Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

Summary

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

Abstract

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Introduction

The extraction of integral membrane proteins from their native lipid environment can dramatically affect their structure and function^1,2,3,4. The complex lipid composition of biological membranes⁵ ensures that critically important protein-lipid interactions can occur⁶. Lipids maintain the structural integrity of membrane proteins, thus enabling them to function correctly in their membrane compartment destination(s)^7,8. Therefore, a critical first step in the membrane protein purification is the extraction of the protein from its native environment without affecting its structure and/or function.

There are many obstacles to characterizing the structure of membrane proteins, most of which are related to their hydrophobic nature, and the difficulties of expressing properly folded and functional membrane proteins in the quantities required for X-ray crystallography or cryo-electron microscopy (cryo-EM)^9,10,11,12. There are three types of membrane protein expression systems: homologous⁹, heterologous^13,14,15, and in vitro expression systems^16,17. The often-low expression levels, or the prohibitive costs, of many expression systems leave only a few hosts as the preferred option to produce membrane proteins. They include the bacterial host, Escherichia coli, the yeasts S. cerevisiae and Pichia pastoris, and higher eukaryotes such as Sf9 insect cells or mammalian cell lines¹⁸. All membrane protein expression technologies have advantages and disadvantages; however, S. cerevisiae is perhaps the best studied eukaryotic model organism suitable for membrane protein production. It is highly versatile with applications in genetic engineering, drug discovery, synthetic biology, and the expression of eukaryotic membrane proteins^14,19,20,21.

In this study, a patented S. cerevisiae membrane protein expression technology²¹ was used, with S. cerevisiae ADΔ¹⁴ and ADΔΔ²² as the preferred hosts (Figure 1A), to overexpress and study the major C. albicans multidrug efflux pump Cdr1. Both the S. cerevisiae strains are derivatives of AD1-8u^- 23 that have either the ura3 (ADΔ) or both the ura3 and his1 (ADΔΔ) genes deleted to eliminate any false positive uracil or histidine prototroph transformants arising through the unwanted integration at the URA3 or the HIS1 genomic loci. The deletion of the 7 major multidrug efflux pumps²³, indicated in Figure 1A, makes ADΔΔ exquisitely sensitive to most xenobiotics. The gain-of-function mutant transcription factor Pdr1-3 causes the constitutive overexpression of heterologous membrane proteins such as Cdr1 (red octagons in Figure 1A) after integration of the heterologous-ORF-containing transformation cassette (Figure 1A) at the genomic PDR5 locus (blue rectangle in Figure 1A) via two homologous recombination events. Proper plasma membrane localization of C-terminally mGFPHis tagged proteins can be confirmed by confocal microscopy (Figure 1A), and the His tag can be used for nickel-affinity purification of the tagged protein. Cloning some fungal ABC transporters (e.g., Candida krusei ABC1) into pABC3-derived plasmids was, however, not possible because they could not be propagated in Escherichia coli due to cell toxicity. This prompted the development of the one-step cloning of membrane proteins^14,24 tagged at either their N- or C-terminus with various affinity, epitope, or reporter tags directly into S. cerevisiae ADΔΔ (Figure 1C). S. cerevisiae ADΔΔ strains overexpressing various CDR1 mutants can also be created efficiently this way by using up to five individual PCR fragments that overlap by 25 bp (Figure 1C). Employing this protocol, many ORFs of interest can be cloned, expressed, and characterized at low cost and at high efficiency within a very short time span. The transformation efficiency reduces only ~2-fold with each additional PCR fragment.

If desired, expression levels can also be readily manipulated by primer design to predictably tune expression levels down to anywhere between 0.1%-50% of the usually high, constitutive expression levels²⁵. The optimized, multifunctional, pABC3¹⁴ derivative cloning vector, pABC3-XLmGFPHis²⁶ (Figure 1B) contains a HRV-3C protease cleavage site (X; LEVLFQ|GP), a protease that performs better at 4 °C than the frequently used tobacco etch virus (TEV) protease²⁷. L is a five amino acid (GSGGS) linker, mGFP is a monomeric mutant (A206K)^28,29 version of the yeast enhanced green-fluorescence protein variant yEGFP3³⁰, and His is a three amino acid linker (GGS) followed by the six-histidine (HHHHHH) nickel-affinity protein purification tag.

