Stephan Vissers

A Family of Ammonium Transporters in Saccharomyces cerevisiae

Ammonium is a nitrogen source supporting growth of yeast cells at an optimal rate. We recently reported the first characterization of an NH4+ transport protein (Mep1p) in Saccharomyces cerevisiae. Here we describe the characterization of two additional NH4+ transporters, Mep2p and Mep3p, both of which are highly similar to Mep1p. The Mep2 protein displays the highest affinity for NH4+ (Km, 1 to 2 microM), followed closely by Mep1p (Km, 5 to 10 microM) and finally by Mep3p, whose affinity is much lower (Km, approximately 1.4 to 2.1 mM). A strain lacking all three MEP genes cannot grow on media containing less than 5 mM NH4+ as the sole nitrogen source, while the presence of individual NH4+ transporters enables growth on these media. Yet, the three Mep proteins are not essential for growth on NH4+ at high concentrations (>20 mM). Feeding experiments further indicate that the Mep transporters are also required to retain NH4+ inside cells during growth on at least some nitrogen sources other than NH4+. The MEP genes are subject to nitrogen control. In the presence of a good nitrogen source, all three MEP genes are repressed. On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression. High-level MEP2 transcription requires at least one of the two GATA family factors Gln3p and Nil1p, which are involved in transcriptional activation of many other nitrogen-regulated genes. In contrast, expression of either MEP1 or MEP3 requires only Gln3p and is unexpectedly down-regulated in a Nil1p-dependent manner. Analysis of databases suggests that families of NH4+ transporters exist in other organisms as well.

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

ABSTRACT The SSY1 gene of Saccharomyces cerevisiaeencodes 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 theAGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in thessy1Δ 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 AGP1requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1by 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.

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.

Complete DNA sequence of yeast chromosome II

In the framework of the EU genome‐sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37‐45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT‐rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT‐rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Effect of 21 different nitrogen sources on global gene expression in the yeast Saccharomyces cerevisiae.

ABSTRACT We compared the transcriptomes of Saccharomyces cerevisiae cells growing under steady-state conditions on 21 unique sources of nitrogen. We found 506 genes differentially regulated by nitrogen and estimated the activation degrees of all identified nitrogen-responding transcriptional controls according to the nitrogen source. One main group of nitrogenous compounds supports fast growth and a highly active nitrogen catabolite repression (NCR) control. Catabolism of these compounds typically yields carbon derivatives directly assimilable by a cells metabolism. Another group of nitrogen compounds supports slower growth, is associated with excretion by cells of nonmetabolizable carbon compounds such as fusel oils, and is characterized by activation of the general control of amino acid biosynthesis (GAAC). Furthermore, NCR and GAAC appear interlinked, since expression of the GCN4 gene encoding the transcription factor that mediates GAAC is subject to NCR. We also observed that several transcriptional-regulation systems are active under a wider range of nitrogen supply conditions than anticipated. Other transcriptional-regulation systems acting on genes not involved in nitrogen metabolism, e.g., the pleiotropic-drug resistance and the unfolded-protein response systems, also respond to nitrogen. We have completed the lists of target genes of several nitrogen-sensitive regulons and have used sequence comparison tools to propose functions for about 20 orphan genes. Similar studies conducted for other nutrients should provide a more complete view of alternative metabolic pathways in yeast and contribute to the attribution of functions to many other orphan genes.

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

Abstract The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases 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 α-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.

Transcriptional Induction by Aromatic Amino Acids in Saccharomyces cerevisiae

ABSTRACT 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 abolishingARO9 induction affect a gene called ARO80(YDR421w), coding for a Zn2Cys6 family transcription factor. A sequence highly similar to UASaro was found upstream from theYDR380w 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 Yeast: Casein Kinase I and the Ssy5 Endoprotease Are Key Determinants of Endoproteolytic Activation of the Membrane-Bound Stp1 Transcription Factor

ABSTRACT Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease. This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membrane-bound Stp1 transcription factor. The SCFGrr1 ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear. Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation. In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids. Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing. Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription. We further show that Stp1 processing also requires the SCFGrr1 complex but is insensitive to proteasome inhibition. However, Stp1 processing does not require SCFGrr1, Ssy1, or Ptr3 when Ssy5 is overproduced. Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation. We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor. Upon detection of external amino acids, Ssy1-Ptr3 likely allows—in a manner dependent on SCFGrr1—the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.

The Stimulation of Yeast Phosphofructokinase By Fructose 2,6-bisphosphate

1. INTRODUCTION Fructose 2,6-bisphosphate was discovered as a stimulator of rat liver PFK [l-3]. Its effect on liver and muscle PFKs is to increase the affinity of the enzyme for Fru-6-P and to relieve the inhibition by ATP with no effect on Vmax [4,5]. It is the most potent positive effector of this enzyme known at the present time. It exerts its effect at concentrations which are lOOO-fold smaller than those of Fru-1,6-P2, a classical positive effector of PFK, re- quired for the same effect 141. Fru-2,6-P2 is present in glucose-grown

Fructose 2,6-bisphosphate in yeast.

Abstract Fructose 2,6-bisphosphate was identified in Saccharomyces cerevisiae grown on glucose both by its property to be an acid-labile stimulator of 6-phosphofructo 1-kinase and by its ability to be quantitatively converted into fructose 6-phosphate under mild acid conditions. Fructose 2,6-bisphosphate was undetectable in cells grown on non-glucose sources. When glucose was added to the culture, fructose 2,6-bisphosphate was rapidly synthesized, reaching within 1 min concentrations able to cause a profound inhibition of fructose 1,6-bisphosphatase and a great stimulation of 6-phosphofructo 1-kinase.