Membrane potential independent transport of NH 3 in the absence of ammonium permeases in Saccharomyces cerevisiae

BMC Syst Biol. 2017 Apr 17;11(1):49. doi: 10.1186/s12918-016-0381-1.

Abstract

Background: Microbial production of nitrogen containing compounds requires a high uptake flux and assimilation of the N-source (commonly ammonium), which is generally coupled with ATP consumption and negatively influences the product yield. In the industrial workhorse Saccharomyces cerevisiae, ammonium (NH4+) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH4+ ion, resulting in ATP expenditure to maintain the intracellular charge balance and pH by proton export using the plasma membrane-bound H+-ATPase.

Results: To decrease the ATP costs for nitrogen assimilation, the Mep genes were removed, resulting in a strain unable to uptake the NH4+ ion. Subsequent analysis revealed that growth of this ∆mep strain was dependent on the extracellular NH3 concentrations. Metabolomic analysis revealed a significantly higher intracellular NHX concentration (3.3-fold) in the ∆mep strain than in the reference strain. Further proteomic analysis revealed significant up-regulation of vacuolar proteases and genes involved in various stress responses.

Conclusions: Our results suggest that the uncharged species, NH3, is able to diffuse into the cell. The measured intracellular/extracellular NHX ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NHx compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the ∆mep strain than in the reference strain, which suggests that the lower biomass yield of the ∆mep strain was related to higher turnover rates of biomass components.

Keywords: Ammonia passive diffusion; Ammonium transport; Central nitrogen metabolism; Intracellular ammonium; Metabolomics; Thermodynamics.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Aerobiosis
  • Ammonium Compounds / metabolism*
  • Biological Transport
  • Cation Transport Proteins / deficiency
  • Cation Transport Proteins / genetics*
  • Cation Transport Proteins / metabolism
  • Diffusion
  • Extracellular Space / metabolism
  • Gene Deletion*
  • Hydrogen-Ion Concentration
  • Intracellular Space / metabolism
  • Membrane Potentials*
  • Metabolomics
  • Nitrogen / metabolism
  • Permeability
  • Proteomics
  • Saccharomyces cerevisiae / cytology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Ammonium Compounds
  • Cation Transport Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphate
  • Nitrogen