This expression technology has been successfully used in drug discovery and the study of membrane proteins. The first structure for a fungal azole drug target, S. cerevisiae Erg11³¹, was solved using this technology. It also enabled the detailed characterization of C. albicans Cdr1^32,33,34 and the creation of a cysteine-deficient Cdr1 molecule³⁵ suitable for cysteine-crosslinking studies to verify any future high-resolution structure. Many other ABC transporters from major human fungal pathogens (i.e., C. albicans, Candida glabrata, Candida auris, Candida krusei, Candida utilis, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, and the Fusarium solani species complex) have also been studied in detail using this expression platform^{24,36,37,38,39}. This has enabled the generation of a panel of S. cerevisiae strains overexpressing efflux pumps that has been used in high-throughput screens to discover the novel fluorescent efflux pump substrate Nile red⁴⁰ and specific⁴¹ and broad-spectrum^{14,33,42,43,44} efflux pump inhibitors. The use of this system also enabled the discovery of clorgyline as the first of its kind broad-spectrum fungal multidrug efflux pump inhibitor⁴².

Complete solubilization of membrane proteins and the creation of a homogeneous membrane protein-micelle preparation devoid of endogenous lipids, requires high detergent concentrations⁴⁵. But unfortunately, this also often inactivates the membrane protein^5,8,45,46. The properties of detergent monomers and their aggregation in solution are affected by the physical properties of the hydrophobic tail, the length and branching of the alkyl chain, the presence of an aromatic nucleus or fluoroalkyl side chain, or the number of polyoxyethylene units. Thus, detergent screening is an important first step to determine the most suitable detergent for membrane protein solubilization and purification.

C. albicans is a major human fungal pathogen of immunocompromised individuals that can cause serious, life threatening invasive infections⁴⁷, and it can become resistant to azole antifungal drugs^48,49. One of the main mechanisms of C. albicans multidrug resistance is the overexpression of Cdr1⁵⁰, which is a type II ATP-binding cassette (ABC) transporter⁵¹ of the ABCG subfamily located in the plasma membrane. Full-size fungal ABCG transporters (consisting of two nucleotide binding domains [NBDs] and two transmembrane domains [TMDs]) are more commonly known as pleiotropic drug resistance (PDR) transporters and are characterized by their unique inverted domain topology [NBD-TMD]₂. PDR transporters are only found in plants^52,53 and fungi⁵⁴. Despite their importance, there are no structures for PDR transporters, although structures for human half-size ABCG transporters have recently been solved which helped create the first tentative model for Cdr1³³. Our recent experimental evidence suggests, however, that this model is flawed possibly because fungal PDR transporters have characteristic asymmetric NBDs resulting quite possibly in a unique transport mechanism. A high-resolution structure of Cdr1 is, therefore, required for both the rational design of novel efflux pump inhibitors that may help overcome efflux-mediated drug resistance, and to provide insights into the mechanism of action of this important ABC transporter family.

The objective of this study was to develop reliable protocols for the expression, solubilization, and purification of Cdr1 in the genetically modified S. cerevisiae expression host, with the ultimate aim of obtaining a high-resolution structure for Cdr1. As part of this process, a protease-cleavable mGFPHis double tag (Figure 1B) was designed with a 16-residue linker separating the tag from the C-terminus of Cdr1, which improved binding of the attached 6x His affinity tag to the nickel-affinity resin and enabled the monitoring of Cdr1 expression levels in living cells and during the entire purification process. A reproducible protocol for small-scale yeast plasma membrane protein preparations containing about 10% C. albicans Cdr1 (as estimated by Coomassie staining after SDS-PAGE) was also developed, which could be used for the biochemical characterization of Cdr1.

