Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels.
The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.
Martin
P E
PE
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. martinpe@cf.ac.uk
Blundell
G
G
Ahmad
S
S
Errington
R J
RJ
Evans
W H
WH
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000097002:Gap Junction beta-1 Protein
D020126:Brefeldin A; D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D015739:Nocodazole; D011993:Recombinant Fusion Proteins; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human