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Introduction

Protocol

Representative Results

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Acknowledgements

Materials

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Biology

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

Published: June 13th, 2021

DOI:

10.3791/62592

1Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, 2Department of Microbiology, Faculty of Medicine, Chulalongkorn University, 3School of Biological Sciences, University of Auckland
* These authors contributed equally

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.

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.

The extraction of integral membrane proteins from their native lipid environment can dramatically affect their structure and function1,2,3,4. 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 crit....

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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 .......

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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. .......

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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.......

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The authors gratefully acknowledge funding from the New Zealand Marsden Fund (Grant UOO1305), and a block grant from Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (M. Niimi). They wish to thank the University of Otago for providing G. Madani with a PhD Scholarship. The authors also wish to express their gratitude to Professor Stefan Raunser and his colleagues, Dr Amir Apelbaum, and Dr Deivanayagabarathy Vinayagam, for their support and supervision during a 6-month visit of G. Madani at the Max Planck Institute of Molecular Physiology (MPIMP), Dortmund, Germany. The authors also thank the German Academic Exchange Service (DAAD) for providing G. Madan....

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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

  1. Arachea, B. T., et al. Detergent selection for enhanced extraction of membrane proteins. Protein Expression and Purification. 86 (1), 12-20 (2012).
  2. Guo, Y. Be cautious with crystal structures of membrane proteins or complexes prepared in detergents. Crystals (Basel). 10 (2), 86 (2020).
  3. Lewinson, O., Orelle, C., Seeger, M. A. Structures of ABC transporters: handle with care. FEBS Letters. 594 (23), 3799-3814 (2020).
  4. Luckey, M. . Membrane Structural Biology: With Biochemical and Biophysical Foundations. , 69-105 (2014).
  5. Opekarova, M., Tanner, W. Specific lipid requirements of membrane proteins--a putative bottleneck in heterologous expression. Biochimica et Biophysica Acta. 1610 (1), 11-22 (2003).
  6. Qiu, W., et al. Structure and activity of lipid bilayer within a membrane-protein transporter. Proceedings of the National Academy of Sciences of the United States of America. 115 (51), 12985-12990 (2018).
  7. Dowhan, W. Molecular basis for membrane phospholipid diversity: why are there so many lipids. Annual Review of Biochemistry. 66, 199-232 (1997).
  8. Lee, A. G. Lipid-protein interactions in biological membranes: a structural perspective. Biochimica et Biophysica Acta. 1612 (1), 1-40 (2003).
  9. Ahn, J. H., Pan, J. G., Rhee, J. S. Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp. Applied and Environmental Microbiology. 67 (12), 5506-5511 (2001).
  10. Newby, Z. E., et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nature Protocols. 4 (5), 619-637 (2009).
  11. Parker, J. L., Newstead, S. Membrane protein crystallisation: current trends and future perspectives. Advances in Experimental Medicine and Biology. 922, 61-72 (2016).
  12. Wiener, M. C. A pedestrian guide to membrane protein crystallization. Methods. 34 (3), 364-372 (2004).
  13. Grisshammer, R. Understanding recombinant expression of membrane proteins. Current Opinion in Biotechnology. 17 (4), 337-340 (2006).
  14. Lamping, E., et al. Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Eukaryot Cell. 6 (7), 1150-1165 (2007).
  15. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., Harvey, L. M. Heterologous protein production using the Pichia pastoris expression system. Yeast. 22 (4), 249-270 (2005).
  16. Focke, P. J., et al. Combining in vitro folding with cell free protein synthesis for membrane protein expression. Biochemistry. 55 (30), 4212-4219 (2016).
  17. Reckel, S., et al. Strategies for the cell-free expression of membrane proteins. Methods in Molecular Biology. 607, 187-212 (2010).
  18. Pandey, A., Shin, K., Patterson, R. E., Liu, X. Q., Rainey, J. K. Current strategies for protein production and purification enabling membrane protein structural biology. Biochemistry and Cell Biology. 94 (6), 507-527 (2016).
  19. Harvey, C. J. B., et al. HEx: A heterologous expression platform for the discovery of fungal natural products. Science Advances. 4 (4), (2018).
  20. Kingsman, S. M., Kingsman, A. J., Dobson, M. J., Mellor, J., Roberts, N. A. Heterologous gene expression in Saccharomyces cerevisiae. Biotechnology & Genetic Engineering Reviews. 3, 377-416 (1985).
  21. Monk, B. C., et al. Yeast membrane protein expression system and its application in drug screening. US patent. , (2002).
  22. Sagatova, A. A., Keniya, M. V., Wilson, R. K., Monk, B. C., Tyndall, J. D. Structural insights into binding of the antifungal drug fluconazole to Saccharomyces cerevisiae lanosterol 14alpha-demethylase. Antimicrobial Agents and Chemotherapy. 59 (8), 4982-4989 (2015).
  23. Decottignies, A., et al. ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. The Journal of Biological Chemistry. 273 (20), 12612-12622 (1998).
  24. Lamping, E., Zhu, J. Y., Niimi, M., Cannon, R. D. Role of ectopic gene conversion in the evolution of a Candida krusei pleiotropic drug resistance transporter family. Genetics. 205 (4), 1619-1639 (2017).
  25. Lamping, E., Niimi, M., Cannon, R. D. Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast. Microbial Cell Factories. 12, 74 (2013).