Protocol

1. Preparation of fresh or frozen stocks of transformation competent ADΔ and ADΔΔ cells

1. Inoculate 25 mL of 2x YPCD [i.e., 2x YPD; 2% (w/v) yeast extract, 2% (w/v) peptone, 4% (w/v) dextrose), 0.079 % (w/v) CSM (complete supplement mixture)]³⁵ medium with a single yeast colony and incubate overnight (o/n) for 16 h at 30 °C with shaking at 200 revolutions per minute (rpm).
2. Inoculate 225 mL of 2x YPCD medium with the 25 mL o/n culture and check the cell optical density at 600 nm (OD₆₀₀); the OD₆₀₀ is usually ~0.5-1.0.
   NOTE: At this stage make sure that all materials that are required for the following transformation experiment are readily available.
3. Grow the culture at 30 °C for a further ~6-8 h with shaking at 200 rpm until the cell density reaches an OD₆₀₀ of ~6-8.
   NOTE: The following steps are performed at room temperature (RT) unless otherwise stated.
4. Harvest these logarithmic-phase cells by centrifugation at 3,000 x g for 3 min.
5. Resuspend the cells and wash them twice with sterile double-distilled water (ddH₂O; i.e., 200 mL and then 20 mL).
6. Harvest the cells at 3,000 x g for 3 min.
7. Slowly (i.e., add 30 equal aliquots of frozen competent cell (FCC) solution every minute for 30 min) resuspend the cell pellet in X mL of FCC [5% (w/v) glycerol, 10% (v/v) dimethyl sulfoxide (DMSO)] on ice (where X = OD₆₀₀; e.g., if OD₆₀₀ = 6 resuspend in 6 mL, or if OD₆₀₀ = 3 resuspend in 3 mL).
   NOTE: The correct FCC composition is critical for the transformation success.
8. Keep cells on ice for 2 h before transformation or store aliquots at -80 °C until required.
   NOTE: ADΔ and ADΔΔ cells are very sensitive to freezing. Thus, cells must be cooled down slowly to -80 °C: place ice-cold microcentrifuge tubes containing 50-600 µL cell aliquots into a plastic storage box (RT). Place the box in a larger polystyrene container (RT) and close the container with a fitting polystyrene lid. Then, put the container into the -80 °C freezer.
   ​CAUTION: Slow freezing is critical for cell survival.

2. Transformation of ADΔ and ADΔΔ with CaCDR1-XLmGFPHis and confirmation of correct transformants by colony PCR and DNA sequencing

NOTE: Plasmid pABC3-CDR1-mGFPHis was created using conventional cloning strategies described in detail in Lamping et al., 2010⁵⁵ and is illustrated in Figure 1B. Wild-type C. albicans CDR1 was isolated as a PacI/NotI fragment from plasmid pABC3-CaCDR1A-GFP¹⁴ and cloned into pABC3-XLmGFPHis²⁶.

