| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
Correspondence
Rajendra Prasad
rp47{at}mail.jnu.ac.in
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
A supplementary figure showing the amino acid sequence alignment of Cdr1p and Cdr3p is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Additionally, a high level of expression of Cdr1p invariably contributes to an increased efflux of fluconazole, thus corroborating its direct involvement in drug efflux (Sanglard et al., 1995; White et al., 1998). Cdr1p thus has not only acquired significant clinical importance but is considered an important player in any design of strategies to combat antifungal resistance.
The CDR1 gene encodes an integral plasma membrane (PM) protein of 1501 amino acids, with a predicted molecular mass of 169·9 kDa. The topology of Cdr1p exhibits typical characteristic features of an ABC transporter: two highly hydrophobic transmembrane domains (TMDs) and two cytoplasmically localized nucleotide-binding domains (NBDs). Each TMD comprises six transmembrane segments (TMSs), which are envisaged to confer substrate specificity (Shukla et al., 2003, 2004). The NBDs of ABC-type transporter proteins, on the other hand, are the sites of ATP hydrolysis and hence the hub of energy generation to facilitate drug efflux. According to our current understanding, Cdr1p and Cdr2p drug extrusion proteins not only mediate the efflux of azoles and their derivatives but also extrude a variety of structurally unrelated drugs and compounds (Dogra et al., 1999; Krishnamurthy et al., 1998a; Prasad et al., 1998; Sanglard et al., 1997; Smriti et al., 2002). However, the molecular mechanism of drug transport mediated by Cdr1p is not yet known.
Although analysis of the C. albicans genome reveals that it has 28 putative ABC transporters, only CDR1 and CDR2 have been shown to be drug transporters (Braun et al., 2005; Gaur et al., 2005). The well-characterized Cdr3p and Cdr4p, despite having 56 % and 62 % identity, respectively, with Cdr1p, and 55 % and 59 % identity with Cdr2p, and similar predicted domain organization, are not involved in drug efflux and antifungal resistance (Balan et al., 1997; Franz et al., 1998; Sanglard et al., 1999; Smriti et al., 2002). The non-drug transporter Cdr3p is a general phospholipid flippase and translocates membrane phospholipids (Smriti et al., 2002). This situation is reminiscent of the mammalian MDR gene family, which encodes the three highly homologous P-gp isoforms in mouse (mdr1, mdr2 and mdr3) and two in humans (MDR1 and MDR2) (Ng et al., 1989). While the overexpression of mouse mdr1 and mdr3 and human MDR1 is closely linked to the cell's ability to exhibit multidrug resistance, no such role for mouse mdr2 or human MDR2 has been observed. Notably, MDR2, which displays 78 % overall amino acid sequence identity with MDR1, rather functions as a flippase and translocates membrane phospholipids between two monolayers of the lipid bilayer (Ruetz & Gros, 1994; Smit et al., 1993).
To understand the structure and function of individual domains of drug transporter proteins, in the present study we focused on the role and distinctiveness of the NBDs and TMDs of Cdr1p in drug transport. For this we employed two approaches: first we constructed chimeras between Cdr1p and its close homologue Cdr3p and second, to study the functional equivalence of the two cytoplasmic domains, we constructed variants of Cdr1p molecules comprising either the two N-terminal or two C-terminal NBDs of the same protein. Our results demonstrate for the first time that the N- and the C-terminal halves of Cdr1p are largely non-exchangeable. TMS12 was the only part of Cdr1p that could be functionally replaced with the equivalent TMS region of the non-drug transporter Cdr3p. On the other hand, the recombinant strain expressing a Cdr1p variant with two N-terminal NBDs produced a chimeric protein that even upon rescuing to the PM remained non-functional, thus implying that the two NBDs display functional asymmetry and non-equivalence.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Media and strains.
Plasmids were maintained in Escherichia coli DH5
. E. coli was cultured in Luria–Bertani medium (Difco) to which ampicillin was added (100 µg ml–1). The bacterial and Saccharomyces cerevisiae strains used in this study are listed in Table 1. The yeast strains were cultured in YEPD broth (Bio 101) or SD-ura– (Bio 101). For agar plates 2 % (w/v) Bacto agar (Difco) was added to the medium.
