Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast

Teem, John L.; Berger, Herbert A.; Ostedgaard, Lynda S.; Rich, Devra P.; Tsui, Lap‐Chee; Welsh, Michael J.

doi:10.1016/0092-8674(93)90233-g

Cited by 176 publications

(161 citation statements)

References 33 publications

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“…In both cases, the results indicate that the mutations are kinetic in nature but with opposite characteristics; R553M is a superfolder, whereas ⌬F508 is an ineffective folder. Results in vivo indicate that the R553M/⌬F508 CFTR double mutant only partially corrects the ⌬F508 maturation defect, and the fully mature mutant protein is only partially functional (16). The current results indicate that the diminished function of R553M observed in vivo is not due to a loss of the ability of NBD1 to bind ATP.…”

Section: Temperature-dependent Folding Yield and Off Pathway Kineticsmentioning

confidence: 50%

“…Altered function may then be due to deficient catalysis or coupling between the nucleotide site and other parts of the protein. The fact that in the in vitro folding system the ATP binding function of the double mutant is normal and the suppression is complete, as opposed to the partial function and suppression in vivo (16), indicates that although NBD1 folding reliably reports the fundamental effects of the mutations the situation is more complex in the cell, and subtle secondary effects may not always be evident in the in vitro system. For example, the reduction of the folding yield by the ⌬F508 mutation is not as pronounced as in vivo, perhaps indicating that cellular mechanisms for identifying malfolded conformers are more efficient than in vitro aggregation.…”

Section: Temperature-dependent Folding Yield and Off Pathway Kineticsmentioning

confidence: 99%

“…Two mutations at this position, R553M and R553Q, revert the mating phenotype of a ⌬F508 STE6-CFTR chimera in yeast (16). Moreover, introduction of the second site mutations into human ⌬F508-CFTR partially corrected the maturation defect, resulting in an increase of functional CFTR at the membrane (16). Recently two groups have demonstrated that when cells expressing ⌬F508-CFTR are grown in the presence of glycerol the maturation and functional defects can be largely rescued (17,18).…”

mentioning

confidence: 99%

“…In a German pancreas-insufficient patient homozygous for ⌬F508 with typical gastrointestinal and pulmonary disease but sweat chloride in the normal range a second site mutation of R553Q was found on one ⌬F508-CFTR allele (15). Two mutations at this position, R553M and R553Q, revert the mating phenotype of a ⌬F508 STE6-CFTR chimera in yeast (16). Moreover, introduction of the second site mutations into human ⌬F508-CFTR partially corrected the maturation defect, resulting in an increase of functional CFTR at the membrane (16).…”

mentioning

confidence: 99%

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Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

Strickland

Thomas

1997

Journal of Biological Chemistry

133

121

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A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, ⌬F508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane. Here we present evidence that the ⌬F508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD). Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant ⌬F508 NBD1 in vitro. In contrast, glycerol, which promotes ⌬F508-CFTR maturation in vivo, increases the folding yield of NBD1⌬F and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability. Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of ⌬F508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. These results demonstrate that ⌬F508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding. The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.

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Section: Temperature-dependent Folding Yield and Off Pathway Kineticsmentioning

confidence: 50%

Section: Temperature-dependent Folding Yield and Off Pathway Kineticsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

Strickland

Thomas

1997

Journal of Biological Chemistry

133

121

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“…A chimeric protein formed by replacing NBD1 of the ABC transporter Ste6p with the analogous region of the CFTR was able to complement the mating defect of a ste6 strain, but only if the F508 position was intact (Teem et al, 1993). A. Complementation of ⌬ycf1 by CF-associated Ycf1p alleles.…”

Section: Mutagenesis Of the Ycf1p Nbd1 Regionmentioning

confidence: 99%

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Wemmie

Moye‐Rowley

1997

Molecular Microbiology

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SummaryYcf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein.These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

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Complex cystic fibrosis allele R334W-R1158X results in reduced levels of correctly processed mRNA in a pancreatic sufficient patient

et al. 1996

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CFTR alleles containing two mutations have been very rarely found in cystic fibrosis (CF) patients. They provide an opportunity to study the effect of two in cis‐interacting gene defects on gene expression. Here, we describe a three‐generation CF family with a complex CFTR allele that has not been previously described, containing the missense mutation R334W in exon 7 and the nonsense mutation R1158X in exon 19. Lymphocyte RNA analysis showed that (1) the mRNA corresponding to the complex allele is present although at markedly reduced levels; and (2) the nonsense mutation does not lead to detectable skipping of exon 19. The clinical picture of the patients with the genotype R334W‐R1158X/ΔF508 is characterized by pancreatic sufficiency and an atypical course of the disease. © 1996 Wiley‐Liss, Inc.

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Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast

Cited by 176 publications

References 33 publications

Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Complex cystic fibrosis allele R334W-R1158X results in reduced levels of correctly processed mRNA in a pancreatic sufficient patient

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