Transcriptional induction by aromatic amino acids in Saccharomyces cerevisiae.

Aromatic aminotransferase II, product of the ARO9 gene, catalyzes the first step of tryptophan, phenylalanine, and tyrosine catabolism in Saccharomyces cerevisiae. ARO9 expression is under the dual control of specific induction and nitrogen source regulation. We have here identified UASaro, a 36-bp upstream element necessary and sufficient to promote transcriptional induction of reporter gene expression in response to tryptophan, phenylalanine, or tyrosine. We then isolated mutants in which UASaro-mediated ARO9 transcription is partially or totally impaired. Mutations abolishing ARO9 induction affect a gene called ARO80 (YDR421w), coding for a Zn2Cys6 family transcription factor. A sequence highly similar to UASaro was found upstream from the YDR380w gene encoding a homolog of bacterial indolepyruvate decarboxylase. In yeast, this enzyme is postulated to catalyze the second step of tryptophan catabolism to tryptophol. We show that ARO9 and YDR380w (named ARO10) have similar patterns of transcriptional regulation and are both under the positive control of Aro80p. Nitrogen regulation of ARO9 expression seems not directly to involve the general factor Ure2p, Gln3p, Nil1p, Uga43p, or Gzf3p. ARO9 expression appears, rather, to be mainly regulated by inducer exclusion. Finally, we show that Gap1p, the general amino acid permease, and Wap1p (Ycl025p), a newly discovered inducible amino acid permease with broad specificity, are the main aromatic amino acid transporters for catabolic purposes.

Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-Box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease.

The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in the ssy1Delta mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination.

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 and aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9 affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, alpha-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known alpha-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium.

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily.

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 and aro9 double-mutant strain that is defective in aromatic aminotransferase I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and alpha-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.

Gzf3p, a fourth GATA factor involved in nitrogen-regulated transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two positive transcription factors of the GATA family, Gln3p and Nil1p/Gat1p, upregulate the expression of multiple nitrogen pathway genes via upstream 5'-GATA-3' sequences. Another GATA factor, Uga43p/Dal80p, downregulates to varying degrees the expression of some nitrogen-regulated genes. Here, we report the functional analysis of a fourth GATA factor, Gzf3p/Nil2p, whose gene was discovered by systematic sequencing of chromosome X. The Gzf3 protein most closely resembles Uga43p. Similar to Uga43p, Gzf3p has the properties of a negative GATA factor. While Uga43p is active specifically under nitrogen-depression conditions, Gzf3p exerts its negative regulatory function specifically on preferred nitrogen sources: It is involved in nitrogen repression of Nil1p-dependent transcription. At least one positive GATA factor is required for the UGA43 and GZF3 genes to be expressed. The Uga43p factor negatively regulates GZF3 expression and vice versa. In addition, both Uga43p and Gzf3p moderately regulate expression of their own genes. These two proteins seem to be parts of a complex network of GATA factors which probably play a determining role in nitrogen-regulated transcription.

Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transport of ammonium across the plasma membrane for use as a nitrogen source is mediated by at least two functionally distinct transport systems whose respective encoding genes are called MEP1 and MEP2. Mutations in the MEP2 gene affect high affinity, low capacity ammonium transport while mutations in the MEP1 gene disrupt a lower affinity, higher capacity system. In this work, the MEP1 gene has been cloned and sequenced and its expression analyzed. The predicted amino acid sequence reveals a highly hydrophobic, 54 kDa protein with 10 or 11 putative membrane-spanning regions. The predicted Mep1p protein shares high sequence similarity with several bacterial proteins of unknown function, notably the product of the nitrogen-regulated nrgA gene of Bacillus subtilis, and with that of a partial cDNA sequence derived from Caenorhabditis elegans. The Mep1p and related proteins appear to define a new family of transmembrane proteins evolutionarily conserved in at least bacteria, fungi and animals. The MEP1 gene is most highly expressed when the cells are grown on low concentrations of ammonium or on 'poor' nitrogen sources like urea or proline. It is down-regulated, on the other hand, when the concentration of ammonium is high or when other 'good' nitrogen sources like glutamine or asparagine are supplied in the culture medium. The overall properties of Mep1p indicate that it is a transporter of ammonium. Its main function appears to be to enable cells grown under nitrogen-limiting conditions to incorporate ammonium present at relatively low concentrations in the growth medium.