Amino acid transport through the Saccharomyces cerevisiae Gap1 permease is controlled by the Ras/cAMP pathway

Abstract

The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is a broad range, low affinity permease that imports amino acids in cells growing on poor nitrogen sources. This permease also signals the presence of amino acids through the fermentable growth medium pathway allowing the cell to respond to new sources of nitrogen in the surrounding medium. Yeast with an activated Ras2/cAMP pathway show many phenotypes indicative of altered nitrogen uptake and metabolism; sensitivity to nitrogen starvation, low amino acid pools. We have shown that Gap1p activity is lowered in cells with an activated RAS2(val19) allele or elevated cAMP levels whereas cells with inactive ras2 allele lose ammonia repression of Gap1p-mediated transport. This regulation is through a post-transcriptional mechanism; transcription of GAP1 is not affected by cAMP level. A mechanism by which the Ras2/cAMP/PKA pathway controls the ubiquitin-dependent degradation of Gap1p is most consistent with the data.

Figure 1. General amino acid permease activity in yeast RAS2 mutants

Strains were grown in either minimal ammonia (black bars) or minimal proline (grey bars) medium and assayed for ¹⁴C-citrulline uptake (2.0mM) as described in the methods. A. S288C-derived strains. B. Σ1278B-derived strains. Gap1p activity is measured as nmoles ¹⁴C-citrulline/min⁻¹/OD550⁻¹.Results are the average of 4–10 independent assays, error bars denote the standard error.

Figure 1. General amino acid permease activity in yeast RAS2 mutants

Strains were grown in either minimal ammonia (black bars) or minimal proline (grey bars) medium and assayed for ¹⁴C-citrulline uptake (2.0mM) as described in the methods. A. S288C-derived strains. B. Σ1278B-derived strains. Gap1p activity is measured as nmoles ¹⁴C-citrulline/min⁻¹/OD550⁻¹.Results are the average of 4–10 independent assays, error bars denote the standard error.

Figure 2. The effect of external cAMP concentration on Gap1p activity in a cyr1 cAMP-sensitive strain

Strains were grown in minimal ammonia medium containing the cAMP concentration given and assayed as described for Fig. 1. Black bars – HR125 (wild-type), grey bars –TC41-1 (cyr1 mutant). Results are the average of three independent assays.

Figure 3. The control of Gap1p activity by cAMP is not through the regulation of transcription

TC41-1 cells containing the expression plasmid pSE (promoter of GAP1 fused to the E. coli lacz gene) was grown in ammonia medium containing the cAMP levels given. β-galactosidase levels were assayed by the method of Miller (1972). Gap1p activity (filled squares) and β-galactosidase levels (filled triangles, dashed line) are given as a percentage of the level obtained in 0.5mM cAMP where 100% Gap1p activity = 6.2 nmoles ¹⁴C-citrulline/min⁻¹/OD₅₅₀⁻¹ and 100% β-galactosidase = 750 Units.