Carbon Catabolite Repression Regulates Amino Acid Permeases in Saccharomyces cerevisiae via the TOR Signaling Pathway

Abstract

We have identified carbon catabolite repression (CCR) as a regulator of amino acid permeases in Saccharomyces cerevisiae, elucidated the permeases regulated by CCR, and identified the mechanisms involved in amino acid permease regulation by CCR. Transport of L-arginine and L-leucine was increased by ϳ10 -25-fold in yeast grown in carbon sources alternate to glucose, indicating regulation by CCR. In wild type yeast the uptake (pmol/10 6 cells/ h), in glucose versus galactose medium, of L-[ 14 C]arginine was (0.24 ؎ 0.04 versus 6.11 ؎ 0.42) and L-[ 14 C]leucine was (0.30 ؎ 0.02 versus 3.60 ؎ 0.50). The increase in amino acid uptake was maintained when galactose was replaced with glycerol. Deletion of gap1⌬ and agp1⌬ from the wild type strain did not alter CCR induced increase in L-leucine uptake; however, deletion of further amino acid permeases reduced the increase in L-leucine uptake in the following manner: 36% (gnp1⌬), 62% (bap2⌬), 83% (⌬(bap2-tat1)). Direct immunofluorescence showed large increases in the expression of Gnp1 and Bap2 proteins when grown in galactose compared with glucose medium. By extending the functional genomic approach to include major nutritional transducers of CCR in yeast, we concluded that SNF/MIG, GCN, or PSK pathways were not involved in the regulation of amino acid permeases by CCR. Strikingly, the deletion of TOR1, which regulates cellular response to changes in nitrogen availability, from the wild type strain abolished the CCR-induced amino acid uptake. Our results provide novel insights into the regulation of yeast amino acid permeases and signaling mechanisms involved in this regulation.The preferred mode of metabolism in Saccharomyces cerevisiae is fermentative, and the preferred carbon source is glucose; other carbohydrates such as galactose or maltose and non-fermentable carbon sources such as ethanol and glycerol could also be utilized by yeast (1). Substitution, reduction, or removal of glucose from the laboratory growth medium, however, is known to have important physiological and genomic consequences in yeast (1-3).The presence of a high concentration of glucose in the growth medium represses transcription of multiple genes including those involved in alternative carbohydrate and mitochondrial metabolism (1, 3). This phenomenon is known as carbon catabolite repression (CCR, 2 gene regulation processes that are initiated when glucose is removed as a source of carbon in the growth medium; reviewed by Refs. 1 and 4). Two nutrient signaling transducers, namely SNF1 and GCN2, are known to play a major role in global gene regulation by CCR. SNF1 is a yeast homologue of the AMP-activated protein kinase that regulates CCR through the MIG1 protein (5). During the conditions of low glucose and starvation of amino acids, GCN2 kinase is also involved in nutrient signaling in yeast (4). TOR is another important nutritional transducer that regulates various cellular processes including protein synthesis and autophagy (6) but in response to the availability of nitrogen. The TOR pathway ...

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