45NISR Research GRANT
2007 Research Grant

Role of the fission yeast ammonium transporters in nitrogensensing
Hiroshi MITSUZAWA
Department of Bioresource Science, Nihon University Junior College

Research aims
Ammonium is an important source of nitrogen for many
microorganisms and plants and is taken up by them fromthe environment to synthesize amino acids. Transport ofammonium across the cell membrane is mediated by ammonium transporters, membrane proteins conserved in bacteria, archaea, and eukarya (1). Interestingly, in the budding
yeast Saccharomyces cerevisiae and in the plant and humanpathogenic fungi Ustilago maydis and Candida albicans,ammonium transporters have been shown to be involved ina switch from yeast-form growth to filamentous growth induced by nitrogen limitation (2–4). Recently, I have functionally characterized three ammonium transporters in thefission yeast Schizosaccharomyces pombe and have foundthat one of them, Amt1, is required for ammonium-limitation induced morphological differentiation (filamentous
growth underneath the surface of agar medium; Fig. 1A) (5,6). There are two models for the role of Amt1 in the morphological differentiation in response to ammonium limitation. Amt1 might function as an ammonium sensor to generate a signal to induce filamentous invasive growth (Fig.
1B left). Alternatively, ammonium taken up by ammoniumtransporter or its metabolite might be a signal (Fig. 1B

Fig. 1. Role for Amt1 in morphological differentiation of S. pombe. (A)Morphology of cells growing filamentously underneath the surface of theagar. (B) Two models for induction of invasive growth.

right). The purpose of this study is to elucidate physiological roles of ammonium transporters in S. pombe, focusing
on their roles in nitrogen sensing.
Methods
S. pombe strains used in this study were described previously (5). Invasive growth of S. pombe was examined on
LNB medium by washing cells off the agar surface withrunning water (5). EMM-N medium and EMM-N medium
supplemented with a nitrogen source other than ammonium
were also used. Sporulation was assessed under microscope. S. cerevisiae mep2D/mep2D strain was constructed
through crosses between haploid strains whose MEP2 gene
was disrupted with the URA3 marker by a PCR-based
method. Pseudohyphal growth of S. cerevisiae was examined on SLAD medium (2) containing various concentrations of ammonium.
Results
Role of ammonium transporter in morphological differentiation
On agar medium containing 1 mM ammonium, wild-type
S. pombe cells exhibited invasive growth, whereas amt1Dcells did not show any growth underneath the surface of theagar (Fig. 2). The model shown in Fig. 1B right suggeststhat the invasive growth defect of the amt1D cells could berecovered at higher concentrations of ammonium. As expected, the amt1D cells underwent invasive growth on
5 mM ammonium (Fig. 2). Next, diploid S. cerevisiae strain
was constructed carrying disruption of the MEP2 gene,Fig. 2. Effect of ammonium concentration on the invasive growth defectof amt1D cells.

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which encodes an ammonium transporter that is requiredfor pseudohyphal growth and is proposed to function as anammonium sensor. Unlike the invasive growth defect of theS. pombe amt1D strain, the pseudohyphal growth defect of
this strain was not relieved by raising ammonium concentration in the medium (data not shown).
Role of ammonium transporter in the retention of ammonium in the cell
S. pombe amt1D amt2D amt3D cells show a defect ingrowth on low ammonium (5; Fig. 3). In the present study,
the amt1D amt2D amt3D cells were found to have a growthdefect on a nitrogen source other than ammonium as well.
For example, the triple mutant grew more slowly than wild
type on medium containing tryptophan as the nitrogensource (Fig. 3). Furthermore, the amt1D amt2D amt3Dcells displayed slower growth compared with wild type on
nitrogen-free (EMM-N) medium (Fig. 3). These results canbe explained by assuming that ammonium produced byamino acid metabolism is exported or leaks out from thecell and then is taken up into the cell by ammonium transporters. The ability of the amt1D amt2D amt3D cells to undergo autophagy was assessed by testing them for sporula-tion. The triple mutant exhibited sporulation, although to alesser extent than wild type, suggesting that the triple mutant can undergo autophagy. This observation supports themodel in which the impaired growth of amt1D amt2D
amt3D cells on EMM-N results not from a defect in autophagy but from a defect in the retention of intracellularammonium.
Conclusion
Raising ammonium concentration in the medium relieved the invasive growth defect of S. pombe amt1D cells,
but not the pseudohyphal growth defect of S. cerevisiae
mep2D /mep2D cells, suggesting that the roles of ammonium transporter in nitrogen sensing are different between
the yeasts. S. pombe ammonium transporters seem to function in the retention of intracellular ammonium producedby cellular metabolism, suggesting their role in growth on anitrogen source other than ammonium.
References1)
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Fig. 3. Growth defects of amt1D amt2D amt3D cells on medium containing a nitrogen source other than ammonium and on nitrogen-freemedium.

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