1. PCR amplify the entire CDR1 transformation cassette with a high-fidelity DNA polymerase and primer pair PDR5-pro/PDR5-ter³⁵ using 1-10 ng of pABC3-CaCDR1-XLmGFPHis as a DNA template or, alternatively, digest 2 µg of pABC3-CaCDR1-XLmGFPHis to completion with 10 U restriction enzyme AscI at 37 °C (Figure 1A,B).
   NOTE: The CDR1 transformation cassette (Figure 1A) comprises the PDR5 promoter - CaCDR1-mGFPHis - PGK1 terminator - URA3 selection marker - PDR5 downstream region.
2. Perform agarose gel electrophoresis and gel extract the ~8 kb transformation cassette.
   NOTE: Gel purification of the ~8 kb CaCDR1 transformation cassette removes any possible undigested plasmid DNA, which could lead to incorrect transformants that have the entire plasmid, rather than the linear transformation cassette, integrated at the genomic PDR5 locus.
3. Denature the required amount of salmon sperm carrier DNA (2 mg/mL; 10 mM Tris, 1 mM EDTA; pH 7.5) for 10 min in a boiling water bath and keep on ice. Use screw-capped tubes for boiling salmon sperm DNA to avoid opening of the lid.
4. Mix 50 µL of denatured salmon sperm DNA with 14 µL of the transformation cassette (500-2,000 ng) and keep the 64 µL of DNA mixture on ice until further use.
   NOTE: Use 10 ng of an E. coli-yeast shuttle plasmid (e.g., pYES2) as a positive transformation control (Figure 2B).
5. Resuspend fresh or frozen competent cells quickly defrosted for 5 min in a 30 °C water bath and divide them into 50 µL aliquots in 1.5 mL microcentrifuge tubes.
6. Harvest cells by centrifugation for 1 min in a microfuge at maximum speed (18,000 x g).
7. Remove the supernatant and keep the cell pellet at RT.
8. For each transformation, mix 296 µL combinations (RT) of 260 µL of 50% (w/v) polyethylene glycol (PEG 3350) and 36 µL of 1 M lithium acetate (LiAc), by repeat pipetting, with the appropriate 64 µL of ice-cold DNA mixtures.
9. Add the appropriate mixture immediately to a 50 µL competent cell pellet aliquot.
10. Resuspend the cell pellet in the 360 µL of PEG-LiAc-DNA mixture by thoroughly vortexing for about 30 s.
11. Incubate the cell mixture in a 30 °C water bath for 1 h.
    NOTE: ADΔ and ADΔΔ cells transform better at 30 °C than at 42 °C³⁵.
12. Harvest the cells at 18,000 x g for 10 s. Discard the supernatant and resuspend the cell pellet in 80 µL of ddH₂O.
13. Spread the cells onto a CSM-URA agar plate³⁵ [i.e., 0.67% (w/v) yeast nitrogen base without amino acids, 0.077% (w/v) CSM minus uracil, 2% (w/v) glucose, and 2% (w/v) agar].
14. Incubate the plates for 2-3 days at 30 °C until uracil prototroph transformants are clearly visible.
    NOTE: Expect ~100 transformants per µg of the linear ~8 kb CaCDR1 transformation cassette and ~4 x 10⁴ transformants per µg pYES2.
15. Pick five independent transformants and spread them on a fresh CSM-URA plate to separate the uracil prototroph transformants from remnants of non-transformed host cells.
16. Remove any possible petite mutants by growing the transformants on YPG-agar plates [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (v/v) glycerol, and 2% (w/v) agar] (Figure 2D).
    NOTE: Petite mutants have defective mitochondria and, thus, cannot grow on non-fermentable carbon sources. They are quite common in S. cerevisiae⁵⁶. S. cerevisiae ADΔ and ADΔΔ are particularly prone to acquire the petite phenotype.
17. Perform yeast colony PCR and confirm at least three independent transformants to be correctly integrated into the genomic PDR5 locus (Figure 1C) by amplifying the entire ~8 kb CaCDR1 transformation cassette with a specific DNA polymerase that is optimized for amplifying PCR products from impure DNA template sources. Use a set of primers that bind just outside the integration site and 1 µL aliquots of cell suspensions (in ddH₂O) derived from single colonies as DNA templates.
    NOTE: This particular DNA polymerase reliably amplifies ~8 kb PCR fragments from intact yeast cells. However, for reliable amplification, 45 PCR cycles are required, and the yeast cells must be resuspended at OD₆₀₀ 1-10 in ddH₂O.
18. Confirm the correct ~8 kb PCR amplification product by DNA agarose gel electrophoresis of a 1 µL portion of the PCR reaction.
19. Remove excess amplification primers from a 10 µL portion of the PCR reaction with an enzyme mixture of a single-strand DNA exonuclease and a phosphatase following the manufacturer's instructions before sequencing the entire ORF using portions of the treated DNA sample with appropriate primers.

