(1), it has only recently been established that there are at least four Rh proteins in mammals, Rh30 and RhAG in red cells and RhBG and RhCG in other tissues (2-7). Rh homologues have also been found in simpler organisms, but relatively few have been identified and hence the origin and evolutionary history of Rh proteins remains elusive.Rh proteins have 12 transmembrane (TM)-passing segments indicative of a transport function (2-7) with limited homology to microbial ammonium transporter (Amt) proteins first noticed by Marini et al. (8). Many research groups think that Amt proteins concentrate the NH 4 ϩ ion against a gradient, i.e., that they are NH 4 ϩ active transporters (9). Likewise, several groups think that human and mouse Rh proteins also transport ammonium and are Amt functional equivalents in mammals (10-16). Both findings have been challenged. think that Amt proteins are gas channels for NH 3 , a view that has been substantiated by the high-resolution protein structures of Escherichia coli AmtB (EcAmtB) (21, 22). Moreover, Soupene et al. find that the substrate for the Rh1 protein of the green alga Chlamydomonas reinhardtii, is apparently CO 2 (23-25). They focused on this organism because it was one of the few microbes previously known to have an Rh protein (7, 23).To probe the evolutionary history of Rh and Amt genes in depth we assembled the sequences of 111 Rh and 260 Amt and analyzed them phylogenetically and bioinformatically. Using this large data set, we explored particularly (i) the organismal distribution of Rh genes as to how often and widespread they coexisted with Amt in the same species (paralogous occurrence); (ii) whether there were distinct differences between Rh and Amt proteins, supporting physiological and genetic evidence that they have different substrate specificities (24, 25); and (iii) proliferation of Rh genes over evolutionary time and the degree of their conservation. Our data are consistent with functional conservation within the Rh family and functional diversification of Rh proteins from the distantly related Amt proteins. Materials and MethodsData Sets. Accession numbers and identifiers of Rh and Amt can be found in the supporting information, which is published on the PNAS web site. The Rh data set contains 111 nonredundant genes mostly of full-length cDNAs (see Table 1, which is published as supporting information on the PNAS web site). Mammalian Rh30 and RhAG were from GenBank via BLAST search (26); other Rh genes were mainly cloned in our laboratory. The Amt data set contains 260 nonredundant genes mostly retrieved from annotated GenBank entries (see the Amt data set, which is published as supporting information on the PNAS web site).Sequence Alignment. Rh and͞or Amt protein sequence alignments were obtained by using MUSCLE (Version 3.52; ref. 27) and were used to derive codon-based nucleotide sequence alignments. Homogeneities of amino acid or codon composition were measured by disparity index (28) as described in MEGA 3.0 (29). A biased codon usage in the first a...

Ammonium transporters play a key functional role in nitrogen uptake and assimilation in microorganisms and plants; however, little is known about their structural counterpart in mammals. Here, we report the molecular cloning and biochemical characterization of Rh type B glycoproteins, human RhBG and mouse Rhbg, two new members of the Rh family with distinct tissue specificities. The RhBG orthologues possess a conserved 12-transmembrane topology and most resemble bacterial and archaeal ammonium transporters. Human RHBG resides at chromosome 1q21.3, which harbors candidate genes for medullary cystic kidney disease, whereas mouse Rhbg is syntenic on chromosome 3. Northern blot and in situ hybridization revealed that RHBG and Rhbg are predominantly expressed in liver, kidney, and skin, the specialized organs involving ammonia genesis, excretion, or secretion. Confocal microscopy showed that RhBG is located in the plasma membrane and in some intracellular granules. Western blots of membrane proteins from stable HEK293 cells and from mouse kidney and liver confirmed this distribution. N-Glycanase digestion showed that RhBG/Rhbg has a carbohydrate moiety probably attached at the NHS motif on exoloop 1. Phylogenetic clustering, tissuespecific expression, and plasma membrane location suggest that RhBG homologous proteins are the long sought major ammonium transporters in mammalians.

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