Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast.
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (delta F508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when delta F508 was introduced into NBD1. We isolated two delta F508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the delta F508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by delta F508 can be partially corrected by additional alterations in the protein.
Teem
J L
JL
Howard Hughes Medical Institute, University of Iowa College of Medicine, Department of Internal Medicine, Iowa City 52242.
Berger
H A
HA
Ostedgaard
L S
LS
Rich
D P
DP
Tsui
L C
LC
Welsh
M J
MJ
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D003550:Cystic Fibrosis / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005656:Fungal Proteins / Q000235:genetics*; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D006023:Glycoproteins; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D000838:Anions; C505032:CFTR protein, human; D005656:Fungal Proteins; D006023:Glycoproteins; D007473:Ion Channels; D008565:Membrane Proteins; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011494:Protein Kinases; D000251:Adenosine Triphosphatases