RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

Isolation of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae deficient in both high- and low-affinity L-leucine transport.

A yeast mutant defective in permeases S1 and S2 which transport L-leucine was isolated from a parental strain already deficient in the general amino acid permease, GAP1. The mutant was selected as a spontaneous, trifluoroleucine-resistant (TFLR) strain. Full resistance depended upon the presence of two unlinked mutant genes designated let1 and let2. The let1 mutation completely inactivates the high-affinity leucine transport system defined kinetically as S1. Although the let2 mutation caused a marked decrease in the Jmax of the low-affinity transport system, S2, residual leucine transport in the let1 let2 gap1 mutant had the same KT as in the LET1 LET2 gap1 parent. The mutant exhibited a marked decrease in growth on minimal medium containing leucine, isoleucine or valine as a sole nitrogen source. Moreover, assimilation of methionine, phenylalanine, serine and threonine was decreased, whereas basic and acidic amino acids supported normal growth. This indicates that at least one of the leucine permeases has a fairly broad, but still limited, specificity. Reversion of the gap1 gene restored leucine transport. The revertant was sensitive to TFL when grown on proline but resistant when NH4+ was the nitrogen source. The previously published mutations (shr3, aat1, lup1 or raa) would not be related to either LET1 or LET2.

Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae.

It has been previously reported that 5-aminolevulinic acid (ALA) and 4-aminobutyric acid (GABA) share a common permease in Saccharomyces cerevisiae (Bermúdez Moretti et al., 1993). The aim of the present work was to determine the relationship between the transport of these compounds in isolated cells. Assessment of amino acid incorporation was performed in S. cerevisiae using 14C-ALA or 3H-GABA. Initial rates of ALA incorporation in cells grown in the presence of 5 mM ALA and 5 mM GABA, were three to four times lower than in cells grown without supplements. Kinetic studies indicate that GABA competitively inhibits ALA transport. During the growth phase GABA uptake was also inhibited by 74% and 60% in the presence of ALA and GABA, respectively. These findings indicate that in S. cerevisiae the structurally related compounds, ALA and GABA, may be incorporated into the cells by a common carrier protein. Should this occur in other lukaryotic cells it may explain the neurotoxic effect attributed to ALA in the pathogenesis of acute porphyrias.