|
) using either pPSCDR1GFP (Shukla et al., 2003) or p425-GPD-CDR3* (Table 1
) as template and Pwo DNA polymerase (Roche Molecular Biochemicals) or Pfu DNA polymerase (Stratagene). For construction of pBS1N/3C, primers PS1 (having a XbaI restriction site) and 1NR2 were used to amplify the N-terminal half of CDR1 using pPSCDR1GFP as template, and primers 3CF and PS4 (having a SalI restriction site) were used to amplify the C-terminal half of CDR3 without the stop codon using p425-GPD-CDR3* as template. The two amplicons were then ligated and the ligated mix was used as template for the final amplification of 1N/3C using primers PS1 and PS4. The amplified fragment was then digested with XbaI and SalI (restriction sites introduced in the primers) and cloned into the XbaI- and SalI-digested pBS. The GFP ORF was amplified from pPSCDR1GFP using primers GFP-F (having a SalI restriction site) and GFP-R (having SalI and XbaI restriction sites). This GFP amplicon was then digested with SalI and cloned into SalI-digested plasmid pBS1N/3C, resulting in plasmid pBS1N/3CGFP. The 1N/3CGFP ORF from plasmid pPBS1N/3CGFP was then taken out with the convenient restriction site XbaI and recloned into SpeI-digested pSK-PDR5PPUS, resulting in plasmid pPS1N/3CGFP.
|
Construction of pPS1(1–1173)/3(1154–1501)GFP.
For constructing pPS1(1–1173)/3(1154–1501)GFP vector, first the endogenous NarI site at amino acid position 823 in pPS1N/3CGFP was modified by site-directed mutagenesis using primers 1N/3C(NarI823)F and 1N/3C(NarI823)R and the Quickchange mutagenesis system (Stratagene), thus resulting in pPS1N/3CGFP*. Subsequently, NarI sites were created at amino acid positions 789 and 1173 [using primers CDR1P(NarI789)F, CDR1P(NarI789)R and CDR1P(NarI1173)F, CDR1P(NarI1173)R respectively] in pPSCDR1GFP, thus forming plasmid pPSCDR1GFP3, and at amino acid positions 789 and 1165 [using primers 1N/3C(NarI789)F, 1N/3C(NarI789)R and 1N/3C(NarI1165)F, 1N/3C(NarI1165)R respectively] in pPS1N/3CGFP*, resulting in plasmid pPS1N/3CGFP*3 without changing the existing codon by site-directed mutagenesis. The resulting NarI-digested 1·152 kb fragment was taken out from plasmid pPSCDR1GFP3 and ligated to NarI-digested plasmid pPS1N/3CGFP*3.
Construction of pPS1(1–1436)/3(1420–1501)GFP.
To construct pPS1(1–1436)/3(1420–1501)GFP vector, NarI and MluI sites were introduced at amino acid positions 789 and 1436 respectively [using primers CDR1P(NarI789)F, CDR1P(NarI789)R and CDR1P(MluI1436)F, CDR1P(MluI1436)R] in pPSCDR1GFP, thus forming plasmid pPSCDR1GFP2, and similarly, NarI and MluI sites were introduced at amino acid positions 789 and 1431 respectively [using primers 1N/3C(NarI789)F, 1N/3C(NarI789)R and 1N/3C(MluI1431)F, 1N/3C(MluI1431)R] in plasmid pPS1N/3CGFP*, resulting in plasmid pPS1N/3CGFP*2 by site-directed mutagenesis without changing the existing codons. The resulting NarI- and MluI-digested 1·941 kb fragment was taken out from plasmid pPSCDR1GFP2 and ligated to NarI- and MluI-digested plasmid pPS1N/3CGFP*2.
Construction of pCdr1-1N/1NGFP.
For constructing pCdr1-1N/1NGFP vector, the DNA encoding a hydrophilic region including the N-terminal NBD was amplified using pPSCDR1GFP as template and primers CDR1F(1210) and CDR1R(2657), which allowed the introduction of a NarI restriction site at the 5' and 3' ends of the amplicon. The resultant amplicon was then digested with NarI-and ligated to NarI digested pPSCDR1GFP3 vector (NarI digestion of pPSCDR1GFP3 results in deletion of the C-terminal NBD).
Construction of pCdr1-1C/1CGFP.
For constructing pCdr1-1C/1CGFP, a HindIII restriction site was introduced at amino acid position 494 (after the N-terminal NBD) in plasmid pPSCDR1GFP by site-directed mutagenesis using primers HindIII 494F and HindIII 494R, resulting in plasmid pPSCDR1GFPHindIII. The DNA encoding a hydrophilic region including the C-terminal NBD was amplified using pPSCDR1GFP as template and primers 1CF HindIII and 1CR HindIII, which allowed the introduction of a HindIII restriction site at the 5' and 3' ends of the amplicon. The resultant amplicon was then digested with HindIII and ligated to HindIII-digested pPSCDR1GFPHindIII (HindIII digestion of pPSCDR1GFP HindIII results in deletion of the N-terminal NBD).
After every cloning, the entire chimeric construct was sequenced by using the Big Dye Terminator Cycle sequencing kit (ABI) and an ABI 310 DNA sequencer to confirm that the construct remained in-frame and no mutation had been introduced. Restriction enzyme digestions further confirmed the orientation of each construct. Each plasmid, after linearizing with XbaI, was used to transform AD1-8u– cells as described previously (Shukla et al., 2003). Transformation of yeast cells was performed by the lithium acetate method using routine laboratory protocols (Shukla et al., 2003). Single-copy integration of each transformant at the PDR5 locus was confirmed by Southern hybridization (data not shown). Two positive clones of each chimera were selected for initial screening to rule out clonal variations.
