title: Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold.
abstract: The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.
authors.affiliation: Unité de biochimie physiologique, Institut des sciences de la vie, Université catholique de Louvain, Croix du Sud 5-15, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Cédric Pety
authors.initials: CP
authors.lastname: de Thozée
authors.forename: Susan
authors.initials: S
authors.lastname: Cronin
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Goj
authors.forename: John
authors.initials: J
authors.lastname: Golin
authors.forename: Michel
authors.initials: M
authors.lastname: Ghislain
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism*; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000494:pharmacology; D025141:Drug Resistance, Fungal; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011755:Pyrophosphatases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D018389:Codon, Nonsense; C092438:PDR5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide; D011755:Pyrophosphatases; C023278:uridine triphosphatase