Mutational Analysis of the Candida albicans Ammonium Permease Mep2p Reveals Residues Required for Ammonium Transport and Signaling

Abstract

The ammonium permease Mep2p mediates ammonium uptake and also induces filamentous growth in the human-pathogenic yeast Candida albicans in response to nitrogen limitation. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is not required for ammonium transport but is essential for Mep2p-dependent morphogenesis. Progressive C-terminal truncations showed Y433 to be the last amino acid that is essential for the induction of filamentous growth, thereby delimiting the Mep2p signaling domain. To understand in more detail how the signaling activity of Mep2p is regulated by ammonium availability and transport, we mutated conserved amino acid residues that have been implicated in ammonium binding or uptake. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is thought to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filament formation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filament formation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filament formation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. These results provide important insights into ammonium transport and control of morphogenesis by Mep2p in C. albicans.