Immunodetection of Cdr1p.
Plasma membranes (PMs) were prepared from S. cerevisiae as described previously (Shukla et al., 2003). Briefly, cells were broken with glass beads. The crude membranes (CM) were recovered by centrifugation at 1000 g to remove unbroken cells and pelleting the CM by ultracentrifugation at 100 000 g for 1 h. The CM were then resuspended in resuspension buffer and applied to a discontinuous gradient made of an equal volume of 53·5 % (w/v) sucrose and 43·5 % (w/v) sucrose. The purified PM was recovered at the interface of the 43·5 % and 53·5 % sucrose layers, following centrifugation for 5 h at 100 000 g. The Western blot analysis was done using anti-GFP monoclonal antibody (1 : 1000 dilution) and anti-Pma1p polyclonal antibody (1 : 10 000 dilution) as described previously (Shukla et al., 2003). Proteins on immunoblots were visualized using the enhanced chemiluminescence assay system (ECL kit, Amersham Biosciences).
Confocal microscopy.
The cells were grown to late exponential phase in SD-ura– medium, except for AD1-8u–, where uridine (0·02 %) was supplemented to the SD-ura– medium. The cells were then washed and resuspended in an appropriate volume of 50 mM HEPES pH 7·0. The cells were placed on glass slides and directly viewed with a 100x oil-immersion objective on a confocal microscope (Radiance 2100, AGR, 3Q/BLD; Bio-Rad).
Drug susceptibility and other functional parameters.
The susceptibilities of S. cerevisiae cells to different drugs were tested by microtitre plate assay and spot assay as described earlier (Mukhopadhyay et al., 2002). The Cdr1p-associated ATPase activity of the purified PM was measured as oligomycin-sensitive release of inorganic phosphate as described previously (Shukla et al., 2003). Efflux of rhodamine 6G and accumulation of [3H]fluconazole were determined essentially as described elsewhere (Kohli et al., 2002; Shukla et al., 2003). Approximately 107 cells from an overnight culture were inoculated in 100 ml YPD and grown for 5–6 h at 30 °C with shaking. The cells were pelleted and washed three times with phosphate-buffered saline (PBS) buffer without glucose. The cells were subsequently resuspended as a 2 % cell suspension in de-energization buffer (5 mM dinitrophenol and 5 mM 2-deoxy-D-glucose in PBS without glucose) and incubated for 2 h at 30 °C with shaking. The cells were then washed, resuspended in PBS without glucose and divided into four parts; rhodamine 6G was added to a final concentration of 10 µM to each part, followed by incubation for 2 h at 30 °C. After washing, the cells were suspended in PBS with 2 % glucose. An aliquot of 1 ml was taken after 45 min and centrifuged at 9000 g for 2 min. The absorbance of the supernatant was measured at 527 nm. To check the accumulation of [3H]fluconazole, the de-energized cells were incubated with 100 nM [3H]fluconazole (0·7 TBq mmol–1) for 2 h. The cells were then washed, and suspended in PBS with 2 % glucose. An aliquot of 1 ml was removed after 45 min, rapidly filtered and washed three times with ice-cold PBS without glucose. The radioactivity that accumulated in filtered cells and that adhered to filter disks was measured in a liquid scintillation counter with a scintillation liquid (tri-Carb 2900TR; Packard). The radioactivity that adhered to the filter disk, which was not significant, was subtracted from the experimental values.
Photoaffinity labelling with [3H]azidopine.
PM (25 µg) protein was photoaffinity labelled with 0·5 µM [3H]azidopine (60 Ci mmol–1; 2·2 TBq mmol–1) as described previously (Shukla et al., 2003).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Shukla et al., 2003) (Fig. 1A) and sequence similarity, the two proteins are functionally distinct (Balan et al., 1997; Prasad et al., 1995; Smriti et al., 2002). For example, we as well as others (Sanglard et al., 1995; Prasad et al., 1995; Shukla et al., 2003) have shown a direct involvement of Cdr1p in efflux of drugs while Cdr3p has no such activity and is thus a non-drug transporter (Smriti et al., 2002). Cdr3p does possess ATPase activity but this is not coupled to drug efflux; rather it powers translocation of phospholipids between the two lipid monolayers of the PM. The inhibitors which block ATPase activity of Cdr3p also block phospholipid translocation mediated by it. Interestingly, Cdr3p appears to be functionally oppositely oriented in the PM as compared to Cdr1p (Smriti et al., 2002).
|
) by exchanging the homologous regions of CDR1 with CDR3 as described in Methods. In order to check the localization of chimeric proteins, each construct, at the C-terminus, was also tagged with the green fluorescent protein (GFP). For functional analysis of the CDR1/CDR3 chimeras, a heterologous hyper-expression system, where Cdr1p is stably overexpressed from a genomic PDR5 locus in a S. cerevisiae mutant AD1-8u–, was used (Nakamura et al., 2002). The host AD1-8u–, from which seven major ABC transporters have been deleted, was derived from a Pdr1-3 mutant strain with a gain-of-function mutation in the transcription factor Pdr1p, resulting in a constitutive hyperinduction of the PDR5 promoter. The new overexpressing strains harbouring CDR1/CDR3 chimeras were designated PS3N/1CGFP, PS1N/3CGFP, PS1(1–1173)/3(1154–1501)GFP and PS1(1–1436)/3(1420–1501)GFP (see Table 1 for details).
