Phosphorylation of Candida glabrata ATP-binding Cassette Transporter Cdr1p Regulates Drug Efflux Activity and ATPase Stability

Shun-Ichi Wada,Koichi Tanabe,Akiko Yamazaki,Masakazu Niimi,Yoshimasa Uehara,Kyoko Niimi,Erwin Lamping,Richard D Cannon,Brian C Monk

doi:10.1074/jbc.m408252200

Shun-Ichi Wada, Koichi Tanabe + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m408252200

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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.

Highlights

Infections caused by Candida sp. are most frequently seen in immunocompromised individuals, including AIDS and leukemia patients
We have shown that the ATPase activity of Cdr1p and the drug efflux activity of Pdh1p are regulated by phosphorylation [7]
Cdr1p cannot be phosphorylated at the position equivalent to Ser420, whereas Pdh1p phosphorylation was regulated by protein kinase A (PKA) at one or more sites not homologous to Pdr5p Ser420

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Growth Media—Growth and selection media, and the methods used to prepare and mutate S. cerevisiae strains expressing Pdh1p and Cdr1p, are described in the Supplemental Data section. Analysis of Pump Protein Phosphorylation—Glucose-starved yeast were obtained by incubation in CSM minus glucose for 3.5 h, and crude plasma membrane fractions were prepared as previously described [7] using GTED-20 buffer (10 mM Tris-HCl, pH 7.0, 0.5 mM EDTA, and 20% (v/v) glycerol) instead of the previous homogenization buffer. ATPase Assay—Purified plasma membrane fractions were prepared from cells grown in YPD to early stationary phase (A600 nm ϭ 7.0 –9.0) and oligomycin-sensitive ATPase activities of samples were measured as previously described [7]. Fluorometric Assay of Rhodamine 6G Efflux—Log phase (A600 nm ϭ 1.5) cells grown in CSM-URA (Qbiogene, Inc., Irvine, CA) medium were stored overnight on ice. The cells were harvested by centrifugation, washed twice with distilled water, and incubated in HEPES buffer (50 mM HEPES-NaOH, pH 7.0) containing 5 mM 2-deoxyglucose at 30 °C for 30 min to deplete intracellular energy levels. The Rh6G content of the eluate, combined with two 80-␮l washes with ice-cold HEPES buffer, was quantitated using a POLARstar OPTIMA (BMG Labtechnologies) fluorometer (excitation and emission wavelengths of 485 and 520 nm, respectively) with Fluostar OPTIMA software and a standard curve of Rh6G in the HEPES buffer

RESULTS

DISCUSSION

Amphotericin B