Targeting motifs and functional parameters governing the assembly of connexins into gap junctions.
To study the assembly of gap junctions, connexin--green-fluorescent-protein (Cx--GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26-- and Cx32--GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32--GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx--GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.
Martin
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PE
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. martinpe@cardiff.ac.uk
Steggles
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J
Wilson
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C
Ahmad
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Evans
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WH
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017629:Gap Junctions / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D017630:Connexins; D018076:DNA, Complementary; C000607042:GJB2 protein, human; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000072259:Connexin 26; D049452:Green Fluorescent Proteins