Dabas Neelam, Schneider Sabrina, Morschhäuser Joachim
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
Eukaryot Cell. 2009 Feb;8(2):147-60. doi: 10.1128/EC.00229-08. Epub 2008 Dec 5.
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.
铵通透酶Mep2p介导铵的摄取，并在氮限制条件下诱导人类致病酵母白色念珠菌的丝状生长。Mep2p的C端细胞质尾巴包含一个信号结构域，该结构域对于铵转运不是必需的，但对于Mep2p依赖的形态发生至关重要。C端渐进性截短显示Y433是诱导丝状生长所必需的最后一个氨基酸，从而界定了Mep2p信号结构域。为了更详细地了解Mep2p的信号活性如何受铵可用性和转运的调节，我们对与铵结合或摄取有关的保守氨基酸残基进行了突变。已提出介导与细胞外铵初始接触的D180或孔内衬残基H188和H342的突变消除了Mep2p的表达，表明这些残基对于蛋白质稳定性很重要。F239与F126一起被认为形成了通向电导通道的胞外门，其突变消除了铵摄取和Mep2p依赖的丝状形成，尽管该蛋白定位正常。另一方面，假定与Y122、F126和S243一起参与细胞膜胞外侧铵离子募集和配位的W167的突变也消除了丝状形成，而对铵转运没有强烈影响，表明Mep2p的胞外改变可影响细胞内信号传导。Y122的突变比W167的突变更强烈地降低了铵摄取，但仍允许有效的丝状形成，表明Mep2p的信号活性与其转运活性不直接相关。这些结果为白色念珠菌中Mep2p的铵转运和形态发生控制提供了重要见解。
