The nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator are extracellularly accessible.
Analysis of the primary sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) has suggested the presence of two predicted cytoplasmic regions of the protein which are thought to be nucleotide binding folds (NBF1 and NBF2). Previous studies have shown that purified recombinant NBF1 can form anion conducting channels in planar phospholipid bilayers [Arispe et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1539-1543] and that the bacterial His P protein (analogous to a NBF) can be extracellularly labeled with a membrane-impermeant reagent [Baichwal et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 620-624]. Based on these observations, it is reasonable to hypothesize that the NBFs from the CFTR are associated with the plasma membrane and have extracellularly-accessible regions. Direct biochemical evidence for this was obtained by determining the ability of the individual NBFs, expressed in intact Hi5 cells, to be chemically modified with the membrane-impermeant reagent NHS-biotin. The results indicate that both NBF1 and NBF2, in intact cells, can be chemically modified by extracellular NHS-biotin. The negative control, the cytosolic enzyme beta-galactosidase, was not significantly labeled under these conditions, verifying the extracellular nature of the labeling reaction. When the surface-accessibility of a NBF1 construct containing the CF-causing mutation deltaF508 was analyzed, similar labeling was observed, suggesting that the mutation does not affect this aspect of the CFTR's structure. These data support the conclusion that, under certain conditions, the NBFs are capable of spanning the plasma membrane, perhaps constituting a portion of the CFTR's ion conducting channel.
Gruis
D B
DB
Department of Veterinary Biomedical Sciences and Dalton Cardiovascular Research Center, Research Park, University of Missouri-Columbia, 65211, USA.
Price
E M
EM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012756:Sheep
D009711:Nucleotides; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator