Degradation of connexin43 gap junctions involves both the proteasome and the lysosome.
Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1-3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of approximately 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.
Laing
J G
JG
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Tadros
P N
PN
Westphale
E M
EM
Beyer
E C
EC
D000818:Animals; D020126:Brefeldin A; D002478:Cells, Cultured; D018031:Connexin 43 / Q000302:isolation & purification / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008954:Models, Biological; D008985:Monensin / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats
D018031:Connexin 43; D003517:Cyclopentanes; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D020126:Brefeldin A; D008985:Monensin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex