Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTR delta F508.
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.
Paddon
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C
Cell Biology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, England.
Loayza
D
D
Vangelista
L
L
Solari
R
R
Michaelis
S
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins; D006207:Half-Life; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D005656:Fungal Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator