Elvira Carvajal

Genome‐wide studies on the nuclear PDR3‐controlled response to mitochondrial dysfunction in yeast

Gain‐of‐function mutations in the transcription factors Pdr1p and Pdr3p lead to the up‐regulation of genes controlling plasma membrane properties. Pdr3p is involved in a retrograde response in which mitochondrial dysfunctions activate PDR5, a gene encoding an ABC membrane transporter. We carried out genome‐wide analyses of the PDR3‐controlled genes activated by the deletion of the mitochondrial DNA. We present evidence showing that PDR1 does not interfere with this PDR3 response. We also showed that the mitochondrially activated PDR3 response is highly sensitive to both yeast strain variations and carbon sources. These observations explain the apparent discrepancies in published studies and better describe the connections between the mitochondrial state and plasma membrane properties.

Asparaginase II of Saccharomyces cerevisiae

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Nitrogen regulation of Saccharomyces cerevisiae invertase: role of the URE2 gene.

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

The role of the GATA factors Gln3p, Nil1p, Dal80p and the Ure2p on ASP3 regulation in Saccharomyces cerevisiae

The role of Gln3p, Nil1p, Dal80p and Ure2p in the nitrogen regulation of ASP3, which codes for the periplasmic Saccharomyces cerevisiae asparaginase II, was investigated. Analysis of enzyme levels and mRNAASP3 in two wild‐type strains and gln3, nil1, gln3nil1, gln3ure2, nil1ure2, nil1dal80, ure2, dal80 and ure2dal80 mutant cells allowed the study of the qualitative and quantitative regulatory role of the GATA factors and Ure2p on ASP3 expression. The simultaneous presence of Gln3p and Nil1p is a required condition for full gene transcription. Enzyme activity doubled upon nitrogen starvation of either ammonium‐grown (possibly due to Nil2p/Deh1p derepression) or proline‐grown (due to Dal80p derepression) cells. The ure2 mutation increased enzyme levels five‐fold in fresh ammonium‐grown cells and ten‐fold in fresh proline‐grown cells. The combined effects of the ure2 mutation and nitrogen starvation on ammonium‐ or proline‐grown cells resulted in an overall 10–20‐fold enzyme activity increase, respectively, in comparison with the wild‐type cells. Copyright

Asparaginase II ofSaccharomyces cerevisiae: GLN3/JURE2 Regulation of a Periplasmic Enzyme

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme ofSaccharomyces cerevisiae encoded by theASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functionalGLN3 gene and that the response to nitrogen availability is under the control of theURE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates theGLN1, GDH2, GAP1, andPUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Nitrogen Regulation of Saccharomyces cerevisiae Invertase

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

L-asparaginase II of saccharomyces cerevisiae. Activity profile during growth using an ure2 mutant P40-3C and a P40-3C + URE2p strain.

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.

L-Asparaginase II of Saccharomyces cerevisiae

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.

Asparaginase II-GFP fusion as a tool for studying the secretion of the enzyme under nitrogen starvation

Production of asparaginase II of Saccharomyces cerevisiae is regulated by nitrogen and can be used as a model system for studying other secreted proteins in yeast. Green fluorescent protein (GFP) from Aequorea victoria was fused to the carboxy-terminus of the enzyme by genomic integration to the locus ASP3 of S. cerevisiae. We determined asparaginase II activity, mRNAASP3, mRNAASP3-GFP and GFP fluorescence. Nitrogen starvation in cells carrying the chimera ASP3-GFP caused an increase in fluorescence and in the expression of ASP3. We have shown that cells producing the chimera Asp3-GFPp displayed the same response to nitrogen starvation as control cells. We demonstrated that Asp3-GFPp can be used for studying asparaginase II secretion under nitrogen starvation in vivo.