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. 2004 Dec;3(6):1492-503.
doi: 10.1128/EC.3.6.1492-1503.2004.

Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids

Isabel Velasco  1 , Sandra Tenreiro, Isabel L Calderon, Bruno André
Affiliations

Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids

Isabel Velasco et al. Eukaryot Cell. 2004 Dec.

Abstract

Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H(+) antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

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Figures

FIG. 1.
FIG. 1.
Excretion of Hom and Thr by yeast cells grown on solid medium. Cell suspensions of wild-type (23344c) or gap1 agp1 gnp1 (JA248) strains transformed with the pMACR7 plasmid (HOM3-R) were dropped over a lawn of Thr (thr4; strain F4) or Hom (hom3; strain Σ-A3hu) auxotroph mutants, and the petri dishes were incubated for 4 days at 29°C. Proline or NH4+ was the sole source of nitrogen in the medium.
FIG. 2.
FIG. 2.
The Gap1, Agp1, and Gnp1 permeases are involved in Thr and Hom utilization. (A) Growth test on increasing concentrations of Thr as the sole nitrogen source. The strains were 23344c (wild type), 30633c (gap1 agp1), JA248 (gap1 agp1 gnp1), and FA010 (gap1 ssy1). (B) The gap1 agp1 gnp1 mutations reduce the ability of external Thr to complement the growth defect of a Thr auxotroph strain. Cells were grown on minimal media containing proline or NH4+ as nitrogen source and increasing concentrations of Thr. The strains were 23344c (wild type), IVU41 (thr1), JA248 (gap1 agp1 gnp1), and IVU61-1B (gap1 agp1 gnp1 thr1). (C) The gap1 agp1 gnp1 mutations reduce the ability of external Hom to complement the growth defect of a Hom auxotroph strain. Cells were grown on minimal media containing proline or NH4+ as nitrogen source or on YPD medium. Hom was eventually added to the media at the indicated final concentrations. The strains were JA248 (gap1 agp1 gnp1), Σ-A3hu (hom3), and IVU441 (gap1 agp1 gnp1 hom3).
FIG. 3.
FIG. 3.
Gap1, Agp1 and Gnp1 are the main Thr uptake systems. Time course accumulation of [14C]Thr added at time zero at a 0.1 mM initial concentration to cells growing on minimal medium with proline (A) or NH4+ (B) as the sole nitrogen source. The strains were gap1 (•, strain 30629c), gap1 agp1 (♦, strain 30633c), gap1 gnp1 (○, strain EK041), and gap1 agp1 gnp1 (⋄, strain JA248).
FIG. 4.
FIG. 4.
Hom inhibits Gap1, Agp1, and Gnp1 uptake activities. (A) Initial velocity of 0.1 mM [14C]Thr uptake in wild-type (○, •) and gap1 (×) strains. The cells were grown on minimal proline medium, and Hom (10 mM final concentration) was added (○) or not (•, ×) to the medium at time zero of the experiment. (B) Initial velocity of 0.1 mM [14C]Thr uptake in gap1 gnp1 (○, •) and gap1 agp1 gnp1 (×) strains. The cells were grown on minimal proline medium to which citrulline (5 mM final concentration) was added 30 min before the uptake assay to induce AGP1 transcription. Hom (10 mM final concentration) was added (○) or not (•, ×) to the medium at time zero of the experiment. (C) Initial velocity of 0.1 mM [14C]Thr uptake in gap1 agp1 (○, •) and gap1 agp1 gnp1 (×) strains. The cells were grown on minimal NH4+ medium to which citrulline was added (5 mM final concentration) 30 min before the uptake assay to induce GNP1 transcription. Hom (10 mM final concentration) was added (○) or not (•, ×) to the medium at time zero of the experiment. The strains were 23344c (wild type), 30629c (gap1), 30633c (gap1 agp1), EK041 (gap1 gnp1), and JA248 (gap1 agp1 gnp1).
FIG. 5.
FIG. 5.
AQR1 is required for Hom excretion. Cell suspensions of wild-type (23344c), gap1 agp1 gnp1 (JA248), and gap1 agp1 gnp1 aqr1 (IVU271) strains transformed with the pMACR7 plasmid (HOM3-R) were dropped over a lawn of Hom (hom3, strain Σ-A3-hu) auxotroph mutants and the petri dishes were incubated for 4 days at 29°C. Proline was the sole source of nitrogen in the medium.
FIG. 6.
FIG. 6.
AQR1 is involved in excretion of aspartate, alanine, and glutamate. Cells of the gap1 agp1 gnp1 aqr1 strain (IV271) transformed with the 2μm plasmid pIVU13 (Tet-AQR1) were grown on minimal medium containing NH4+ as the sole nitrogen source plus doxycycline to repress AQR1 transcription. At time zero of the experiment (early exponential phase, 0.2 to 0.4 mg of dry weight ml−1), the culture was filtered and cells were resuspended in the same medium with (Aqr1 not expressed) or without (Aqr1 expressed) doxycycline. Cells were harvested after 22 h, i.e., at late-exponential/early-stationary phase (1 to 2 mg of dry weight ml−1), and the concentrations of free amino acids inside cells (int.) and in the external medium (ext.) were measured by HPLC as detailed in Materials and Methods. Data are shown for aspartate (Asp), glutamate (Glt), alanine (Ala), glycine (Gly), Thr, and valine (Val).
FIG. 7.
FIG. 7.
Expression of an AQR1-lacZ fusion gene. (A) Cells of the wild-type strain (23344c) transformed with the YCpAQR1-lacZ plasmid were grown on minimal medium with urea as the sole nitrogen source. At time zero of the experiment, the indicated amino acids were added at a 2 mM final concentration. After 2 h, cells were collected and β-galactosidase activity was assayed as described in Materials and Methods. (B) Cells of the wild type (23344c) and gnc4 mutant (IHI120) transformed with the YCpAQR1-lacZ plasmid were grown on minimal medium containing urea as the sole nitrogen source. β-Galactosidase activity was assayed as described in Materials and Methods.
FIG. 8.
FIG. 8.
Mutations impairing endocytosis affect Aqr1 subcellular location and Aqr1-mediated excretion of amino acids. (A) Subcellular location of an Aqr1-GFP fusion protein. Cells of the JA551 (AQR1-GFP ura3), JA618 (AQR1-GFP npi1 ura3), and JA629 (AQR1-GFP end3Δ ura3) strains were grown on minimal glutamate medium and examined by fluorescence microscopy. Cells were labeled with FM4-64 to stain the vacuolar membrane and endosomal compartments (not shown). (B) Cell suspensions of wild-type (23344c), gap1 agp1 gnp1 (JA248), gap1 agp1 gnp1 aqr1 (IVU271), and gap1 agp1 gnp1 npi1 (32600a) strains transformed with the pMACR7 plasmid (HOM3-R) were dropped over a lawn of Thr (thr4, strain F4) or Hom (hom3, strain Σ-A3hu) auxotroph mutants and the petri dishes were incubated for 4 days at 29°C. Glutamate was the sole source of nitrogen in the medium.
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