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      Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTR delta F508.

      Molecular Microbiology
      ATP-Binding Cassette Transporters, genetics, metabolism, Amino Acid Sequence, Base Sequence, Biological Transport, Cloning, Molecular, Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator, physiology, DNA Primers, Endoplasmic Reticulum, Fungal Proteins, Glycoproteins, Half-Life, Molecular Sequence Data, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins

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          Abstract

          The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al. (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTR delta F508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of delta F508, providing a yeast phenotype for this mutation. In human cells, the delta F508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTR delta F508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTR delta F508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.

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