Metabolic fate of the increased yeast amino Acid uptake subsequent to catabolite derepression

Abstract

Catabolite repression (CCR) regulates amino acid permeases in Saccharomyces cerevisiae via a TOR-kinase mediated mechanism. When glucose, the preferred fuel in S. cerevisiae, is substituted by galactose, amino acid uptake is increased. Here we have assessed the contribution and metabolic significance of this surfeit of amino acid in yeast undergoing catabolite derepression (CDR). L-[U-(14)C]leucine oxidation was increased 15 ± 1 fold in wild type (WT) strain grown in galactose compared to glucose. Under CDR, leucine oxidation was (i) proportional to uptake, as demonstrated by decreased uptake and oxidation of leucine in strains deleted of major leucine permeases and (ii) entirely dependent upon the TCA cycle, as cytochrome c1 (Cyt1) deleted strains could not grow in galactose. A regulator of amino acid carbon entry into the TCA cycle, branched chain ketoacid dehydrogenase, was also increased 29 ± 3 fold under CCR in WT strain. Protein expression of key TCA cycle enzymes, citrate synthase (Cs), and Cyt1 was increased during CDR. In summary, CDR upregulation of amino acid uptake is accompanied by increased utilization of amino acids for yeast growth. The mechanism for this is likely to be an increase in protein expression of key regulators of the TCA cycle.

Figure 1

(a) L-[¹⁴C]leucine (750 μM) oxidation in WT and amino acid permease deleted yeast strains. Yeast was grown in either 2% glucose (filled bars) or 2% galactose (hollow bars). L-[¹⁴C]leucine uptake and oxidation were performed as described in Materials and Methods. Deletion of various amino acid permeases (M4055 : MATa ura3 gap1 D D(bap2-tat1), M4581: MATa ura3 gap1D agp1D gnp1D D(bap2-tat1), M4582: MATa ura3 gap1D D(bap2-tat1) bap3D tat2D) from the WT (M3750) reduced the rate of L-[¹⁴C]leucine oxidation observed when yeast are grown in galactose. Results are mean ± SEM from 3 different preparations each measured in quadruplicate. (b) Correlation between carbon catabolite derepressed L-leucine uptake (shaded bars) and oxidation (hatched bars). For correlation analysis data (nmol/million cells/h), oxidation (0.7 ± 0.02) and uptake (1.2 ± 0.12) in the WT M3750 strain were normalized to 1. In deleted strains bars represent the proportion of either uptake or oxidation observed in the WT strain.

Figure 2

Presence of glucose or galactose in the medium is essential to measure L-[¹⁴C]leucine (750 μM) oxidation in WT (M3750) yeast. Yeast was grown in 2% glucose (filled bars) or 2% galactose (hollow bars) and incubated in the assay medium with glucose, galactose (± carbohydrate, CHO), or the nonmetabolizable glucose analogue deoxyglucose (DOG) as described in experimental procedures. (Oxidation in yeast grown in glucose in the presence of DOG was not measured.) Results are means ± SEM for 3 different preparations measured in quadruplicate.

Figure 3

Time course of BckDH basal activity in WT yeast, grown in 2% glucose (dashed line) or 2% galactose (solid line). Representative trace of NADH formation in cell homogenates using α-ketoisovalerate as a BCKD substrate (added at arrow). Dashed line represents glucose and solid line represents galactose grown yeast. Inset represents mean calculated activity (n = 4) in mU (nmoles NADH formed/min). BckDH activity in yeast grown in glucose YNB medium (filled bars) or galactose YNB medium (hollow bars), n = 3.

Figure 4

Activity of the TCA cycle is essential when WT yeast is grown with galactose as sole carbon source. Image of WT (BY4741) and Dcyt1 grown on agar plates with 2% glucose or galactose added to the YNB medium.

Figure 5

Direct fluorescence was measured in yeast strains with GFP-tagged CYT1 and CIT1 using Zeiss LSM510 and Bio-Rad Radiance 2100 confocal systems, respectively. (a) Cyt1-GFP fluorescence in yeast grown in 2% glucose YNB compared to (c) in 2% galactose YNB medium. Micrographs (b) and (d) are corresponding Nomarski differential interference contrast images of (a) and (c), respectively. (e) Cit1-GFP fluorescence of yeast grown in 2% glucose YNB compared to (g) grown in 2% galactose YNB medium. (f) and (h) are corresponding bright-field images of (e) and (g), respectively. Representative pictures of experiments in triplicate are presented.