3. Small-scale yeast plasma membrane isolation protocol

1. Growing yeast cells
   1. Pre-culture a single yeast colony in 10 mL of YPD at 30 °C for ~7-8 h with shaking at 200 rpm.
   2. Inoculate 40 mL of YPD medium with the 10 mL pre-culture and incubate the cells at 30 °C o/n (~16 h) with shaking at 200 rpm until the cell density reaches an OD₆₀₀ of 1-3.
2. Harvesting yeast cells
   1. Harvest 40 OD units (ODU; e.g., 1 mL at an OD₆₀₀ of 1 = 1 ODU) of logarithmic-phase cells at 4,200 x g for 5 min at 4 °C.
   2. Resuspend and wash cells twice with ice cold sterile ddH₂O (i.e., 40 mL and then 1 mL; harvest cells in between steps by centrifugation at 4,200 x g for 5 min at 4 °C).
   3. Resuspend the pellet in 1 mL of ice cold sterile ddH₂O and transfer the cell suspension into a pre-cooled (on ice) 1.5 mL microcentrifuge tube.
   4. Harvest cells at 3,300 x g for 3 min at 4 °C.
   5. Aspirate the supernatant.
   6. Resuspend the cell pellet in 0.5 mL homogenizing buffer [HB; 50 mM Tris, 0.5 mM EDTA, 20% (v/v) glycerol; pH 7.5] freshly supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF).
      NOTE: Always add PMSF fresh because PMSF is inactivated quickly upon exposure to water.
      CAUTION: PMSF is a serine protease inhibitor, which is extremely corrosive and destructive to tissues. It may cause irreversible eye damage.
   7. Store the cell suspension at -80 °C or use immediately.
3. Isolation of plasma membranes
   1. If frozen, defrost the cells on ice for ~1 h.
   2. Add ice-cold 0.5 mm diameter silica beads to the 0.5 mL cell suspension to reach a total volume of 1 mL.
   3. Break cells with 6 cycles of vortexing at maximum shaking intensity for 1 min interspersed with 3 min cooling periods on ice.
   4. Make a thin hole at the bottom of the tube with a heated scalpel blade.
   5. Collect the broken cell homogenate through the bottom of the tube fitted into another ice-cold 1.5 mL microcentrifuge tube with a 10 s low-speed (~200 rpm) spin.
      NOTE: This ensures that the silica beads remain in the original tube.
   6. Centrifuge the cell homogenate at 5,156 x g for 5 min at 4 °C to remove cell debris, unbroken cells, and nuclei.
   7. Transfer 450 µL of supernatant into an ice cold 1.5 mL microcentrifuge tube and add an additional 1 mL of ice-cold HB supplemented with fresh PMSF (1 mM).
      ​NOTE: This dilution step is critical for the high-quality plasma membrane protein recovery.
   8. Harvest plasma membranes at 17,968 x g for 1 h at 4 °C and resuspend the plasma membrane pellet, by repeat pipetting, in 100 µL of HB freshly supplemented with 1 mM PMSF. Loosen the cell pellet for proper plasma membrane homogenization by stirring the cell pellet with the 100 µL pipette tip before releasing the 100 µL of HB and up and down pipetting.
   9. Measure the protein concentration of the plasma membrane preparation with a protein assay kit that is compatible with buffers containing reducing agent and detergent.
   10. Store the plasma membranes at -80 °C or keep on ice for immediate use.

4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

1. Assemble the apparatus for preparing polyacrylamide gels.
2. For two separating gels (7% polyacrylamide), mix 2.1 mL of 40% acrylamide/bis-acrylamide, 3 mL of 4x separating buffer (1.5 M Tris, 0.4% sodium dodecyl sulfate [SDS] (w/v); pH 8.8), and 6.9 mL of ddH₂O. Add 8 µL of tetramethylethylenediamine (TEMED) and 60 µL of 10% ammonium persulphate (APS) to initiate polymerization of acrylamide.
   CAUTION: Acrylamide/bis-acrylamide is very toxic. It causes skin irritation, peripheral neuropathy and is a carcinogen. TEMED is harmful if swallowed or inhaled. APS is harmful if swallowed. It causes serious eye and skin irritations.
3. Pour ~4-5 mL of this mixture into the assembled gel apparatus, up to ~2 cm from the top.
4. Carefully layer ~1-2 mL of 0.1% SDS on top to create a planar meniscus.
5. Allow the polyacrylamide to set for ~60 min at RT.
6. Prepare a stacking gel mixture for two gels by mixing 0.5 mL of 40% acrylamide/bis-acrylamide, 1 mL of 4x stacking buffer (0.5 M Tris, 0.4% SDS (w/v); pH 6.8), and 6.9 mL of ddH₂O. Add 2 µL of TEMED and 30 µL of 10% APS to initiate polymerization of acrylamide.
7. Remove the 0.1% SDS layer from the polymerized separating gel and rinse with ddH₂O to remove traces of SDS.
8. Pour the stacking gel mix onto the separating gel.
9. Place a comb into the stacking gel and remove any air bubbles from around the comb.
10. Allow the stacking gel to set for ~60 min at RT.
11. Remove the comb and rinse the gel slots with water. Put the gel into the gel tank and fill the gel tank to the top with 1x running buffer (24.8 mM Tris, 190 mM glycine, 0.1% SDS).
    NOTE: Prepare 1x running buffer from a 10x buffer stock (248 mM Tris, 1.9 M glycine, 1% SDS (w/v) in ddH₂O) stored at RT.
12. Mix 5-10 µL plasma membrane samples (i.e., 10-20 µg protein) with equal volumes of 2x protein loading dye [120 mM Tris-HCl (pH 6.8), 20% glycerol, 0.02% bromophenol blue, 4% SDS, 200 mM dithiothreitol (DTT)] and immediately load into individual gel slots submerged in running buffer.
    CAUTION: DTT is harmful if swallowed. It causes serious eye and skin irritations.
    NOTE: Make a 10 mL stock of 2x protein loading dye, aliquot and store at -20 °C. Do not heat the mixed samples but immediately load them into individual gel slots so that the GFP tag is not denatured, and the in-gel fluorescence signals can be detected.
13. Load protein molecular weight markers (10-245 kDa range) into a separate slot to enable the size estimation of individual protein fragments.
14. Perform gel electrophoresis at 200 V until the blue loading dye reaches the bottom of the gel (usually 45-55 min).
15. Examine the gel for in-gel GFP-fluorescence with a gel imaging system (excitation and emission wavelengths are 475-485 nm and 520 nm, respectively).
16. Following in-gel fluorescence imaging, fix proteins by gentle agitation of the gel in ~10-20 mL Protein Gel Fixing Solution (40% ethanol, 10% acetic acid) for 15 min at RT.
17. Rinse the gel twice for 10 min with 10 mL of ddH₂O and visualize protein bands by placing the gel in 10 mL colloidal Coomassie stain solution with gentle shaking for ~1 h at RT.
18. For improved visualization of protein bands, de-stain the gel once or twice in ~20 mL of ddH₂O for ~1 h before recording images with the gel imaging system.

