Table 1.
Buffers.
Fig 1.
Putative membrane topology of studied transporters:
(A) MntH2 from the Nramp family, (B) AXZIP, PFZIP from the ZIP family, (C) CBZIP, GSZIP, DVZIP from the ZIP family and (D) EcNupC, VcCNT, from the CNT family investigated in the present study. The TMs of the human homologues are shown in black. The putative additional TM helixes are shown in white.
Fig 2.
Schematic representation of the constructs used in this study is depicted in the panel above.
The summary of the constructs details is shown in the table of the panel below.
Fig 3.
(A) Western blot analysis of expression levels yielded by 3 different His-tagged MntH2 constructs. The indicated constructs in E. coli strain BL21 Star™ (DE3) cells harbouring pRARE2 were autoinduced for 24 h as detailed in the text and then cell lysates analysed by SDS-PAGE/western blotting. Each lane contained 20 μg total protein. The amounts of His-tagged TEV protease standards blotted in parallel are indicated. The blot was stained for the presence of oligohistidine tags with HRP-labelled monoclonal anti-6 × polyhistidine antibody. The mobilities of marker proteins of known molecular mass are shown on the left. (B) Western blot analysis of samples from expression trials performed using pL21-AXZIP. Expression was performed in E. coli strains BL21Star either with IPTG induction (IPTG) or autinduction for amount of times indicated under the figure. The blot was stained with HRP-labelled monoclonal anti-6 × polyhistidine antibody. (C) Purification of MntH2 and AXZIP. Purified proteins were loaded on a gel and either stained with Coomassie Blue (Co) or transferred on a nitrocellulose membrane and stained with anti-His antibody (IB).
Fig 4.
(A) Analysis of the secondary structure of His-tagged MntH2, expressed from construct pL33-MntH2, by circular dichroism spectroscopy (top pannel). The analysis of the spectrum using the SELCON3 algorithm indicated that the composition were 81.0% ± 0.2% α-helix, 0.5% ± 0.3% β-sheet and 21.4% ± 0.7% turns plus random coil. The thermal stability of MntH2 (Bottom pannel). IMAC purified pL33-MntH2 (0.125 mg/mL) was dissolved in 50 mM MES buffer, pH 6.0 containing 5% glycerol and 0.05% DDM. Thermal stability was measured by ramping the temperature from 10°C to 90°C, at a rate of 1°C per second. CD measurements were performed at 222 nm following each 1°C increase in temperature, and are plotted following correction for the buffer blank. The solid line shows a non-linear fit of these data to the Boltzmann equation. (B) Purification steps of MntH2. Pure MntH2 protein before (P) or after cleavage with HRV3C (C) was loaded on a gel and either stained with Coomassie Blue (Co) or transferred on a membrane and immunoblotted with anti-His antibody (IB). (C) Separation of HRV-3C protease-cleaved MntH2 from the protease by size-exclusion chromatography. Left panel: A280nm profiles of the HRV-3C protease-cleaved MntH2 on a Superdex 200 10/300 column (“MntH2 cleaved with HRV”, solid blue line) and of HRV-3C protease alone (“HRV”, dotted green line). Right panel: Samples containing 20 μg of MntH2 before and after cleavage with HRV-3C protease, and of the indicated peak fractions from the elution profile (40 μL) were subjected to SDS-PAGE and then either stained with Coomassie blue (top) or transferred to nitrocellulose membrane and stained for the presence of oligohistidine tags with HRP-labelled monoclonal anti-6× polyhistidine antibody (bottom). The mobilities of marker proteins of known molecular mass (kDa) are shown on the left.
Fig 5.
Comparison of zinc uptake by protein-free liposomes and proteoliposomes containing either MntH2 or AXZIP.
(A) Liposomes and MntH2-containing proteoliposomes containing 200 μM FluoZin™-1 were mixed with zinc-containing assay buffer to yield a final [Zn2+] of 2 mM, and the resultant fluorescence changes recorded using a stopped flow fluorimeter. The normalized fluorescence change (ΔF/ΔFmax) was determined by dividing the observed fluorescence change (ΔF) by that induced by adding 1% β-OG to the extravesicular medium (ΔFmax). The results shown are the means of three measurements. (B) Fluorescent assays of zinc uptake were performed as described in the legend above, except that the extravesicular concentration of Zn2+ was varied from 100 μM to 1 mM. (C) same as in (B) but with AXZIP proteoliposomes and a Zn2+ concentration was from 50 μM to 2 mM (D) Initial rates of zinc uptake versus the extravesicular zinc concentration was subjected to linear regression. Initial rates (s-1) were estimated by fitting the first five data points (0–0.1 s) for each Zn2+ concentration in (C). All the results shown are the means of three measurements ± standard deviation.
Fig 6.
(A) Expression tests of pL53-CBZIP in various strains: C41, C43, BL21 Star (S) or BL21 Gold (G). Total extracted were loaded on a gel and stained with Coomassie Blue. (B) pL53-CBZIP was expressed in BL21 Star (S) and purified with an affinity column. Pure CBZIP was loaded on a gel and stained with Coomassie Blue (Co) or transferred onto a membrane and immunoblotted with Anti-His antibody (IB). (C) Expression test of p40-NupC. Cells expressing NupC were induced with IPTG. After 3h induction, cells were lysed by water-lysis technique as described in material and methods, loaded on a gel and stained with Coomassie Blue (I). Uninduced cells were loaded as a control (C). (D) Purified NupC protein was loaded on gel before (P) and after cleavage by TEV (C), and submitted to a negative purification to remove His-TEV and collect NupC in the flow through (F). Comparison between Coomassie Blue (Co) stained and immunoblot (IB) with antiHis Antibody is shown. The size of the purified protein (P) or the TEV protein (T) is shown on the right. (E) Effect of promoter on expression of NupC and VcCNT. Whole cellular extracts expressing NupC or VcCNT on either a pL53 (ptac promoter) or pL55 vector (T7 promoter). (F) Purification of NupC and VcCNT as described in (D).
Fig 7.
(A) Concentration dependence of Zn2+ uptake by proteoliposomes containing CBZIP. Fluorescent assays of zinc uptake were performed as described in Fig 5D with extravesicular Zn2+ concentration from 50 μM to 2 mM. (B) Membrane potentials drive bidirectional zinc fluxes. The effect on the fluorescence of encapsulated FluoZin™-1 caused by dilution of CBZIP proteoliposomes containing 100 mM K+ and 50 μM Zn2+ into medium containing 50 μM Zn2+ with varying K+ concentrations as indicated. (C) Analysis of the secondary structure of NupC expressed from construct pL55 by circular dichroism spectroscopy. The analysis of the spectrum using the SELCON3 algorithm indicated that the composition were 81.0% ± 0.2% α-helix, 0.5% ± 0.3% β-sheet and 21.4% ± 0.7% turns plus random coil. Thermal stability was measured by ramping the temperature from 10°C to 90°C, at a rate of 1°C per second. CD measurements were performed at 222 nm following each 1°C increase in temperature, and are plotted following correction for the buffer blank. The solid line shows a non-linear fit of these data to the Boltzmann equation. (D) Transport assay by NupC. Radioactively labelled Uridine import was measured on intact cells expressing NupC from either pL55 or pL40. Transport was stopped after 15s or 120s by filtration has described in material and methods. As a control, mock cells were also tested (control).