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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.
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.
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....
1. Generation of the E. coli Double Knock Out Mutant with P1 Transduction
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.......
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 .......
Support for this project came from the BGSU Graduate College, and the BGSU Office of Sponsored Programs and Research.
....Name | Company | Catalog Number | Comments |
2-mercaptoethanol | Sigma-Aldrich | M6250 | |
3H-putrescine | PerkinElmer | NET185001MC | |
3H-spermidine | PerkinElmer | NET522001MC | |
4-chloro-1-naphthol | Sigma-Aldrich | C8890 | |
14C arginine | Moravek Inc. | MC137 | |
Arginine | Sigma-Aldrich | A-5006 | |
Anti-His (C-term)-HRP antibody | ThermoFisher | R931-25 | Detects the C-terminal polyhistidine (6xHis) tag, requires the free carboxyl group for detection |
Arabinose | Sigma-Aldrich | A3256 | |
BCA protein assay kit | ThermoFisher | 23227 | Pierce BCA protein asay kit. |
Bromophenol blue | Bio-Rad | 161-0404 | |
Carboxypeptidase B | Sigma-Aldrich | C9584-1mg | |
Centrifuge | Sorvall | SS-34 fixed angle rotor and GA-6 fixed angle rotor | |
Dounce tissue grinder | LabGenome | 7777-7 | Corning 7777-7 pyrex homogenizer with pour spout. |
Ecoscint-H | National Diagnostics | LS275 | scintillation cocktail |
EDTA | Sigma-Aldrich | ||
Filtration manifold | Hoefer | FH225V | |
French Pressure Cell | Glen Mills | FA-080A120 | |
GABA | Sigma-Aldrich | A2129 | |
Glutamate | Sigma-Aldrich | G6904 | |
Glycerol | |||
GraphPad Prism software | http://www.graphpad.com/prism/Prism.htm | ||
Hydrogen peroxide | KROGER | ||
Potassium Chloride | J.T. Baker | 3040-01 | |
Liquid scintillation counter | Beckman | LS-6500 | |
Maleate | Sigma-Aldrich | M0375 | |
Nanodrop | ThermoFisher | ||
Nitrocellulose membrane filters | Merck Millipore | hawp02500 | 0.45 µM |
PCR clean up kit | Genscript | QuickClean II | |
Potassium Phosphate dibasic | ThermoFisher | P290-500 | |
putrescine | fluka | 32810 | |
Potassium Phosphate monobasic | J.T.Baker | 4008 | |
Spermidine | Sigma-aldrich | S2501 | |
Strains :E. coli ΔpotE740(del)::kan, ΔcadB2231::Tn10 | This manuscript | Available upon request. | Strain is deficient in the PotE and CadB polyamine exchangers. |
Tris-base | Research Products | T60040-1000 | |
Ultracentrifuge | Sorvall MTX 150 | 46960 | Thermo Fisher S150-AT fixed angle rotor |
Ultracentrifuge tubes | ThermoFisher | 45237 | Centrifuge tubes for S150-AT rotor |
Vector: pBAD-DEST49 | ThermoFisher | Gateway expression vector for E. coli |
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