5. Determination of Cdr1 ATPase Activities ⁵⁷

1. Dilute plasma membrane samples >2.2 mg/mL to 1-2 mg/mL in HB.
2. Equilibrate the ATPase assay cocktail (75 mM MES-Tris, 75 mM potassium nitrate, 0.3 mM ammonium molybdate, 7.5 mM sodium azide; pH 7.5) and Mg-ATP (28.8 mM ATP disodium salt, 28.8 mM MgCl₂; pH 7.0) to 30 °C in a 30 °C incubator.
   CAUTION: Sodium azide is highly toxic.
   NOTE: Ensure all buffer stocks and bottles are phosphate free; i.e., wash glassware with 1% (vol/vol) HCl and rinse it several times with ddH₂O. Also, keep it separate from other glassware that likely contains traces of phosphate commonly present in detergents used to wash glassware.
3. Add 90 µL of assay cocktail with or without Cdr1-ATPase inhibitor (20 µM of oligomycin) into individual wells of a 96-well microtiter plate.
   NOTE: The assay is performed in triplicate.
4. Add 5 µL of the isolated plasma membranes (~5-10 µg protein) or phosphate standards (0-100 nmoles Pi) into the appropriate wells of the microtiter plate.
   NOTE: Keep the first and last columns for two separate sets of phosphate standards.
5. Start the assay by adding 25 µL of prewarmed 28.8 mM Mg-ATP (6 mM final concentration) with a multi-channel pipette into individual wells and incubate at 30 °C for 30 min.
6. Stop the reaction by adding 130 µL of development reagent (1.6% sodium L-ascorbate, 1% SDS, 12% ammonium molybdate in 6 M sulfuric acid).
   CAUTION: Concentrated sulfuric acid reacts violently with water. It is corrosive and may cause skin and lung damage.
7. Incubate at RT for 10 min.
8. Read the absorbance of the microtiter plate wells at 750 nm with a microtiter plate reader.
   ​NOTE: Sticking to the 10 min incubation time for blue dye development (i.e., a reduced phosphor-molybdenum complex) is critical for assay accuracy because the blue dye development continues with time.
9. Obtain the Cdr1-specific ATPase activity (i.e., the oligomycin-sensitive ATPase activity) by subtracting the ATPase activity in the presence of oligomycin from the total ATPase activity in the absence of oligomycin.

