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Published on: May 4, 2018
Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with
Brendon J Monahan1, Shiela E Unkles, Tchuc Tsing I
1Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia.
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.
Insights
Understanding ammonium transport proteins is crucial. This study characterizes mutations in Aspergillus nidulans meaA, revealing key regions for ammonium uptake and protein interactions, essential for cellular ammonium retention.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- Ammonium transport across biological membranes is facilitated by the AMT/MEP protein family.
- Human and mouse Rhesus proteins show relatedness to AMT/MEP sequences, suggesting potential ammonium transport function.
Purpose of the Study:
- To functionally characterize ammonium transport proteins by analyzing loss-of-function mutations in Aspergillus nidulans meaA.
- To identify conserved residues and regions critical for ammonium permease activity and protein interactions.
Main Methods:
- Characterization of 37 loss-of-function mutations in the meaA gene.
- Mutational analysis to predict important regions for uptake activity.
- Introduction of a specific Gly447 to Asp mutation in MeaA and assessment of its effect on protein activity.
- Cross-feeding studies to evaluate the role of MeaA and MepA in intracellular ammonium retention.
Main Results:
- Identified conserved residues and regions within AMT/MEP proteins important for ammonium uptake.
- A major facilitator superfamily-like motif (161-GAVAERGR-168) and Pro186 were predicted as crucial for ammonium translocation.
- A G447D mutation in MeaA led to trans-inhibition of both MeaA and MepA, suggesting self-interaction and interaction with MepA.
- MeaA and MepA are essential for intracellular ammonium retention.
Conclusions:
- MeaA and MepA likely interact with themselves and each other, though hetero-interaction is not essential for transport.
- These ammonium transporters play a vital role in maintaining intracellular ammonium homeostasis.
