title: Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension.
abstract: Overexpression of ATP-binding cassette (ABC) transporters is a frequent cause of multidrug resistance in cancer cells and pathogenic microorganisms. One example is the Cdr1p transporter from the human fungal pathogen Candida albicans that belongs to the pleiotropic drug resistance (PDR) subfamily of ABC transporters found in fungi and plants. Cdr1p is overexpressed in several azole-resistant clinical isolates, causing azole efflux and treatment failure. Cdr1p appears as a doublet band in western blot analyses, suggesting that the protein is post-translationally modified. We investigated whether Cdr1p is phosphorylated and the function of this modification.
  
  
  
  Phosphorylated residues were identified by MS. Their function was investigated by site-directed mutagenesis and expression of the mutants in a C. albicans endogenous system that exploits a hyperactive allele of the Tac1p transcription factor to drive high levels of Cdr1p expression. Fluconazole resistance was measured by microtitre plate and spot assays and transport activity by Nile red accumulation.
  
  
  
  We identified a cluster of seven phosphorylated amino acids in the N-terminal extension (NTE) of Cdr1p. Mutating all seven sites to alanine dramatically diminished the ability of Cdr1p to confer fluconazole resistance and transport Nile red, without affecting Cdr1p localization. Conversely, a Cdr1p mutant in which the seven amino acids were replaced by glutamate was able to confer high levels of fluconazole resistance and to export Nile red.
  
  
  
  Our results demonstrate that the NTE of Cdr1p is phosphorylated and that NTE phosphorylation plays a major role in regulating Cdr1p and possibly other PDR transporter function.
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada.
authors.forename: Sarah
authors.initials: S
authors.lastname: Tsao
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada.
authors.forename: Sandra
authors.initials: S
authors.lastname: Weber
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Cameron
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Nehme
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada.
authors.forename: Elaheh
authors.initials: E
authors.lastname: Ahmadzadeh
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC H3T 1J4, Canada martine.raymond@umontreal.ca.|Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada.
authors.forename: Martine
authors.initials: M
authors.lastname: Raymond
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
chemical_list: D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D015725:Fluconazole