title: Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability.
abstract: Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.
authors.affiliation: Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shin-juku-ku, Tokyo 162-8640, Japan.
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Wada
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanabe
authors.forename: Akiko
authors.initials: A
authors.lastname: Yamazaki
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Uehara
authors.forename: Kyoko
authors.initials: K
authors.lastname: Niimi
authors.forename: Erwin
authors.initials: E
authors.lastname: Lamping
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D001692:Biological Transport; D041221:Candida glabrata / Q000187:drug effects / Q000378:metabolism*; D026141:Drug Resistance, Multiple, Fungal; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases