Phosphorylation of Candida glabrata ATP-binding Cassette Transporter Cdr1p Regulates Drug Efflux Activity and ATPase Stability
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m408252200
ISSN1083-351X
AutoresShun‐ichi Wada, Koichi Tanabe, Akiko Yamazaki, Masakazu Niimi, Yoshimasa Uehara, Kyoko Niimi, Erwin Lamping, Richard D. Cannon, Brian C. Monk,
Tópico(s)Cancer therapeutics and mechanisms
ResumoFungal 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 Ser307, which borders the nucleotide binding domain 1 ABC signature motif, and Ser484, 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 Ser484 and Ser307. 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. 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 Ser307, which borders the nucleotide binding domain 1 ABC signature motif, and Ser484, 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 Ser484 and Ser307. 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. Infections caused by Candida sp. are most frequently seen in immunocompromised individuals, including AIDS and leukemia patients. Candida albicans remains the leading cause of candidiasis, but the incidence of drug-resistant non-albicans Candida infections has become an increasingly significant clinical problem. Candida glabrata is among the most common of these pathogens (1Fidel Jr., P.L. Vazquez J.A. Sobel J.D. Clin. Microbiol. Rev. 1999; 12: 80-96Crossref PubMed Google Scholar), with many clinical isolates showing a 16- to 64-fold higher minimum inhibitory concentration (MIC) 1The abbreviations used are: MIC, minimum inhibitory concentration; FLC, fluconazole; PDR, pleiotropic drug resistance; ABC, ATP-binding cassette; PKA, protein kinase A; NBD, nucleotide binding domain; CSM, complete synthetic medium; Rh6G, rhodamine 6G; p-PKAs, phospho-(Ser/Thr) PKA substrate; p-Akts, phospho-Akt substrate; p-Thr, phospho-Threonine; BIM, bisindolylmaleimide I. of fluconazole (FLC) than C. albicans (2Diekema D.J. Messer S.A. Brueggemann A.B. Coffman S.L. Doern G.V. Herwaldt L.A. Pfaller M.A. J. Clin. Microbiol. 2002; 40: 1298-1302Crossref PubMed Scopus (357) Google Scholar). Azole drugs such as FLC and itraconazole, which target lanosterol 14α-demethylase and block the synthesis of ergosterol, are well tolerated and widely used in the treatment of fungal disease. They are, however, fungistatic substrates of pleiotropic drug resistance (PDR) family ATP-binding cassette (ABC) transporters, and resistant fungi that overexpress these pumps are frequently isolated in the clinic (3Lupetti A. Danesi R. Campa M. Tacca DelM. Kelly S. Trends Mol. Med. 2002; 8: 76-81Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). The C. glabrata PDR family ABC transporters Cdr1p and Pdh1p, which efflux azole agents and structurally unrelated compounds, are among the primary causes of the intrinsic resistance of C. glabrata to azole drugs (4Miyazaki H. Miyazaki Y. Geber A. Parkinson T. Hitchcock C. Falconer D.J. Ward D.J. Marsden K. Bennett J.E. Antimicrob. Agents. Chemother. 1998; 42: 1695-1701Crossref PubMed Google Scholar, 5Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. Antimicrob. Agents. Chemother. 1999; 43: 2753-2765Crossref PubMed Google Scholar, 6Sanglard D. Ischer F. Bille J. Antimicrob. Agents. Chemother. 2001; 45: 1174-1183Crossref PubMed Scopus (209) Google Scholar, 7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8Izumikawa K. Kakeya H. Tsai H-F. Grimberg B. Bennett J.E. Yeast. 2003; 20: 249-261Crossref PubMed Scopus (54) Google Scholar). The two pumps have >70% amino acid sequence identity and transport a similar spectrum of substrates, but Cdr1p had greater drug efflux activity for most substrates. Structural information on fungal single subunit ABC transporters is rudimentary, and the molecular and regulatory features that determine their enzyme activity and substrate specificity are poorly understood. Such information is required for the rational design of pump inhibitors and antifungal drugs that are not pump substrates. There are few reports of the effects of post-translational modification on the activity of fungal ABC transporters. Serine 420, a casein kinase-dependent phosphorylation site that determines Saccharomyces cerevisiae Pdr5p turnover (9Decottignies A. Owsianik G. Ghislain M. J. Biol. Chem. 1999; 274: 37139-37146Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), is the only experimentally proven fungal ABC transporter phosphorylation site. We have shown that the ATPase activity of Cdr1p and the drug efflux activity of Pdh1p are regulated by phosphorylation (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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. More than one type of phosphorylation therefore occurs in fungal PDR family pumps. This report describes the differential regulation of C. glabrata Cdr1p and Pdh1p expressed in S. cerevisiae. Immunological, physiological, and biochemical methods were applied to site-directed mutants in putative phosphorylation sites and to kinase-deletion mutants. PKA catalytic subunit isoforms differentially affected pump phosphorylation, and the effects of the phosphorylation of two putative novel sites in Cdr1p were determined. A phosphorylation site adjacent to the Cdr1p ABC signature motif in nucleotide binding domain 1 (NBD1) affected the extent of multidrug efflux and the in vitro stability of ATPase activity while mutation of another cytoplasmic site nearer the membrane diminished transport at low glucose concentrations. Mutation in both sites eliminated the pumping activity of Cdr1p, whereas intragenic suppressors obtained by exposing the double mutant to FLC partially restored pump function. The Cdr1p NBD1 is a functional sensor of cell physiology and stress that may regulate interactions between homodimers. 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. Drug Susceptibility Assays—Agar diffusion assays on YPD (Qbiogene, Irvine, CA) agar plates, microplate MIC assays in HEPES and MES-buffered CSM (Qbiogene, Inc.) were performed, and the drugs and chemical compounds used were obtained as previously described (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) 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. The membrane fractions were analyzed by SDS-PAGE and immunoblotting as previously described (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) using 1/2000 dilutions of anti-phospho (serine/threonine) protein kinase A substrate antibody, anti-phospho-(serine/threonine) Akt substrate antibody, and anti-phospho threonine antibody from Cell Signaling Technology (Beverly, MA), together with anti-rabbit IgG conjugated with horseradish peroxidase secondary antibody (Amersham Biosciences). ATPase Assay—Purified plasma membrane fractions were prepared from cells grown in YPD to early stationary phase (A600 nm = 7.0–9.0) and oligomycin-sensitive ATPase activities of samples were measured as previously described (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Fluorometric Assay of Rhodamine 6G Efflux—Log phase (A600 nm = 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 then 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 cells were preloaded with 15 μm Rh6G for 30 min, washed twice, and resuspended in HEPES buffer at A600 nm = 15 (1.5 × 108 cells ml-1). Cell samples (50 μl) were incubated at 30 °C for 5 min and 50 μl of glucose at twice the final concentration added to start the reaction. After 8 min the cells in 80-μl samples were removed by passage through a Multi-well Filter plate (Acro Prep, Pall Corp.) placed on a Multiscreen resist vacuum manifold (Millipore), with a 96-well black flat-bottom microtiter collection plate (BMG Labtechnologies GmbH, Offenburg, Germany) underneath. The Rh6G content of the eluate, combined with two 80-μl 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. Antibodies Recognizing Cdr1p or Pdh1p Phosphorylations—We have previously described the S. cerevisiae strains CDR1-AD and PDH1-AD, which contain a mutant Pdr1p transcriptional regulator and thus constitutively heterologously hyperexpress Cdr1p and Pdh1p, respectively, from the PDR5 promoter (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Decottignies A. Grant A.M. Nichols J.W. de Wet H. McIntosh D.B. Goffeau A. J. Biol. Chem. 1998; 273: 12612-12622Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Cdr1p and Pdh1p were hyperexpressed at comparable levels (∼10% of plasma membrane protein) and were readily distinguished as heterologous plasma membrane proteins on Coomassie Blue-stained SDS-polyacrylamide gels due to the deletion of seven similar-sized, endogenous pump proteins. Each strain also showed the expected high level resistance to azole drugs (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The pumps heterologously expressed in S. cerevisiae were therefore correctly folded and should be post-translationally regulated as in C. glabrata, because these two closely related yeasts have similar intracellular molecular machinery. The phosphorylation of both Cdr1p and Pdh1p was detected by 32P labeling, but the signals were too weak to support more detailed experiments. Neither Cdr1p nor Pdh1p reacted with phospho-protein kinase C substrates antibody or phospho-tyrosine antibodies (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). An anti-phospho-(Ser/Thr) PKA substrate (p-PKAs) antibody detected phosphorylation of Pdh1p but not Cdr1p or background ABC transporters in Western blots of plasma membrane preparations (Fig. 1). Antibodies with different specificities detected novel phosphorylation sites in Cdr1p and Pdh1p. The anti-phospho-Akt substrate (p-Akts) and anti-phospho-threonine (p-Thr) antibodies recognized the phosphorylation of Cdr1p and Pdh1p expressed in all growth phases of YPD culture (Fig. 1, black arrowheads). Cdr1p expression in CDR1-AD decreased slightly in stationary phase (Fig. 1, CBB staining), whereas the p-Akts antibody recognition signal increased. Conversely, the p-Thr antibody gave a stronger signal in the log phase preparation. Cdr1p is therefore multiply phosphorylated at discrete p-Akts and p-Thr antibody recognition sites. Pdh1p phosphorylation in the PDH1-AD strain was readily detected by the p-PKAs, p-Akts, and p-Thr antibodies, with constant signals obtained throughout all growth phases of YPD culture. The recognition of the 100-kDa protein, which co-migrates with the constitutively expressed plasma membrane ATPase Pma1p (Fig. 1, white arrowheads) by the three antibodies provided an internal control for both the CDR1-AD and PDH1-AD preparations. Glucose-sensitive Phosphorylation of Pdh1p and Cdr1p—We previously found that the Cdr1p and Pdh1p pumps were extensively dephosphorylated after a few hours of glucose starvation and immediately rephosphorylated when 2% glucose was added (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The glucose concentration dependence of each rephosphorylation was therefore determined (Fig. 2A). Early stationary phase YPD cultures were glucose-starved for 3.5 h and then treated for 10 min with 10 μm, 1 mm, or 100 mm glucose. Rephosphorylation of Cdr1p was detected with p-Akts and p-Thr antibodies, but only after exposure to 100 mm glucose. Pdh1p remained partially phosphorylated after glucose starvation, but all three antibodies detected glucose-dependent rephosphorylation at a glucose concentration (10 μm) that failed to induce Pma1p phosphorylation. The different phosphorylation patterns detected with each antibody indicated that Pdh1p was also rephosphorylated at multiple sites. The >100-fold differential in the glucose-dependence of Cdr1p and Pdh1p phosphorylation suggested that the two pumps were modified by different kinases/phosphatases or had sites with different susceptibilities to phosphorylation. Pdh1p phosphorylation at p-PKAs sites is inhibited by PKA inhibitors (7Wada S. Niimi M. Niimi K. Holmes A.R. Monk B.C. Cannon R.D. Uehara Y. J. Biol. Chem. 2002; 277: 46809-46821Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). A panel of PKA catalytic subunit (Tpk) single and double deletion mutants was used to identify the TPK gene product that regulated Pdh1p phosphorylation in PDH1-AD. The three genes (TPK1, -2, and -3) encoding these PKA catalytic subunit isoforms can be deleted without lethality, either singly or up to two at a time (11Toda T. Cameron S. Sass P. Zoller M. Wigler M. Cell. 1987; 50: 277-287Abstract Full Text PDF PubMed Scopus (533) Google Scholar). In YPD culture, identical Pdh1p phosphorylation signals were observed, even in the double deletion mutants PDH1-TPK1Δ2Δ, -TPK1Δ3Δ, and -TPK2Δ3Δ (Fig. 2B). In contrast, rephosphorylation at the PKA and Akt sites was not induced by 10 μm glucose in PDH1-TPK2Δ3Δ, and PKA site rephosphorylation was dramatically decreased in PDH1-TPK1Δ3Δ (Fig. 2B). Thus Tpk3p was the main contributor to glucose-dependent rephosphorylation of Pdh1p PKA and Akts sites at low glucose concentrations, and only Tpk2p could augment this process. Stress Sensitivity of Cdr1p and Pdh1p Phosphorylation—The glucose-dependent differences in Cdr1p and Pdh1p rephosphorylation patterns suggested that the two pumps might be phosphorylated in discrete physiological contexts. This hypothesis was initially tested for Cdr1p rephosphorylation in glucose-starved CDR1-AD by adding 1 mm glucose together with a stressor for 10 min (Fig. 3A). The 1 mm glucose supplied sufficient ATP as kinase substrate, because the Pma1p band was strongly phosphorylated even in the absence of Akt site phosphorylation of Cdr1p (Figs. 2A and 3A). The pump substrate FLC (45 μg/ml) did not affect rephosphorylation (data not shown), but oxidative stress (2 mm H2O2), osmotic stress (500 mm NaCl), and heat shock (42 °C) for 10 min induced p-Akt site phosphorylation, albeit to a lesser extent than 100 mm glucose (Fig. 3A). Stress-dependent rephosphorylation was not detected with the p-Thr antibody. Pdh1p in PDH1-AD was highly phosphorylated in 1 mm glucose, and stress treatments caused no additional phosphorylation. In contrast, stress with 2 mm H2O2 alone decreased Pdh1p phosphorylation and gave no rephosphorylation of Cdr1p. However, Cdr1p phosphorylation was not affected when the yeast in YPD culture were stressed (H2O2, NaCl, or 42 °C) for 10 min (data not shown). Under these conditions, the effects of multiple kinases and phosphatases may have been complex and/or compensatory.Table IAntifungal susceptibilities of CDR1-AD and PDH1-AD derivative strainsStrainMIC80aMIC80 values (μg/ml) were the lowest concentration of drug that inhibited the growth yield by at least 80% compared with the growth yield for a non-drug controlFluconazoleMiconazoleKetoconazoleFlucytosineAmphotericin Bμg/mlpSK-AD0.50.0160.0630.50.25CDR1-AD1282420.5CDR1-M1642410.5CDR1-M21282410.5CDR1-M31282410.5CDR1-M41282410.5CDR1-M51282410.5CDR1-M61282410.5CDR1-M71282410.5CDR1-M81282410.5CDR1-M91282410.5CDR1-M1,2321210.5CDR1-TPK1Δ1282420.5CDR1-TPK2Δ2564820.5CDR1-TPK3Δ1282420.5CDR1-TPK1Δ2Δ2564420.5CDR1-TPK1Δ3Δ1282421CDR1-TPK2Δ3Δ2564810.25CDR1-SCH9Δ256480.250.25CDR1-HOG1Δ2564821PDH1-AD160.5110.5PDH1-TPK1Δ160.5110.5PDH1-TPK2Δ160.5110.5PDH1-TPK3Δ160.5111PDH1-TPK1Δ2Δ161120.5PDH1-TPK1Δ3Δ160.5121PDH1-TPK2Δ3Δ160.5110.5a MIC80 values (μg/ml) were the lowest concentration of drug that inhibited the growth yield by at least 80% compared with the growth yield for a non-drug control Open table in a new tab Fig. 1Phosphorylation of Cdr1p and Pdh1p. The Cdr1p and Pdh1p-expressing yeast strains, CDR1-AD and PDH1-AD, and the empty vector integrated strain pSK-AD were cultured in YPD medium from initial A600 nm = 0.005, 0.015, 0.05, and 0.15 values. After 14 h, the A600 nm values of the cultures were measured (indicated at the top), and crude cell membrane fractions were prepared from the cells. Membrane samples containing 5 μg of protein were separated by SDS-PAGE, and the phosphorylation status of pump proteins was analyzed by immunoblotting using phospho-protein kinase A substrate (p-PKAs), phospho-(Ser/Thr), Akt substrate (p-Akts), and phospho-threonine (p-Thr) antibodies. The membrane fraction samples (15 μg of protein) were also separated by SDS-PAGE and stained with Coomassie Brilliant Blue R250 (CBB) to show relative protein expression levels. The 170-kDa Cdr1p and Pdh1p are indicated with a black arrowhead, and the control 100-kDa plasma membrane H+-ATPase (Pma1p) band is indicated with a white arrowhead. Representative data from several experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Stress- and kinase-dependent phosphorylation of Cdr1p.A, glucose-starved pSK-AD, CDR1-AD, or PDH1-AD cells were stressed or treated with 100 mm glucose for 10 min as indicated in the top panel. Stress experiments that included 1 mm glucose are indicated. Phosphorylation patterns and expression levels of pump proteins were determined as in the previous figures. B, glucose-starved CDR1-AD (-glucose) cells were treated with protein kinase inhibitors H-89, H-8, 14–22 amide (14Seibert F.S. Tabcharani J.A. Chang X.B. Dulhanty A.M. Mathews C. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1995; 270: 2158-2162Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Vanoye C.G. Castro A.F. Pourcher T. Reuss L. Altenberg G.A. Am. J. Physiol. 1999; 276: C370-C378Crossref PubMed Google Scholar, 16Decottignies A. Goffeau A. Nat. Genet. 1997; 15: 137-145Crossref PubMed Scopus (405) Google Scholar, 17Taglight D. Michaelis S. Methods Enzymol. 1998; 292: 130-162Crossref PubMed Scopus (85) Google Scholar, 18Kölling R. FEBS Lett. 2002; 531: 548-552Crossref PubMed Scopus (15) Google Scholar, 19Hunter T. Plowman G.D. Trends. Biochem. Sci. 1997; 22: 18-22Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 20Hohmann S. Microbiol. Mol. Biol. Rev. 2002; 66: 300-372Crossref PubMed Scopus (1323) Google Scholar, 21Nanduri J. Tarkakoff A.M. Mol. Biol. Cell. 2001; 12: 1835-1841Crossref PubMed Scopus (28) Google Scholar, 22Hahn J.S. Thiele D.J. J. Biol. Chem. 2002; 277: 21278-21284Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), bisindolylmaleimide I (BIM), or a solvent control (1% Me2SO plus 1% water). After 10 min, 500 mm NaCl plus 1 mm glucose, or 100 mm glucose was added, the cells cultured for 10 min, and crude membrane fractions were prepared for analysis. C, single and double TPK gene deletion mutants were constructed from CDR1-AD as described for PDH1-AD (Fig. 2). Glucose-starved CDR1-AD and the derivative TPK deletion mutants were treated with 500 mm NaCl, and 1 mm glucose, or 100 mm glucose for 10 min. The phosphorylation patterns and protein expression levels of the crude membrane fractions were analyzed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rephosphorylation of Cdr1p Akt sites was inhibited by the PKA inhibitors H-89 and amide 14–22 but not by the H-89 homologue H-8, which has a Ki for PKA 30-fold higher than H-89 (12Hidaka H. Watanabe M. Kobayashi R. Methods Enzymol. 1991; 201: 328-339Crossref PubMed Scopus (123) Google Scholar), or the protein kinase C inhibitor bisindolylmaleimide I (BIM) (Fig. 3B). A panel of TPK single or double deletion mutants constructed in CDR1-AD was tested for the effects of NaCl- and glucose-dependent rephosphorylation of Cdr1p (Fig. 3C). Apart from CDR1-TPK1Δ2Δ and CDR1-TPK2Δ3Δ, which reduced Cdr1p rephosphorylation by about 50% during both treatments, p-Akts site re-phosphorylation was unaffected. Thus Tpk2p and to a lesser extent Tpk1p and Tpk3p may play a role with other kinases susceptible to PKA inhibitors in the rephosphorylation of Cdr1p Akts sites. Identification of Putative Phosphorylation Sites in Cdr1p— Modest rephosphorylation signals suggested the presence of few p-Akts sites in Cdr1p. The p-Akts antibody recognizes phosphorylated Ser or Thr in the -5(K/R)X-3(K/R)XX0(S/T) motif and cross-reacts with the phosphorylated -3(K/R)-2(K/R)X0(S/T) motif (manufacturer's information). The full size, single subunit, ABC transporter Cdr1p comprises two nucleotide binding domains (NBD1 and NBD2) that each contain the Walker A, Walker B, and ABC signature motifs, alternating with two pairs of six transmembrane segments, as illustrated in Fig. 4A. We constructed the yeast strains CDR1-M1 to CDR1-M9, which expressed equivalent amounts of Cdr1p (Fig. 4B), and each contained a point mutation (S/T → A) at each one of the nine putative p-Akts recognition sites. Of these sites, only M2, M4, and M7 may be recognized by the phospho-PKA substrates antibody (phosphorylated Ser or Thr in RXX(S/T)). Unlike the essentially normal phosphorylation of Cdr1p p-Akts sites in the CDR1-M3-CDR1-M9 mutants in 12-h YPD (early stationary phase) cultures, Cdr1p phosphorylation was eliminated in CDR1-M1 and >90% inhibited in CDR1-M2 (Fig. 4B). Rephosphorylation of Cdr1p-M1 and Cdr1p-M2 was not detected in response to stressors plus 1 mm glucose (Fig. 4, C and D), and 100 mm glucose gave rephosphorylation to about 50% of the control level in each mutant (Fig. 4D). These results suggested that Ser307 and Ser484 are the dominant Akts sites in Cdr1p. This hypothesis was confirmed by finding, in the S307A/S484A double mutant strain CDR1-M1,2, that 100 mm glucose gave no Cdr1p p-Akts site rephosphorylation (Fig. 4E). Thus, the phosphorylation of M1 and M2 sites responded comparably to 12-h YPD culture (low glucose) and stressors, whereas glucose-induced rephosphorylation of the two sites occurred independently. Interestingly, the S307A mutation of the M1 site mutation reduced Thr phosphorylation by one-third in 12-h YPD cultures (Fig. 4B), and the S509A mutation at the membrane-associated M3 site similarly affected Akts site rephosphorylation of Cdr1p in 100 mm glucose (Fig. 4E). Rhodamine 6G Efflux from Pdh1p, Cdr1p, and Point Mutant Yeast—The effect of the M1 and M2 mutations on energy-dependent drug efflux by Cdr1p was quantitated by comparably pre-loading CDR1-AD, CDR1-M1, CDR1-M2, CDR1-M1,2 and AD1–8u- cells with the pump substrate rhodamine 6G (Rh6G) after 2-deoxyglucose treatment and then stimulating efflux by adding glucose (Fig. 5). Fluorometric measurements showed that the CDR1-M2 strain (half-maximal rate of Rh6G pumping, 2.5 mm glucose; maximal pumping rate, 5–10 mm glucose) pumped Rh6G at rates up to 80% of the CDR1-AD strain (half-maximal rate of Rh6G pumping, 1 mm glucose; maximal rate, 5–10 mm glucose). In contrast, the CDR1-M1 strain required at least 5 mm glucose for a significant rate of Rh6G efflux and reached a maximal rate at 20 mm glucose, which was only 30% that of the CDR1-AD strain. The CDR1-M1,2 strain showed no glucose-dependent Rh6G efflux, even at 100 mm glucose. All strains that effluxed Rh6G showed a 30% decrease in the pumping rate at glucose concentrations between 20 and 100 mm glucose. The pumping activities of Cdr1p and its point mutants were confirmed by flow cytometric measurement of cellular Rh6G content, which monitors combined dye uptake and efflux (see Supplemental Data, Fig. S1). These data also showed that strain CDR1-M1,2 did not efflux Rh6G even in the presence of 100 mm glucose and that phosphorylation of the Cdr1p-M1 site may be required for Rh6G efflux at low glucose concentrations. Lability of ATPase Activity in CDR1-M1—The Cdr1p drug efflux pumps were functional in CDR1-M1, CDR1-M2, and CDR1-M8 cells and therefore expected to retain significant ATPase activity on cell fractionation. Plasma membrane fractions from these strains contained equivalent amounts of Cdr1p proteins, CDR1-M8 had a normal oligomycin-sensitive ATPase activity with a broad pH profile, but CDR1-M2 showed a decrease of ∼50% in the ATPase activity compared with CDR1-AD (Fig. 6). CDR1-M8 was chosen as a control, because the T1007A mutation borders the NBD2 ABC signature motif residue and aligns with Ser307 of NBD1. The membranes from CDR1-M1, however, lacked detectable oligomycin-sensitive ATPase activity. The M1 site may therefore be critical for the in vitro ATPase activity. Experiments that included ATP during membrane isolation allowed the recovery of small amounts (10%) of vanadate and oligomycin-sensitive ATPase activity compared with the control strain (see Supplemental Data Table SIII). A statistically significant proportion of this activity (p < 0.01) can be attributed to S307A Cdr1p that survived membrane isolation due to the protective ATP. Drug Susceptibilities of CDR1-AD and PDH1-AD Derivative Yeasts—The MIC80 values for antifungal agents were determined for PDH1-AD and CDR1-AD derivative strains to assess whether the phosphorylation of Cdr1p and Pdh1p affected drug efflux activity (Table I). As expected, the Pdh1p- and Cdr1p-expressing strains were more resistant to azole agents than the control pSK-AD strain but were equally susceptible to flucytosine and amphotericin B, which are not pump substrates. The flucytosine and amphotericin B susceptibilities for all the strains were within two dilutions of the values for pSK-AD. Among the CDR1-AD derivatives, only CDR1-M1 and CDR1-M1,2 consistently showed lower FLC susceptibilities than CDR1-AD, giving values that were reproducibly 2-fold and 4-fold lower, respectively. The TPK2 deletion mutants CDR1-TPK2Δ, -TPK1Δ2Δ, and -TPK2Δ3Δ gave detectably greater azole resistance than the control Cdr1p-expressing strain. Repeated testing of Cdr1p phosphorylation in TPK2 deletion mutants found no relationship between the phos
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