Interestingly, expression of proteins with homologous substitution of either the N- or the C-terminal half of Cdr1p with Cdr3p (PS3N/1CGFP, PS1N/3CGFP) resulted in non-functional recombinant strains. In contrast to wild-type Cdr1p, cells expressing either of the chimeras showed no resistance to drugs on spot and MIC80 assays (Fig. 2A, B). This enhanced supersensitivity of recombinant strains expressing either of the chimeras may be linked to their poor expression and localization. To check this, we examined the localization of wild-type Cdr1p and CDR1/CDR3GFP chimeric proteins by confocal microscopy. The confocal images revealed that unlike the rimmed appearance of Cdr1pGFP in cells expressing wild-type Cdr1p, small patches of chimeric PS3N/1CGFP protein on the cell surface were apparent, whereas surface localization of PS1N/3CGFP protein was more severely affected since its fluorescence appeared trapped intracellularly (Fig. 3A). Western blot analysis of PM proteins isolated from PS3N/1CGFP and PS1N/3CGFP chimeras confirmed the confocal microscopy results. None of the PM fractions isolated from these chimeras showed any detectable protein (Fig. 3B, a). However, a faint band was observed with CM preparations of PS3N/1CGFP (Fig. 3B, c). PM-ATPase was used as a marker to check the purity of PM fraction (Fig. 3B, b).
|
|
), we sequentially replaced the Cdr3p region with Cdr1p, selecting the inactive chimera, PS1N/3CGFP, as a framework to restore Cdr1p-like function. Consequently, two chimeras were constructed, one comprising Cdr1p up to the C-terminal NBD [PS1(1–1173)/3(1154–1501)GFP] and the other comprising Cdr1p up to TMS11 [PS1(1–1436)/3(1420–1501)GFP]. As shown in Fig. 2, recombinant strains expressing a chimeric protein comprising the Cdr1p segment up to the C-terminal NBD in PS1(1–1173)/3(1154–1501)GFP remained non-functional. The poor functioning of the PS1(1–1173)/3(1154–1501)GFP chimera in the recombinant strain was apparently due to its poor cell surface localization as was revealed by confocal and Western analysis (Fig. 3). These results further emphasize the importance of the entire TMD region for the synthesis and assembly of active protein. Interestingly, the replacement of TMS12 of Cdr1p with the homologous TMS of Cdr3p, as in PS1(1–1436)/3(1420–1501)GFP, resulted in complete restoration of Cdr1p activity, which was evident from the drug resistance profile of the recombinant strain (Fig. 2). Thus cells expressing the PS1(1–1436)/3(1420–1501)GFP chimera displayed increased resistance to all the tested drugs, which was comparable to that seen with the wild-type protein (Fig. 2).
The functionality of PS1(1–1436)/3(1420–1501)GFP was further confirmed by analysing ATPase activity, efflux of the fluorescent substrate rhodamine 6G and accumulation of radiolabelled fluconazole. For this, the purified PM protein fractions isolated from PSCDR1GFP and CDR1/CDR3GFP chimeras were analysed for their oligomycin-sensitive ATPase activity. Cells expressing wild-type CDR1GFP and chimeric 1(1–1436)/3(1420–1501)GFP protein showed comparable oligomycin-sensitive ATPase activity, in contrast to the rest of the chimeric proteins (Fig. 4A). In addition, both PSCDR1GFP and PS1(1–1436)/3(1420–1501)GFP cells showed similar levels of rhodamine 6G efflux and fluconazole accumulation (Fig. 4B, C). As expected, confocal images and Western blot analysis of PS1(1–1436)/3(1420–1501)GFP confirmed the proper surface localization and expression of this chimeric protein (Fig. 3).