6. Small scale detergent screen

1. Combine the plasma membrane preparations (i.e., 2.5 mg of plasma membrane protein) of cells overexpressing Cdr1-mGFPHis with GTED-20 buffer [10 mM Tris, 0.5 mM EDTA, 20 % (w/v) glycerol; pH 7.5; freshly supplemented with 1 mM PMSF] containing 5 mg of the test detergent, to reach a total volume of 0.5 mL supplemented with 1% (w/v) detergent.
2. Rotate the mixture at 4-8 °C for 2 h, with a rotation device.
3. Centrifuge the mixture at 141,000 x g at 4 °C for 1 h.
4. Transfer the supernatant containing solubilized material to a fresh microcentrifuge tube.
5. Add 0.5 mL of GTED-20 buffer supplemented with 2% (w/v) SDS to the insoluble pellet fraction and incubate at 30 °C o/n in a shaking incubator to extract all detergent-insoluble membrane protein.
6. Analyze and compare the supernatant and solubilized pellet fractions by SDS-PAGE.
7. Photograph gels containing GFP-tagged proteins for in-gel GFP-fluorescence before Coomassie staining and quantify expression levels with the imaging system.
8. Use the soluble membrane protein fraction for downstream applications such as fluorescence size exclusion chromatography (FSEC)⁵⁸ to identify suitable detergent(s).

Results

A high frequency of transformation of S. cerevisiae ADΔΔ (~4 x 10⁴ transformants/µg) was achieved with pYES2 (Figure 2B). As expected, the no DNA (i.e., ddH₂O only) control gave no transformants, and 1 µg of the linear CDR1-mGFPHis transformation cassette (Figure 1A) gave ~50 transformants (Figure 2C) with the optimized ADΔΔ transformation protocol. The CDR1-mGFPHis ...

Discussion

Despite recent progress in the structural analysis of membrane proteins, no 3D structure for Cdr1, or any other PDR transporter, is currently available. So, gaining knowledge of the Cdr1 structure and its biochemical features is important, as this will not only provide insight into rational design of novel drugs to overcome efflux-mediated drug resistance, but also into the mechanism of function of an important subfamily of ABC proteins.

