Abstract
BackgroundRh 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 NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.Methodology/Principal FindingsAn 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 C12E8 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 NH3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.Conclusions/SignificanceThis study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red blood cells endogenously expressing RhAG (2.18×10−3 µm3.s−1). The major finding of this study is that RhCG protein is active as an NH3 channel and that this function does not require any protein partner.
Highlights
While ammonium movement across the plasma membrane is a fundamental process which provides the principal source of nitrogen for microorganisms, it is known, in animals, to be involved in acido-basic regulation in the kidney [1] and can be associated with cytotoxic effects leading, for example to hepatic encephalopathy [2].The molecular mechanism by which the ammonium crosses the plasma membrane is not completely understood
The use of a double HA tag previously introduced in the second extracellular loop of RhCG [21], immuno affinity capture of the tagged protein and affinity elution with the epitope peptide allowed the obtention of RhCG protein of very high purity without exposing it to deleterious buffer conditions
RhCG could not be detected in the flow through (FT), demonstrating that amount of immobilized anti HA was adequate to capture all extracted protein present in the lysate
Summary
While ammonium movement across the plasma membrane is a fundamental process which provides the principal source of nitrogen for microorganisms, it is known, in animals, to be involved in acido-basic regulation in the kidney [1] and can be associated with cytotoxic effects leading, for example to hepatic encephalopathy [2].The molecular mechanism by which the ammonium crosses the plasma membrane is not completely understood. Functional studies which were carried out in different heterologous systems such as yeasts [12,13], Xenopus oocytes [14,15,16,17,18] and recombinant eukaryotic cells [19,20,21] or in red blood cells [22] provided insights into the mechanisms used by Rh glycoproteins for ammonium transport. 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 In these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties
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