|
, b, 2004) that the unique positioning of the catalytically important residues C193 in Walker A of NBD1 and K901 in Walker A of NBD2 could not be exchanged. Hence a construct like CDR1-1C/1NGFP was not used; instead, to address this question, two constructs were instead made, one having Cdr1p with cytoplasmic hydrophilic domains comprising two N-terminal NBDs and another with two C-terminal NBDs (Fig. 5A). These constructs were integrated in S. cerevisiae mutant AD1-8u– as described previously for CDR1/CDR3 chimeras and checked for single-copy integration (data not shown). The resultant variant proteins Cdr1-1N/1NGFP and Cdr1-1C/1CGFP were analysed for their functionality using two independent drug sensitivity assays. As shown in Fig. 5(B), recombinant strains expressing both types of NBD variants were hypersensitive to all the tested drugs as compared to those expressing wild-type CDR1GFP. The drug sensitivities revealed by spot assay generally matched well with the MIC80 test (data not shown). Although both variants of NBDs were hypersensitive to the tested drugs, confocal images revealed differences in their surface localization. While the variant Cdr1-1N/1NGFP, consisting of hydrophilic domains with two N-terminal NBDs, revealed poor cell surface localization, the variant having two C-terminal NBDs showed altogether very poor GFP fluorescence (Fig. 5C).
|
F774) of Cdr1p could be functionally restored to the cell surface if the cells were exposed to drug substrates which act as powerful ‘chaperones' for processing misfolded proteins (Shukla et al., 2003). We therefore checked whether the mislocalization of the CDR1/CDR3 chimeric proteins and Cdr1p NBDs variants could be similarly improved. For this, we added different drug substrates of Cdr1p separately, immediately after the lag phase of the S. cerevisiae cells (4–5 h). The cells were grown for a further 7 h, then checked by confocal microscopy for the localization of the protein. Of the two NBD variants only Cdr1-1N/1NGFP protein showed improved surface localization with increasing concentrations of drugs (shown for cycloheximide in Fig. 6A, a–f). The surface expression level of the Cdr1-1N/1NGFP variant could be restored to that of wild-type CDR1GFP at 50 ng cycloheximide ml–1 (Fig. 6A, a–h). Interestingly, PM protein isolated from Cdr1-1N/1NGFP cells grown in increasing concentrations of cycloheximide also showed a concentration-dependent increase in the amount of variant protein in the PM fraction (Fig. 6B, a), while the presence of drug had no effect in the cells expressing wild-type CDR1GFP (Fig. 6A, g, h; Fig. 6B, a), thus suggesting that there was no inhibition of protein synthesis at the concentration of cycloheximide used for rescue experiments. Additionally, other substrates of Cdr1p, such as fluconazole, ketoconazole, miconazole, itraconazole and anisomycin, were also tested to improve the localization of chimeric proteins. Of all the substrates tested, only anisomycin could fully rescue the Cdr1-1N/1NGFP variant protein to the PM (Fig. 6A, k), while miconazole and itraconazole were able to rescue the Cdr1-1N/1NGFP variant protein partially to the PM (Fig. 6A, i, j). The presence of drugs in cells expressing variant protein with both C-terminal NBDs (Cdr1-1C/1CGFP) or CDR1/CDR3 chimeric proteins did not improve localization (data not shown). It is noteworthy that no growth difference was observed between strains harbouring variant protein, chimeric protein and wild-type CDR1GFP protein in the absence or presence of the concentrations of the drugs used for the rescue experiments (data not shown).
|
show that rescued Cdr1-1N/1NGFP protein was unable to mediate efflux of rhodamine 6G in the presence of cycloheximide. In contrast to this, as observed earlier, SS6G (F774) cells when grown in the presence of drug showed not only improved localization (Shukla et al., 2003) but also restoration of rhodamine 6G efflux. These results suggest that although Cdr1p substrates can rescue the folding defects in one of the NBD variants Cdr1-1N/1NGFP, the protein remains non-functional. Notably, there was some reduction in the efflux of rhodamine 6G by wild-type CDR1GFP cells grown in the presence of cycloheximide. The decrease in efflux by the cells expressing wild-type Cdr1p is probably due to the fact that cycloheximide, which is also a substrate for the Cdr1p transporter, competes with rhodamine 6G.
Rescued Cdr1-1N/1NGFP protein is capable of binding Cdr1p substrates but unable to hydrolyse ATP
To address the question whether the rescued Cdr1p variant having two identical N-terminal NBDs is non-functional because of impairment in the substrate binding or ATP hydrolysis, we performed photoaffinity labelling with azidopine, a dihydropyridine analogue, and also measured ATPase activity with the PM protein fraction isolated from the wild-type and the Cdr1p N-terminal NBDs variant grown in the presence of cycloheximide (50 ng ml–1). As shown in Fig. 6(D), comparable azidopine labelling was observed with PM isolated from wild-type CDR1GFP and the Cdr1-1N/1NGFP variant. However, the Cdr1-1N/1NGFP variant showed impaired ATPase activity as compared to wild-type CDR1GFP (Fig. 6E).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 2004). We observed that several point mutations resulted in mutant variants of Cdr1p that were hypersensitive to different drugs (Jha et al., 2004; Shukla et al., 2003, 2004).
