Abstract
The Rh blood group proteins are erythrocyte proteins important in neonatal and transfusion medicine. Recent studies have shed new light on the possible biological function of Rh proteins as members of a conserved family of proteins involved in ammonium transport. The erythrocyte Rh-associated glycoprotein (RhAG) mediates uptake of ammonium when expressed in Xenopus laevis oocytes, and functional studies indicate that RhAG might function as an NH(4)(+)-H(+)-exchanger. To further delineate the functional properties of RhAG, in this study we have expressed RhAG in both a Saccharomyces cerevisiae ammonium-transport mutant (mep1Delta mep2Delta mep3Delta) and a wild-type strain. RhAG was able to complement the transport mutant, with complementation strictly pH-dependent, requiring pH 6.2-6.5. RhAG also conferred resistance to methylamine (MA), a toxic analog of ammonium, and expression in wild-type cells revealed that resistance was correlated with efflux of MA. RhAG-mediated resistance was pH-dependent, being optimal at acid pH. The opposite pH dependence of ammonium complementation (uptake) and MA resistance (efflux) is consistent with bidirectional movement of substrate counter to the direction of the proton gradient. This report clarifies and expands previous observations of RhAG-mediated transport in yeast and supports the hypothesis that ammonium transport is coupled to the H(+) gradient and that RhAG functions as a NH(4)(+)/H(+) exchanger.
Highlights
The erythrocyte Rh proteins are well known in clinical medicine as the cause of hemolytic disease of the newborn and blood transfusion incompatibilities (1), but knowledge of their biochemical structure and possible function has only recently become available
The erythrocyte Rh-associated glycoprotein (RhAG) mediates uptake of ammonium when expressed in Xenopus laevis oocytes, and functional studies indicate that RhAG might function as an NH4؉-H؉-exchanger
We reported recently that erythroid RhAG expressed in Xenopus laevis oocytes mediates uptake of ammonium, providing direct evidence for a transport function (10)
Summary
RhAG, Rh-associated glycoprotein; MA, methylamine; WT, wild type; MES, 2-(N-morpholino)ethanesulfonic acid. Protein sequences with similarities to Rh and RhAG were first found in Caenorhabditis elegans; these homologs, in turn, showed a distant similarity with ammonium transporters from bacteria (SNYN, AMT), yeast (MEP), and plants (AMT). In these organisms, transport of ammonia (NH3)/ammonium (NH4ϩ) is critical for acquisition of nitrogen. Characterization in oocytes revealed that uptake was independent of the membrane potential and the Naϩ gradient but that raising extracellular pH or lowering intracellular pH dramatically stimulated uptake These results suggested that uptake was coupled to an outwardly directed Hϩ gradient and led us to hypothesize that RhAG might function by an Hϩ-coupled, counter-transport mechanism.
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