Different Transport Mechanisms in Plant and Human AMT/Rh-type Ammonium Transporters

Abstract

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4 + and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4 + and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4 +, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport.

Molecular mechanisms of ammonium transport and accumulation in plants

“…In addition, the growth of yeast is sensitive to high concentrations of MeA; this effect is more pronounced in wild type yeast than in the triple-mepD mutant. While AMTs increased the sensitivity to MeA, the Rh glycoproteins restored yeast growth on high MeA [20,26].…”

“…The comparison in oocytes and yeast showed that LeAMT1;2 facilitated electrogenic transport, which is coupled to the electrochemical gradient of NH þ 4 , but HsRhCG transported along the chemical gradient of NH 3 without generating ionic currents [26]. When expressed in yeast, AMTs and Rh glycoproteins were both competent to restore growth on low ammonium [20,26]. However, AMTs are generally more efficient and are less dependent on the pH [4,20,26,27].…”

“…It has hence been discussed that these currents by ammonium might reflect NH þ 4 transport through the TIP pores themselves, in addition to NH 3 transport [49]. This topic may need further analysis in the future, keeping in mind that Rh glycoproteins, which appear to facilitate NH 3 , elicited similar, strongly pHdependent electrical currents in oocytes of some laboratories (for discussion see [26]). It should be noted that there were striking similarities of the function of Rh glycoproteins and TIP2 aquaporins in yeast: heterologous expression of both proteins weakly supported growth on low ammonium in the triple-mepD strain [27,48], but alleviated the toxicity of high MeA [27].…”

Rhesus glycoprotein gene expression in the mangrove killifishKryptolebias marmoratusexposed to elevated environmental ammonia levels and air

“…RhAG expression is restricted to the erythrocyte membrane, whereas RhBG and RhCG are expressed in various tissues in the mammalian systems, including liver, kidney and gastrointestinal tract (Handlogten et al, 2005;Liu et al, 2000;Liu et al, 2001;. Studies in mammalian and plant systems reveal that at least some of those Rh genes encode proteins that mediate NH 3 /NH 4 + movement (Khademi et al, 2004;Zheng et al, 2004;Mayer et al, 2006), but the form of ammonia (NH 3 gas or NH 4 + ion) being transported, and whether the transport is active or passive, are still under much debate (Bakouh et al, 2004;Khademi et al, 2004;Nakhoul et al, 2005). A fulllength cDNA of Rh-like protein (Rh-CM) has been identified recently from the gills of the aquatic crab Carcinus maenas, which has a similar predicted transmembrane structure as the mammalian Rh proteins (Weihrauch et al, 2004).…”

Expression of the non-erythroid Rh glycoproteins in mammalian tissues

“…Rhbg and Rhcg are non-erythroid Rh glycoproteins [8,9]. Functional studies has confirmed that both Rhbg and Rhcg transport ammonia, although different studies have identified differing affinities for the two molecular forms of ammonia, NH 3 and NH 4 + , and whether transport is electroneutral or electrogenic [10][11][12][13][14][15][16]. In this review, we will concentrate on the expression of Rhbg and Rhcg in the kidney, liver and gastrointestinal tract.…”