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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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....

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 .......

Representative 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. .......

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.......

Acknowledgements

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....

Materials

NameCompanyCatalog NumberComments
2-(N-Morpholino)ethane-sulphonic acid (MES)Sigma-AldrichM3671
2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base; ultra-pure)Merck77-86-1
2,2-Didecylpropane-1,3-bis-β-D-maltopyranosideAnatraceNG310SLMNG
2,2-Dihexylpropane-1,3-bis-β-D-glucopyranosideAnatraceNG311SOGNG (MNG-OG)
2,2-Dioctylpropane-1,3-bis-β-D-maltopyranosideAnatraceNG322SDMNG
4-Trans-(4-trans-propylcyclohexyl)-cyclohexyl α-D-maltopyranosideGlycon Biochemicals GmbHD99019-CPCC-α-M
40% Acrylamide/Bis-acrylamide (37.5:1)Bio-Rad1610148
Acetic acid (glacial)Merck64-19-7
AgarFormedium 009002-18-0
Ammonium molybdateSigma-Aldrich13106-76-8
Ammonium persulphate (APS)Bio-Rad1610700
ATP disodium saltsigma-AldrichA-6419
Bromophenol blueSERVA Electrophoresis GmbH34725-61-6
CHAPSAnatraceC316S
CHAPSOAnatraceC317S
CSMFormediumDCS0019
CSM minus uracilFormediumDCS0161
Cyclohexyl-1-butyl-β-D-maltopyranosideAnatraceC324SCYMAL-4
Cyclohexyl-1-heptyl-β-D-maltopyranosideAnatraceC327SCYMAL-7
Cyclohexyl-methyl-β-D-maltopyranosideAnatraceC321SCYMAL-1
DigitoninSigma-Aldrich11024-24-1
Dithiothreitol (DTT)Roche Diagnostics10197785103
DMSOMerck67-68-5
EthanolMerck459836
Ethylenediaminetetraacetic acid disodium salt (EDTA; Titriplex III)Merck6381-92-6
ExoSAP-IT PCR Product Cleanup ReagentApplied Biosystems78205A blend of exonuclease and phosphatase
GlucoseFormedium50-99-7
GlycerolMerck56-81-5
GlycineMerckG8898
HEPESFormedium7365-45-9
Hydrochloric acidMerck1003172510
KOD Fx NeoTOYOBO CoKFX-201Use for reliable colony PCR
lithium acetate (LiAc)Sigma-Aldrich546-89-4
Magnesium chloride hexa-hydratesigma-AldrichM2393
MESFormedium145224-94-8
n-Decanoyl-N-hydroxyethyl-glucamideAnatraceH110SHEGA-10
n-Decanoyl-N-methyl-glucamideAnatraceM320SMEGA-10
n-Decyl-phosphocholineAnatraceF304SFos-choline-10
n-Decyl-β-D-maltopyranosideAnatraceD322SDM
n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonateAnatraceAZ312SAnzergent 3-12
n-Dodecyl-N,N-dimethylamine-N-oxideAnatraceD360SLDAO
n-Dodecyl-α-D-maltopyranosideAnatraceD310HAα-DDM
n-Dodecyl-β-D-maltopyranosideAnatraceD310Sβ-DDM
n-Nonyl-β-D-glucopyranosideAnatraceN324SNG
n-Nonyl-β-D-maltopyranosideAnatraceN330SNM
n-Octadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonateAnatraceAZ318SAnzergent 3-18
n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulphonateAnatraceAZ308SAnzergent 3-8
n-Octyl-phosphocholineAnatraceF300SFos-choline-8
n-Octyl-β-D-glucopyranosideAnatraceO311SOG
n-Tetradecyl-phosphocholineAnatraceF312SFos-choline-14
n-Tetradecyl-β-D-maltopyranosideAnatraceT315STDM
n-Tridecyl-phosphocholineAnatraceF310SFos-choline-13
n-Tridecyl-β-D-maltopyranosideAnatraceT323S-
n-Undecyl-β-D-maltopyranosideAnatraceU300SUM (UDM)
N,N,N’,N’-tetramethyl-ethylenediamine (TEMED)Sigma-AldrichT9281
OctylphenoxypolyethoxyethanolSigma-Aldrich9002-93-1TRITON X-100
OligomycinSigma-Aldrich75351
PeptoneFormedium3049-73-7
phenylmethylsulfonyl fluoride (PMSF)Roche Diagnostics329-98-6
Phusion Hot Start Flex DNA PolymeraseNew England BiolabsM0535SHigh-fidelity DNA polymerase
polyethylene glycol (PEG 3350)Sigma-Aldrich25322-68-3
polyoxyethylenesorbitan monooleateSigma-Aldrich9005-65-6TWEEN 80
Potassium nitrateSigma-AldrichP8394
Protein Assay KitBio-Rad5000122RC DC Protein Assay Kit II
QC Colloidal Coomassie StainBio-Rad1610803
Prism Ultra Protein Ladder (10-245 kDa)AbcamAB116028
Sodium azideSigma-Aldrich71289
Sodium dodecyl sulphateSigma-Aldrich151-21-3SDS
Sodium L-ascorbate BioXtraSigma-Aldrich11140
Sucrose MonododecanoateAnatraceS350SDDS
Sulphuric acidSigma-Aldrich339741
Yeast extractFormedium008013-01-2
Yeast nitrogen base without amino acidsFormediumCYN0402
 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 GLGE Healthcare Life SciencesGE17-5172-01
UV/Visible spectrophotometer (Ultraspec 6300 pro)Amersham BioSciences UK Ltd

References

  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 ....

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