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In This Article

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

Summary

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Abstract

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, γ-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Introduction

Proteins involved in the trafficking of metabolites constitute an essential level of physiological regulation, but the vast majority of plant membrane transporters have not yet been functionally characterized. Several strategies have been implemented to characterize novel transport proteins. Heterologous expression in model organisms such as E. coli and eukaryotic cells such as yeast, Xenopus oocytes, mammalian cells, insect cells and plant cells have all been used to determine their transport activity1. Eukaryotic cells are favored for the expression of eukaryotic proteins, because the basic cellular composition, signal trans....

Protocol

1. Generation of the E. coli Double Knock Out Mutant with P1 Transduction

  1. Obtain the E. coli single-knockout mutant strains ΔPotE and ΔCadB from the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu).
    NOTE: The ΔPotE strain is kanamycin resistant26 and the ΔCadB strain is tetracycline resistant27.......

Representative Results

The major steps in this protocol are summarized pictorially in Figure 1. Briefly, E. coli cells deficient in all polyamine exchangers and expressing AtBAT1 are cultured, centrifuged, washed with a buffer and subjected to cell lysis using a French press. Lysis tends to produce vesicles that are mostly inside-out and trap the buffer outside the cells. Cell debris is removed by centrifugation, and a second ultracentifugation step is used to col.......

Discussion

In the present study, we outline a method for the characterization of an antiporter by first expressing the protein in E. coli and then generating membrane vesicles, so that the heterologously-expressed protein can be assayed in a cell-free system. In addition to equipment found in most molecular biology labs, this strategy requires the use of a French press, an ultracentrifuge, and access to a facility to conduct radioisotope assays.

A basic requirement of this technique is that the .......

Acknowledgements

Support for this project came from the BGSU Graduate College, and the BGSU Office of Sponsored Programs and Research.

....

Materials

NameCompanyCatalog NumberComments
2-mercaptoethanolSigma-AldrichM6250
3H-putrescinePerkinElmerNET185001MC
3H-spermidinePerkinElmerNET522001MC
4-chloro-1-naphtholSigma-AldrichC8890
14C arginineMoravek Inc.MC137
ArginineSigma-AldrichA-5006
Anti-His (C-term)-HRP antibodyThermoFisherR931-25Detects the C-terminal polyhistidine (6xHis) tag, requires the free carboxyl
group for detection
ArabinoseSigma-AldrichA3256
BCA protein assay kitThermoFisher23227Pierce BCA protein asay kit.
Bromophenol blueBio-Rad161-0404
Carboxypeptidase BSigma-AldrichC9584-1mg
CentrifugeSorvallSS-34 fixed angle rotor and GA-6 fixed angle rotor
Dounce tissue grinderLabGenome7777-7Corning 7777-7 pyrex homogenizer with pour spout.
Ecoscint-HNational DiagnosticsLS275scintillation cocktail
EDTASigma-Aldrich
Filtration manifoldHoeferFH225V
French Pressure CellGlen MillsFA-080A120
GABASigma-AldrichA2129
GlutamateSigma-AldrichG6904
Glycerol
GraphPad Prism softwarehttp://www.graphpad.com/prism/Prism.htm
Hydrogen peroxideKROGER
Potassium ChlorideJ.T. Baker3040-01
Liquid scintillation counterBeckmanLS-6500
MaleateSigma-AldrichM0375
NanodropThermoFisher
Nitrocellulose membrane filtersMerck Milliporehawp025000.45 µM
PCR clean up kitGenscriptQuickClean II
Potassium Phosphate dibasicThermoFisherP290-500
putrescinefluka32810
Potassium Phosphate monobasicJ.T.Baker4008
SpermidineSigma-aldrichS2501
Strains :E. coli ΔpotE740(del)::kan, ΔcadB2231::Tn10This manuscriptAvailable upon request.Strain is deficient in the PotE and CadB polyamine exchangers.
Tris-baseResearch ProductsT60040-1000
UltracentrifugeSorvall MTX 15046960Thermo Fisher S150-AT fixed angle rotor
Ultracentrifuge tubesThermoFisher45237Centrifuge tubes for S150-AT rotor
Vector: pBAD-DEST49ThermoFisherGateway expression vector for E. coli

References

  1. Haferkamp, I., Linka, N. Functional expression and characterisation of membrane transport proteins. Plant Biology (Stuttgart). 14 (5), 675-690 (2012).
  2. Sauer, N., Caspari, T., Klebl, F., Tanner, W.

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