Our current study focused on examining the functional relevance of the two NBDs of Cdr1p and identifying candidate protein segments that are important for substrate recognition and binding. To address these questions, we exchanged homologous segments of Cdr1p with corresponding segments of the non-drug transporter Cdr3p. This study shows that any exchange of either the N- or the C-terminus of Cdr1p with the homologous portion of Cdr3p results in non-functional recombinant strains (Fig. 2). It seems that the loss of activity of CDR1/CDR3GFP chimeric proteins could be attributed to the poor cell surface localization of these proteins (Figs 2 and 3), which could also contribute to their enhanced degradation. These results, however, do emphasize that although Cdr1p and Cdr3p have great topological and sequence similarities, this resemblance is not symmetrically distributed between the two halves of the proteins; positioning of both the halves of the individual proteins is probably essential for their distinct functions. Although the sequence identity between Cdr1p and Cdr3p at the N-terminus and C-terminus is as high as 54 % and 58 %, respectively, it is distinct enough to make the former a drug transporter and latter a phospholipid translocator (Prasad et al., 1995; Smriti et al., 2002). By employing a similar chimeric approach, Zhou et al. (1999) earlier reported that while the N-terminal halves of human Mdr1p and Mdr2p could be switched, this degree of exchangeability was not found between the C-terminal halves. In the present case, unlike MDR1/MDR2 chimeras, even the replacement of the N-terminal part of Cdr1p with the corresponding region of Cdr3p did not yield functional recombinant strains. This implies that in spite of the high similarities between the two homologous proteins, the TMDs of Cdr1p and of Cdr3p cannot be exchanged.
Interestingly, replacement of residues 1437–1501 (which include TMS12) of Cdr1p by the corresponding stretch of Cdr3p resulted in a recombinant strain expressing a functional chimeric Cdr1p variant. It would thus seem that this extreme C-terminus probably does not contribute to the functional difference detected between Cdr1p and Cdr3p. We had earlier observed that the deletion of a 79 amino acid stretch from the C-terminal end of Cdr1p, which encompasses TMS12 of this transporter, resulted in impaired resistance to certain drugs (Krishnamurthy et al., 1998b). Combining the two observations, it seems likely that TMS12 of Cdr1p does harbour drug-binding site(s), which can be exchanged with TMS12 of Cdr3p in the PS1(1–1436)/3(1420–1501)GFP chimeric protein. A closer look at the TMS12 sequence of the two ABC proteins suggests that this may be the case. Comparison of the amino acid sequence of Cdr1p with that of Cdr3p in this residue 1437–1501 segment revealed an overall 38 % identity and the sequence identity within TMS12 of the two proteins becomes as high as 57 % (Fig. 7A). The evident functional exchangeability of TMS12 between Cdr1p and Cdr3p provides clues about residues present in the TM12 that may be important for substrate-binding funtion. A site-directed mutagenesis approach could identify such common residues between the two homologous proteins.
|
). Interestingly, although NBD1 of Cdr1p contains the conserved Walker A motif (GRPGAGCST), the commonly conserved lysine residue within the Walker A motif is replaced by an uncommon cysteine that appears to be a unique feature of most of the fungal ABC transporters (Jha et al., 2004). Conversely, NBD2 of Cdr1p contains the commonly conserved lysine (GASGAGKT) at the equivalent position in its Walker A motif. There is a complete lack of understanding with regard to the functional equivalence of both the NBDs of Cdr1p and the significance of the variation in their Walker A amino acid sequence (Jha et al., 2003a, b). Our more recent results show that both the NBDs are essential for Cdr1p function, albeit asymmetrically, and the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable (Jha et al., 2004). In order to further probe the role of NBDs in Cdr1p, in this study we constructed Cdr1p variants possessing either both N-terminal NBDs or both C-terminal NBDs. Our results show that the substitution of either NBD yielded non-functional recombinant strains. Such a situation has previously been encountered with mammalian MDR proteins. For example, CFTR chimeric molecules comprising either two N-terminals or C-terminal NBDs having core NBD sequence or the flanking sequence in addition to core NBD were non-functional because of trafficking defects (Pollet et al., 2000). Similarly, mouse mdr3 and human MDR1 chimeric proteins comprising two C-terminal NBDs resulted in the synthesis of non-functional protein owing to decreased accumulation or altered targeting of the protein to the membrane (Beaudet & Gros, 1995; Hrycyna et al., 1999). Of note, recently a Walker A mutation (K86M) of an ABC half-transporter ABCG2 was shown to affect oligomerization and surface targeting followed by retrieval to the endoplamic reticulum (Henriksen et al., 2005). Interestingly, in the present study, one of the NBD variants of Cdr1p (having both N-terminal NBDs) could be rescued to the PM if cells expressing this protein were exposed to drug substrates. The exact mechanism by which these specific drug substrates facilitate processing of probably misfolded protein is not known. A possible explanation, as suggested in case of P-gp and CFTR protein, is that the drug-binding site(s) in the protein are formed early in the folding intermediates during biosynthesis. It is possible that the occupation of these drug-binding site(s) in the early stages of folding may reduce the concentration of the intermediate that is prone to self-aggregation, thus stabilizing the folding intermediates in a conformation that resembles the wild-type protein, which in turn helps in escaping the cell's quality control mechanism (Loo & Clarke, 1997; Morello et al., 2000). It is noteworthy that the non-functionality of rescued N-terminal NBD variant protein was not due to any impairment in substrate binding since [3H]azidopine labelling remained unaffected by this substitution of NBDs. Therefore, the functional defect in spite of proper localization was not due to impaired drug binding but rather to a poor ability to hydrolyse ATP, which could be attributed to the loss of communication between the two NBDs or between NBDs and TMDs when exchanged. One must not ignore the flanking sequences as well as the sequence stretches between the Walker A, Walker B and signature C motifs of NBDs, which if exchanged may also contribute to the non-functionality of the variants.
