Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA

Abstract

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Introduction

Ammonium is a preferred nitrogen source for many microorganisms and plants, and the transport of ammonium into the cell has been studied in various fungal, bacterial, and plant species (Howitt and Udvardi, 2000; Kleiner, 1981; von Wiren et al., 2000a). The ammonium analogue methylammonium is taken into the cell via the ammonium system (Hackette et al., 1970) and at high concentrations methylammonium is toxic to Aspergillus nidulans and Saccharomyces cerevisiae. Selecting for colonies resistant to the methylammonium growth inhibition, mutants defective in ammonium transport activity were isolated from S. cerevisiae (mep mutants; Dubois and Grenson, 1979) and A. nidulans (meaA mutants; Arst and Cove, 1969). Two ammonium permease genes, MEP1 and MEP2, were isolated by functional complementation of the S. cerevisiae Mep mutant strain 26972c (mep1-1 mep2-1) with a third, MEP3, identified by genome sequence (Marini et al., 1994, Marini et al., 1997a). Heterologous complementation of the S. cerevisiae 26972c strain was used to isolate the first ammonium permease gene, At Amt1:1, from Arabidopsis thaliana (Ninnemann et al., 1994), which led to five additional Amt genes being identified from this organism (Howitt and Udvardi, 2000; Sohlenkamp et al., 2000; von Wiren et al., 2000a).

Of the S. cerevisiae ammonium permeases, Mep2p displays the highest affinity for ammonium ($\text{K}{}_{\mathrm{m}}\text{,1–2}\text{μ}$M) followed by Mep1p ($\text{K}{}_{\mathrm{m}}\text{,5–10}\text{μ}$M) and Mep3p $\text{(K}{}_{\mathrm{m}}\text{,1.4–2.1}\text{mM}\text{)}$ (Marini et al., 1994, Marini et al., 1997a). Deletion of all three MEP genes rendered the cell unable to grow on media containing less than 5 mM ammonium as sole nitrogen source, whereas no phenotypic effects were noted in single deletion strains (Marini et al., 1997a). The Mep2p transporter has also been proposed to act as an ammonium sensor generating a signal to regulate pseudohyphal growth in response to ammonium starvation (Lorenz and Heitman, 1998). Unlike Mep1p and Mep3p, the Mep2 protein is N-glycosylated in the N-terminal tail (Marini and Andre, 2000), although glycosylation of Mep2p is not required for ammonium transport activity nor is it essential for signaling pseudohyphal growth (Marini and Andre, 2000). Glycosylation of the Mep2p amino terminus implied that this region of the protein is extracellular which was confirmed by protease treatment of protoplasts that also indicated an intracellular carboxy-terminal tail for this putative 11-transmembrane domain protein (Marini and Andre, 2000). This extracellular N terminus and intracellular C terminus (N_out,C_in) topology, which is also predicted for Mep1p, Mep3p, and other ammonium transporters, is unusual for polytopic integral membrane proteins (Marini and Andre, 2000). Thomas et al. (2000) proposed that the ammonium transporter/methylammonium permease (AMT/MEP) proteins contain a functional core of 11 membrane-spanning helices with conserved topology. Some bacterial ammonium permeases such as the Escherichia coli AmtB transporter have an additional 12th transmembrane domain at the N terminus creating an N_in,C_in topology (Thomas et al., 2000).

The Rhesus (Rh) blood group polypeptides are transmembrane proteins which display significant relatedness to members of the AMT/MEP family (Marini et al., 1997b; Liu et al., 2001). By complementation of the S. cerevisiae MEP1,2,3 triple-deletion mutant ammonium growth defect, human RhAG and RhGK proteins were shown to function as ammonium transporters (Marini et al., 2000a). S. cerevisiae transformants expressing RhAG and RhGK also exhibited increased ammonium efflux and methylammonium resistance, indicating a possible role of these proteins in ammonium export (Marini et al., 2000a). As Rh proteins function as hetero-oligomeric complexes it is possible that AMT/MEP permeases have a similar organization. Interestingly, the mep1-1 mutation, which results in a Gly413→Asp413 substitution in Mep1p, in combination with a mep2 deletion mutation displayed no growth on low ammonium concentrations, whereas the MEP1 MEP2 double-deletion mutant did exhibit growth under these conditions. The altered Mep1-1 protein was shown to negatively interfere with Mep3p function, suggesting that the Mep1p and Mep3p transporters may interact with each other (Marini et al., 2000b). Any hetero-interaction is not required for transport function as a single functional permease can still support growth on ammonium (Marini et al., 1997a).

The A. nidulans MeaA and MepA ammonium permeases are recent additions to the AMT/MEP family (Monahan et al., 2002). The A. nidulans meaA gene was isolated by complementation of the methylammonium-resistant phenotype of the meaA8 mutant and a second ammonium permease gene, mepA, was identified by degenerate PCR (Monahan et al., 2002). MeaA and MepA exhibit 50–55% amino acid identity to each other and to the S. cerevisiae Mep proteins, and both MeaA and MepA are predicted to contain 11 transmembrane helices with an N_out,C_in topology. Whereas a meaA deletion mutant showed reduced growth on ammonium, a mepA deletion mutant exhibited normal growth under these conditions. However, the mepA meaA double-deletion mutant was unable to grow on low ammonium concentrations (Monahan et al., 2002). The MepA permease displayed a higher affinity for methylammonium than MeaA ($\text{K}{}_{\mathrm{m}}\text{,}\text{44.3}$, and 3.04 mM, respectively) and was expressed only under nitrogen-limiting conditions, consistent with the MepA protein having a scavenging role in ammonium uptake. In contrast, the meaA gene was also expressed under nitrogen-sufficient conditions and MeaA appears to serve as the main ammonium transporter (Monahan et al., 2002).

In this report we present the nucleotide sequence changes in 37 meaA mutations that disrupt transport function. Analysis of the predicted protein sequence changes in these mutants indicates regions important for uptake activity. We have also introduced a specific Gly→Asp mutation at Gly447 of MeaA. The equivalent change in the S. cerevisiae Mep1-1 protein trans-inhibited Mep3p activity and our results indicated that MeaA may interact with itself and with MepA. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.