx43-Myc (WT-Myc), WT-Cx43EGFP (WT-GFP), and YA/VD-Cx43-Myc (YA/VD-Myc) either alone or in the various combinations indicated. GFP-tagged proteins were pulled down with immobilized anti-GFP antibody. Note that YA/VD-Cx43-Myc coimmunoprecipitated with WT-Cx43EGFP and WT-Cx43-Myc coimmunoprecipitated with YA/VD-Cx43EGFP. (C) Capan-1 cells, expressing endogenous Cx43 along with retrovirally introduced WT-Cx43 or YA/VD-Cx43, were seeded as single cells and immunostained for Cx43 (red) and either clathrin, Caveolin 1 (Cav1), or Lamp1 (green). Note that puncta composed of both endogenous Cx43 and retrovirally introduced WT-Cx43 colocalize extensively with clathrin and Lamp1 but not with Cav1. In contrast, puncta composed of both endogenous Cx43 and retrovirally introduced YA/VD-Cx43 fail to colocalize with clathrin, but colocalize discernibly with Cav1 at cell borders.