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Functional reconstitution into liposomes of purified human RhCG ammonia channel.

Mouro-Chanteloup I, Cochet S, Chami M, Genetet S, Zidi-Yahiaoui N, Engel A, Colin Y, Bertrand O, Ripoche P - PLoS ONE (2010)

Bottom Line: When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively).This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)).

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR_S 665, Paris, France. isabelle.mouro-chanteloup@inserm.fr

ABSTRACT

Background: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.

Methodology/principal findings: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.

Conclusions/significance: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.

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TEM of negatively stained RhCG particles.The electron micrograph reflects the homogeneity of the purified protein. Scale bar is 50 nm. Arrows indicate some top-views of RhCG. In the insert, class averages corresponding to top-views of RhCG are displayed. Scale bar is 10 nm.
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pone-0008921-g002: TEM of negatively stained RhCG particles.The electron micrograph reflects the homogeneity of the purified protein. Scale bar is 50 nm. Arrows indicate some top-views of RhCG. In the insert, class averages corresponding to top-views of RhCG are displayed. Scale bar is 10 nm.

Mentions: Transmission electron microscopy (TEM) was used to ascertain whether the purified transporter is in a monomeric or oligomeric form. Negatively stained purified HA-tagged RhCG revealed particles with a size of 9 nm homogeneously distributed on the carbon film (Figure 2). Reference-free alignment and classification of 858 such particles yielded class averages corresponding to different orientations of the molecules on the carbon film. The average of the top view class (See insert in Figure 2) revealed 3 domains. By western blot analysis, an oligomeric form of RhCG was observed despite the presence of SDS (0.1% in the gel and 1% in the sample) but, because of these denaturing conditions, this oligomeric state might be a partial one. The TEM results, suggesting a trimeric form of RhCG, are consistent with homology modelling of the human Rh proteins [23] deduced from structural data on AmtB [24], [25] and with the crystal structures of NeRh50, a bacterial homologue [26], [27] and of human RhCG, recently determined by Gruswitz et al., (PDB 3HD6). This form of the purified RhCG-HA without any detectable residual amount of C12E8 detergent as determined by measurement of the surface tension (data not shown) could be efficiently incorporated into liposomes in order to test its activity.


Functional reconstitution into liposomes of purified human RhCG ammonia channel.

Mouro-Chanteloup I, Cochet S, Chami M, Genetet S, Zidi-Yahiaoui N, Engel A, Colin Y, Bertrand O, Ripoche P - PLoS ONE (2010)

TEM of negatively stained RhCG particles.The electron micrograph reflects the homogeneity of the purified protein. Scale bar is 50 nm. Arrows indicate some top-views of RhCG. In the insert, class averages corresponding to top-views of RhCG are displayed. Scale bar is 10 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2812482&req=5

pone-0008921-g002: TEM of negatively stained RhCG particles.The electron micrograph reflects the homogeneity of the purified protein. Scale bar is 50 nm. Arrows indicate some top-views of RhCG. In the insert, class averages corresponding to top-views of RhCG are displayed. Scale bar is 10 nm.
Mentions: Transmission electron microscopy (TEM) was used to ascertain whether the purified transporter is in a monomeric or oligomeric form. Negatively stained purified HA-tagged RhCG revealed particles with a size of 9 nm homogeneously distributed on the carbon film (Figure 2). Reference-free alignment and classification of 858 such particles yielded class averages corresponding to different orientations of the molecules on the carbon film. The average of the top view class (See insert in Figure 2) revealed 3 domains. By western blot analysis, an oligomeric form of RhCG was observed despite the presence of SDS (0.1% in the gel and 1% in the sample) but, because of these denaturing conditions, this oligomeric state might be a partial one. The TEM results, suggesting a trimeric form of RhCG, are consistent with homology modelling of the human Rh proteins [23] deduced from structural data on AmtB [24], [25] and with the crystal structures of NeRh50, a bacterial homologue [26], [27] and of human RhCG, recently determined by Gruswitz et al., (PDB 3HD6). This form of the purified RhCG-HA without any detectable residual amount of C12E8 detergent as determined by measurement of the surface tension (data not shown) could be efficiently incorporated into liposomes in order to test its activity.

Bottom Line: When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively).This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)).

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR_S 665, Paris, France. isabelle.mouro-chanteloup@inserm.fr

ABSTRACT

Background: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.

Methodology/principal findings: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.

Conclusions/significance: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.

Show MeSH
Related in: MedlinePlus