The two NBDs of a number of ABC transporters have been shown to be functionally dissimilar. Interestingly, in the case of human P-gp, which is a close homologue of Cdr1p, the two NBDs were partially interchangeable. For example Pgp-1N/1N, containing two N-terminal NBDs, was functional and was also asymmetric with respect to 8-azido-ATP labelling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. Pgp-1C/1C and Pgp-1C/1N variants were, however, defective in cell surface expression and function (Hrycyna et al., 1999). In prokaryotic ABC-type transporters such as the histidine permease of E. coli, both NBDs are functionally identical and contribute equally to the protein's activity. Inactivation of either one of these NBDs in the full protein resulted in 50 % of the activity (Nikaido & Ames, 1999). On the other hand, there is clear evidence to indicate asymmetry of function of the NBDs of MRP1 and CFTR (Aleksandrov et al., 2002; Gao et al., 2000).
In conclusion, our analysis of Cdr1p/Cdr3p chimeras shows that neither the N- nor the C-terminal half of Cdr1p can be exchanged with the homologous portion of Cdr3p. The exchange of domains results in altered interfaces leading to non-functional recombinant strains expressing chimeric proteins. We have also confirmed our earlier observation that the N- and C-terminal NBDs of Cdr1p are functionally asymmetric. These results provide important clues to our understanding of the complex interactions between the NBDs and their neighbouring TMDs and should help in resolving mechanisms of drug efflux of structurally unrelated compounds mediated by Cdr1p.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Balan, I., Alarco, A. M. & Raymond, M. (1997). The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. J Bacteriol 179, 7210–7218.
Beaudet, L. & Gros, P. (1995). Functional dissection of P-glycoprotein nucleotide binding domains in chimeric and mutant proteins. J Biol Chem 270, 17159–17170.
Braun, B. R., Hoog, M. V. H., d'Enfert, C. & 40 other authors (2005). A human curated annotation of Candida albicans genome. PLoS Genet 1, 36–57.
Dogra, S., Krishnamurthy, S., Gupta, V., Dixit, B. L., Gupta, C. M., Sanglard, D. & Prasad, R. (1999). Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, A multidrug transporter belonging to ATP binding cassette (ABC) superfamily. Yeast 15, 111–121.[CrossRef][Medline]
Franz, R., Michel, S. & Morschhauser, J. (1998). A fourth gene from the Candida albicans CDR family of ABC transporters. Gene 220, 91–98.[CrossRef][Medline]
Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P. C. & Deeley, R. G. (2000). Comparison of the functional characteristics of the nucleotide binding domains of the multidrug resistance protein 1. J Biol Chem 275, 13098–13108.
Gaur, M., Devapriya, C. & Prasad, R. (2005). The complete inventory of ABC proteins in human pathogenic yeast, Candida albicans. J Mol Microbiol Biotechnol 9, 3–15.[CrossRef][Medline]
Henriksen, U., Gether, U. & Litman, T. (2005). Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2. J Cell Sci 118, 1417–1426.
Hrycyna, C. A., Ramachandra, M., Germann, U. A., Cheng, P., Wu, Pastan, I. & Gottesman, M. M. (1999). Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry 38, 13887–13899.[CrossRef][Medline]
Jha, S., Karnani, N., Dhar, S. K., Mukhopadhyay, K., Shukla, S., Saini, P., Mukhopadhyay, G. & Prasad, R. (2003a). Purification and charaterization of N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis. Biochemistry 42, 10822–10832.[CrossRef][Medline]
Jha, S., Karnani, N., Lynn, A. M. & Prasad, R. (2003b). Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump. Biochem Biophys Res Commun 310, 869–875.[CrossRef][Medline]
Jha, S., Dabas, N., Karnani, N., Saini, P. & Prasad, R. (2004). ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry. FEMS Yeast Res 5, 63–72.[CrossRef][Medline]
Kohli, A., Smriti, Mukhopadhyay, K., Rattan, A. & Prasad, R. (2002). In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrob Agents Chemother 46, 1046–1052.