One of the main requirements for the structural characte...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-(N-Morpholino)ethane-sulphonic acid (MES)	Sigma-Aldrich	M3671
2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base; ultra-pure)	Merck	77-86-1
2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside	Anatrace	NG310S	LMNG
2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside	Anatrace	NG311S	OGNG (MNG-OG)
2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside	Anatrace	NG322S	DMNG
4-Trans-(4-trans-propylcyclohexyl)-cyclohexyl α-D-maltopyranoside	Glycon Biochemicals GmbH	D99019-C	PCC-α-M
40% Acrylamide/Bis-acrylamide (37.5:1)	Bio-Rad	1610148
Acetic acid (glacial)	Merck	64-19-7
Agar	Formedium	009002-18-0
Ammonium molybdate	Sigma-Aldrich	13106-76-8
Ammonium persulphate (APS)	Bio-Rad	1610700
ATP disodium salt	sigma-Aldrich	A-6419
Bromophenol blue	SERVA Electrophoresis GmbH	34725-61-6
CHAPS	Anatrace	C316S
CHAPSO	Anatrace	C317S
CSM	Formedium	DCS0019
CSM minus uracil	Formedium	DCS0161
Cyclohexyl-1-butyl-β-D-maltopyranoside	Anatrace	C324S	CYMAL-4
Cyclohexyl-1-heptyl-β-D-maltopyranoside	Anatrace	C327S	CYMAL-7
Cyclohexyl-methyl-β-D-maltopyranoside	Anatrace	C321S	CYMAL-1
Digitonin	Sigma-Aldrich	11024-24-1
Dithiothreitol (DTT)	Roche Diagnostics	10197785103
DMSO	Merck	67-68-5
Ethanol	Merck	459836
Ethylenediaminetetraacetic acid disodium salt (EDTA; Titriplex III)	Merck	6381-92-6
ExoSAP-IT PCR Product Cleanup Reagent	Applied Biosystems	78205	A blend of exonuclease and phosphatase
Glucose	Formedium	50-99-7
Glycerol	Merck	56-81-5
Glycine	Merck	G8898
HEPES	Formedium	7365-45-9
Hydrochloric acid	Merck	1003172510
KOD Fx Neo	TOYOBO Co	KFX-201	Use for reliable colony PCR
lithium acetate (LiAc)	Sigma-Aldrich	546-89-4
Magnesium chloride hexa-hydrate	sigma-Aldrich	M2393
MES	Formedium	145224-94-8
n-Decanoyl-N-hydroxyethyl-glucamide	Anatrace	H110S	HEGA-10
n-Decanoyl-N-methyl-glucamide	Anatrace	M320S	MEGA-10
n-Decyl-phosphocholine	Anatrace	F304S	Fos-choline-10
n-Decyl-β-D-maltopyranoside	Anatrace	D322S	DM
n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate	Anatrace	AZ312S	Anzergent 3-12
n-Dodecyl-N,N-dimethylamine-N-oxide	Anatrace	D360S	LDAO
n-Dodecyl-α-D-maltopyranoside	Anatrace	D310HA	α-DDM
n-Dodecyl-β-D-maltopyranoside	Anatrace	D310S	β-DDM
n-Nonyl-β-D-glucopyranoside	Anatrace	N324S	NG
n-Nonyl-β-D-maltopyranoside	Anatrace	N330S	NM
n-Octadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate	Anatrace	AZ318S	Anzergent 3-18
n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate	Anatrace	AZ308S	Anzergent 3-8
n-Octyl-phosphocholine	Anatrace	F300S	Fos-choline-8
n-Octyl-β-D-glucopyranoside	Anatrace	O311S	OG
n-Tetradecyl-phosphocholine	Anatrace	F312S	Fos-choline-14
n-Tetradecyl-β-D-maltopyranoside	Anatrace	T315S	TDM
n-Tridecyl-phosphocholine	Anatrace	F310S	Fos-choline-13
n-Tridecyl-β-D-maltopyranoside	Anatrace	T323S	-
n-Undecyl-β-D-maltopyranoside	Anatrace	U300S	UM (UDM)
N,N,N’,N’-tetramethyl-ethylenediamine (TEMED)	Sigma-Aldrich	T9281
Octylphenoxypolyethoxyethanol	Sigma-Aldrich	9002-93-1	TRITON X-100
Oligomycin	Sigma-Aldrich	75351
Peptone	Formedium	3049-73-7
phenylmethylsulfonyl fluoride (PMSF)	Roche Diagnostics	329-98-6
Phusion Hot Start Flex DNA Polymerase	New England Biolabs	M0535S	High-fidelity DNA polymerase
polyethylene glycol (PEG 3350)	Sigma-Aldrich	25322-68-3
polyoxyethylenesorbitan monooleate	Sigma-Aldrich	9005-65-6	TWEEN 80
Potassium nitrate	Sigma-Aldrich	P8394
Protein Assay Kit	Bio-Rad	5000122	RC DC Protein Assay Kit II
QC Colloidal Coomassie Stain	Bio-Rad	1610803
Prism Ultra Protein Ladder (10-245 kDa)	Abcam	AB116028
Sodium azide	Sigma-Aldrich	71289
Sodium dodecyl sulphate	Sigma-Aldrich	151-21-3	SDS
Sodium L-ascorbate BioXtra	Sigma-Aldrich	11140
Sucrose Monododecanoate	Anatrace	S350S	DDS
Sulphuric acid	Sigma-Aldrich	339741
Yeast extract	Formedium	008013-01-2
Yeast nitrogen base without amino acids	Formedium	CYN0402
Equipment (type)
Centrifuge  (Eppendorf 5804)	Eppendorf
Centrifuge (Beckman Ultra)	Beckman
Centrifuge (Sorvall RC6)	Sorvall
FSEC apparatus (NGC Chromatography Medium Pressure system equipped with a fluorescence detector, an autosampler, a fractionator)	Bio-Rad
Gel imaging (GelDoc EZ Imager)	Bio-Rad
Microplate reader (Synergy 2 Multi-Detection)	BioTek Instruments
PCR thermal cycler (C1000 Touch)	Bio-Rad
Power supply (PowerPac)	Bio-Rad
SDS PAGE (Mini-PROTEAN Tetra)	Bio-Rad
Shaking incubator (Multitron)	Infors HT, Bottmingen
Superose 6 Increase 10/300 GL	GE Healthcare Life Sciences	GE17-5172-01
UV/Visible spectrophotometer (Ultraspec 6300 pro)	Amersham BioSciences UK Ltd