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GAP1 activity is dependent on cAMP in Saccharomyces cerevisiae

Adrian A Amitrano, Daniel A Saenz, Eugenia H Ramos
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12560.x 131-133 First published online: 1 June 1997

Abstract

General amino acid permease (GAP1) activity was evaluated in adenylate cyclase-deficient Saccharomyces cerevisiae to determine the effect of cAMP levels on GAP1 activity. Lowering cAMP concentrations in the culture media led to a decrease in the initial rates of l-citrulline uptake. Kinetics of the amino acid transport system showed a partial loss of transport capacity, with no apparent modifications in permease affinity.

Keywords
  • Amino acid transport
  • cAMP regulation
  • Saccharomyces cerevisiae

1 Introduction

In Saccharomyces cerevisiae, the general amino acid permease, GAP1, is capable of transporting all l-amino acids found in proteins, related compounds such as citrulline and ornithine, several d-amino acids and toxic amino acid analogs [1]. The GAP1 permease exhibits its highest activity in cells grown on poor nitrogen source (l-proline), and no GAP1 activity is detected in normal cells from media containing ammonium ions as the sole nitrogen source [2]. These different activities are the result of a dual control involving inactivation of permease activity and repression of permease synthesis [3].

The NPR1 gene product appears to be a regulatory protein, exerting a positive control on the different permeases. This gene has been cloned and its sequence was homologous to that found for protein kinases [4].

Stanbrough and Magasanik [5] presented evidence that the active GAP1 permease is phosphorylated and that its inactivation results from its dephosphorylation.

Several yeast metabolic processes including carbohydrate storage [6] and cytochrome synthesis [7] in addition to others [8, 9], are regulated by the RAS2/PKA signal pathway. In a previous study, we described the participation of the RAS2/PKA pathway in the nitrogen regulation of l-leucine uptake in S. cerevisiae[10].

In the present study, in order to investigate the effects of cAMP on the transport activity of GAP1, the transport of l-citrulline, an amino acid essentially transported by GAP1 [2], in an adenylate cyclase-deficient S. cerevisiae mutant, was evaluated [11]. The results presented herein demonstrate an effect, either direct or indirect, of cAMP levels on GAP1 transport activity.

2 Materials and methods

2.1 Strain and media

Yeast strain AM18-5C (MATα cyr1-1) was kindly provided by Dr Kunihiro Matsumoto [11].

MPDG medium was minimal proline medium (MPM) containing 0.1% (w/v) l-proline as the sole nitrogen source [12]. The carbon sources were 0.1% (w/v) d-glucose and 3% (v/v) glycerol.

YPD-agar medium contained 1% yeast extract, 1% peptone, 2%d-glucose and 3% agar (all w/v).

These media were supplemented with different concentrations of cAMP, as described below.

2.2 Culture conditions

After 17 h of growth at 30°C in MPDG+1.0 mM cAMP medium, cells were harvested, washed and transferred aseptically to fresh MPDG medium containing different cAMP concentrations and were further grown for 4 h. Cells were then harvested by centrifugation, washed twice, resuspended in distilled water and immediately used to measure l-14C-citrulline uptake.

Yeast viability was measured by plating on YPD+1.0 mM cAMP plates to obtain viable cell counts.

2.3 Measurement of l-14C-citrulline uptake

l-14C-Citrulline uptake experiments were performed as described by Kotliar et al. using an external concentration of 0.05 mM [12].

In order to determine the kinetic parameters of l-citrulline uptake, samples were obtained at 5, 60 and 120 s, and initial rate values were calculated by linear regression. The kinetic parameters were calculated using the Graph Pad Prism program. The kinetic parameters were determined using l-citrulline concentrations ranging from 0.01 to 1 mM.

The time between recovery and filtering of the samples did not exceed 5 s. Measurements were carried out in duplicate for each experiment. Values presented are the averages of at least three experiments. Deviation of these values from the mean was less than 5%.

3 Results and discussion

3.1 Effects of cAMP on l-14C-citrulline transport

The initial rates of l-citrulline uptake (Ji) were measured in cells grown in MPDG containing different concentrations of cAMP and normalized for viability.

The initial rate of l-citrulline uptake at 0.5 mM cAMP decreased 42% and the viability of the cells was slightly affected with respect to controls (cells grown in MPDG+1.0 mM cAMP medium) (Table 1). In addition, at lower concentrations of the cyclic nucleotide, the initial uptake rates were very low but cellular viability was also drastically decreased.

View this table:
1

Initial rates of l-citrulline uptake

cAMP conc.JiNViability (%)
1.000.94100
0.500.5581
0.250.2958
0.10ND<10
0.00ND<10
  • Cells grown in MPDG medium with different cAMP concentrations, as described in Section 2. JiN: initial rates of l-14C-citrulline uptake normalized for viability, N is normalized for viability and cAMP concentrations in mM.

On the other hand, in cells grown in nitrogen-rich minimal medium or YPD medium, supplemented in both cases with 1.0 mM cAMP, GAP1 activity could not be detected (data not shown).

3.2 Kinetics of l-14C-citrulline transport

Taking in account the initial rate results, we decided to investigate the kinetics of l-citrulline uptake in cells grown in MPDG containing either 1.0 or 0.5 mM cAMP.

The kinetics of l-citrulline uptake as a function of l-citrulline external concentrations, in the presence of 1.0 or 0.5 mM cAMP, are shown in Fig. 1A. Under both types of experimental conditions, the data fitted a single Michaelis-Menten curve. At both cAMP concentrations, a straight line in the Eadie-Hofstee plots for these data is consistent with the presence of a single transport system (Fig. 1B). In cells grown in 1.0 mM cAMP, the estimated KTN (KT normalized for viability) value was 0.05 mM and JmaxN was 1.76 μmol·g−1·min−1. In cells grown in the presence of 0.5 mM cAMP, there was no change in KTN values, whereas the JmaxN decreased 42% with respect to controls, reaching 1.02 μmol·g−1·min−1. The GAP1 affinity values were similar to those reported previously for S. cerevisiae[2].

Figure 1

(A) Initial uptake of l-14C-citrulline as a function of external citrulline concentration (mM) in cells grown in MPDG with 1.0 mM (●) or 0.5 mM cAMP (○). (B) Eadie-Hofstee plot of the data shown in (A). Ji is expressed as μmol·g−1·min−1.

These results are direct evidence that in S. cerevisiae the regulation of the general amino acid permease transport activity is dependent on the cAMP, and are in agreement with the complex regulation mechanism proposed for GAP1 permease [1, 3, 5].

Acknowledgements

This study was supported by grants from the Argentine National Research Council (CONICET). We thank Dr. Kunihiro Matsumoto, Department of Molecular Biology, Faculty of Science, Nagoya University, Nagoya, Japan, for generously providing the yeast strain.

Abbreviations
GAP1
general amino acid permease
cAMP
adenosine 3′,5′-cyclic monophosphate
PKA
cAMP-protein kinase
NPR1
nitrogen-permease reactivation

References

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