Krishnamurthy, S., Gupta, V., Snehlata, P. & Prasad, R. (1998a). Characterisation of human steroid hormone transport mediated by Cdr1p, multidrug transporter of Candida albicans, belonging to the ATP binding cassette super family. FEMS Microbiol Lett 158, 69–74.[CrossRef][Medline]
Krishnamurthy, S., Chatterjee, U., Gupta, V., Prasad, R., Das, P., Snehlata, P., Hasnain, S. E. & Prasad, R. (1998b). Deletion of transmembrane domain 12 of CDR1, a multidrug transporter from Candida albicans, leads to altered drug specificity: expression of a yeast multidrug transporter in Baculovirus expression system. Yeast 14, 535–550.[CrossRef][Medline]
Loo, T. W. & Clarke, D. M. (1997). Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. J Biol Chem 272, 709–712.
Morello, J.-P., Petaja-Repo, U. E., Bichet, D. G. & Bouvier, M. (2000). Pharmacological chaperones: a new twist on receptor folding. Trends Pharmacol Sci 21, 466–468.[CrossRef][Medline]
Mukhopadhyay, K., Kohli, A. K. & Prasad, R. (2002). Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46, 3695–3705.
Nakamura, K., Niimi, M., Niimi, K., Holmes, A. R., Yates, J. E., Decottignies, A., Monk, B. C., Goffeau, A. & Cannon, R. D. (2002). Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob Agents Chemother 45, 3366–3374.
Ng, W. F., Sarangi, F., Zastawny, R. L., Veinot-Drebot, L. & Ling, V. (1989). Identification of members of the P-glycoprotein multigene family. Mol Cell Biol 9, 1224–1232.
Nikaido, K. & Ames, G. F. L. (1999). One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease. J Biol Chem 274, 26727–26735.
Pollet, J.-F., Geffel, J. V., Stevens, E. V., Geffel, R. V., Beauwens, R., Bollen, A. & Jacobs, P. (2000). Expression and intracellular processing of chimeric and mutant CFTR molecules. Biochim Biophys Acta 1500, 59–69.[Medline]
Prasad, R., Worgifosse, P. D., Goffeau, A. & Balzi, E. (1995). Molecular cloning and characterisation of a novel gene of C. albicans, CDR1, conferring multiple resistance to drugs and antifungals. Curr Genet 27, 320–329.[CrossRef][Medline]
Prasad, R., Krishnamurthy, S., Gupta, V. & Panwar, S. L. (1998). Multidrug transporters of Candida albicans. Folia Microbiol 43, 228.
Rice, P., Longden, I. & Bleasby, A. (2000). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276–277.[CrossRef][Medline]
Ruetz, S. & Gros, P. (1994). Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 77, 1071–1081.[CrossRef][Medline]
Sanglard, D., Kuchler, K., Ischer, F., Pagani, J.-L., Monod, M. & Bille, J. (1995). Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39, 2378–2386.[Abstract]
Sanglard, D., Ischer, F., Monod, M. & Bille, J. (1997). Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143, 405–416.[Abstract]
Sanglard, D., Ischer, F., Monod, M., Dogra, S., Prasad, R. & Bille, J. (1999). Analysis of the ATP-binding cassette (ABC)-transporter gene CDR4 from Candida albicans. In ASM Conference on Candida and Candidiasis, Charleston, SC, USA, March 1–4, p. 56.
Shukla, S., Saini, P., Smriti, Jha, S., Ambudkar, S. V. & Prasad, R. (2003). Functional characterization of Candida albicans ABC transporter Cdr1p. Eukaryot Cell 2, 1361–1375.
Shukla, S., Ambudkar, S. V. & Prasad, R. (2004). Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520. J Antimicrob Chemother 54, 38–45.
Smit, J. J., Schinkel, A. H., Oude Elferink, R. P. J. & 11 other authors (1993). Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to complete absence of phospholipid from bile and to liver disease. Cell 75, 451–462.[CrossRef][Medline]
Smriti, Krishnamurthy, S., Dixit, B. L., Gupta, C. M., Milewski, S. & Prasad, R. (2002). ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators. Yeast 19, 303–318.[CrossRef][Medline]
Tusnady, G. E. & Simon, I. (1998). Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J Mol Biol 283, 489–506.[CrossRef][Medline]
Tusnady, G. E. & Simon, I. (2001). The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849–850.
Walmsley, M. B., Mckeegan, K. S. & Walmsley, A. R. (2003). Structure and function in efflux pumps that confer resistance to drugs. Biochem J 376, 313–338.[CrossRef][Medline]
White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11, 382–402.
Zhou, Y., Gottesman, M. M. & Pastan, I. (1999). Domain exchangeability between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2). Mol Pharmacol 56, 997–1004.
Received 26 August 2005;
revised 16 January 2006;
accepted 17 January 2006.
| ||||||||||||||||||||||||||||||||||