  26. James, J. E., Lamping, E., Santhanam, J., Cannon, R. D. PDR transporter ABC1 is involved in the innate azole resistance of the human fungal pathogen Fusarium keratoplasticum. Frontiers in Microbiology. , (2021).
  27. Ullah, R., et al. Activity of the human rhinovirus 3C protease studied in various buffers, additives and detergent solutions for recombinant protein production. PLoS One. 11 (4), 0153436 (2016).
  28. von Stetten, D., Noirclerc-Savoye, M., Goedhart, J., Gadella, T. W., Royant, A. Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K. Acta Crystallographica Section F. Structural Biology and Crystalization Communications. 68, 878-882 (2012).
  29. Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 296 (5569), 913-916 (2002).
  30. Cormack, B. P., et al. Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology (Reading). 143, 303-311 (1997).
  31. Monk, B. C., et al. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer. Proceedings of the National Academy of Sciences of the United States of America. 111 (10), 3865-3870 (2014).
  32. Holmes, A. R., et al. ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrobial Agents and Chemotherapy. 52 (11), 3851-3862 (2008).
  33. Tanabe, K., et al. FK506 resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 involves mutations in the transmembrane domains and extracellular loops. Antimicrobial Agents and Chemotherapy. 63 (1), 01146 (2019).
  34. Tanabe, K., et al. Chimeras of Candida albicans Cdr1p and Cdr2p reveal features of pleiotropic drug resistance transporter structure and function. Molecular Microbiology. 82 (2), 416-433 (2011).
  35. Madani, G., Lamping, E., Cannon, R. D. Engineering a cysteine-deficient functional Candida albicans Cdr1 molecule reveals a conserved region at the cytosolic apex of ABCG transporters important for correct folding and frafficking of Cdr1. mSphere. 6 (1), (2021).
  36. Lamping, E., et al. Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrobial Agents and Chemotherapy. 53 (2), 354-369 (2009).
  37. Panapruksachat, S., et al. Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2. Medical Mycology. 54 (5), 478-491 (2016).
  38. Wada, S., et al. Phosphorylation of Candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability. The Journal of Biological Chemistry. 280 (1), 94-103 (2005).
  39. Watanasrisin, W., et al. Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p. FEMS Yeast Research. 16 (4), (2016).
  40. Ivnitski-Steele, I., et al. Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p. Analytical Biochemistry. 394 (1), 87-91 (2009).
  41. Niimi, K., et al. Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor. Molecular Microbiology. 85 (4), 747-767 (2012).
  42. Holmes, A. R., et al. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates. Antimicrobial Agents and Chemotherapy. 56 (3), 1508-1515 (2012).
  43. Reis de Sa, L. F., et al. Synthetic organotellurium compounds sensitize drug-resistant Candida albicans clinical isolates to fluconazole. Antimicrobial Agents and Chemotherapy. 61 (1), 01231 (2017).
  44. Tanabe, K., et al. Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium. Biochemical and Biophysical Research Communications. 364 (4), 990-995 (2007).
  45. le Maire, M., Champeil, P., Moller, J. V. Interaction of membrane proteins and lipids with solubilizing detergents. Biochimica et Biophysica Acta. 1508 (1-2), 86-111 (2000).
  46. Seddon, A. M., Curnow, P., Booth, P. J. Membrane proteins, lipids and detergents: not just a soap opera. Biochimica et Biophysica Acta. 1666 (1-2), 105-117 (2004).
  47. Pfaller, M. A. Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission. Clinical Infectious Diseases. 22, 89-94 (1996).
  48. Cannon, R. D., et al. Efflux-mediated antifungal drug resistance. Clinical Microbiology Reviews. 22 (2), 291-321 (2009).
  49. Sanglard, D., et al. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrobial Agents and Chemotherapy. 39 (11), 2378-2386 (1995).
  50. Prasad, R., De Wergifosse, P., Goffeau, A., Balzi, E. Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Current Genetics. 27 (4), 320-329 (1995).
  51. Lamping, E., Madani, G., Lee, H. J., Niimi, M., Cannon, R. D., Prasad, R. . Candida albicans: Cellular and Molecular Biology. , 379-406 (2017).
  52. Crouzet, J., Trombik, T., Fraysse, A. S., Boutry, M. Organization and function of the plant pleiotropic drug resistance ABC transporter family. FEBS Letters. 580 (4), 1123-1130 (2006).
  53. Kang, J., et al. Plant ABC Transporters. Arabidopsis Book. 9, 0153 (2011).
  54. Lamping, E., et al. Fungal PDR transporters: phylogeny, topology, motifs and function. Fungal Genetics and Biology. 47 (2), 127-142 (2010).
  55. Lamping, E., Cannon, R. D. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps. Methods in Molecular Biology. 666, 219-250 (2010).
  56. Day, M. Yeast petites and small colony variants: for everything there is a season. Advances in Applied Microbiology. 85, 1-41 (2013).
  57. Niimi, K., et al. Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative. Antimicrobial Agents and Chemotherapy. 48 (4), 1256-1271 (2004).
  58. Kawate, T., Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure. 14 (4), 673-681 (2006).
  59. Hao, Z., et al. A novel and fast purification method for nucleoside transporters. Frontiers in Molecular Biosciences. 3, 23 (2016).
  60. Nakamura, K., et al. Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrobial Agents and Chemotherapy. 45 (12), 3366-3374 (2001).
  61. Bernaudat, F., et al. Heterologous expression of membrane proteins: choosing the appropriate host. PLoS One. 6 (12), 29191 (2011).
  62. Byrne, B. Pichia pastoris as an expression host for membrane protein structural biology. Current Opinion in Structural Biology. 32, 9-17 (2015).
  63. Holmes, A. R., et al. Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2. Molecular Microbiology. 62 (1), 170-186 (2006).
  64. Keniya, M. V., et al. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5. Molecular Pharmaceutics. 11 (10), 3452-3462 (2014).

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