15 research outputs found
Mitochondrial Metabolism in the Spotlight:Maintaining Balanced RNAP III Activity Ensures Cellular Homeostasis
Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1.
Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III)-driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose-rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae In a mutant compromised in RNAP III activity, there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flu
Tup1 occupancy is significantly increased on the transcriptionally active <i>HXT2</i> promoter in <i>rpc128-1007</i>.
<p>(A) Schematic representation of the <i>HXT2</i> promoter region. The Rgt1 binding site is indicated by a circle. The Mig1 binding site is denoted by a square. The horizontal lines represent cDNA fragments amplified by real-time PCR in the ChIP assay (numbered in squares). Arrows facing each other correspond to primer pairs hybridization. Coordinates represent the position of the aforementioned elements in the region with respect to the translation start site. The black arrow above <i>HXT2</i> indicates the translation start site. The WT (MB159-4D) and <i>rpc128-1007</i> strain expressing HA-tagged Mig1 (B), HA-tagged Rgt1 (C) and HA-tagged Tup1 (D) were grown in 2% glucose or 2% glycerol-rich medium. Crosslinked chromatin was immunoprecipitated with antibodies against the HA epitope, followed by real-time PCR. The signals are presented as the percent of the INPUT signal from three separate experiments with standard deviations. Numbers from 1 to 6 correspond to real-time PCR amplification products in the <i>HXT2</i> promoter region on the A panel. (B) Mig1 shows the same degree of occupancy on the <i>HXT2</i> promoter in the wild-type strain and mutant strain with low RNAP III activity at high glucose conditions. (C) Rgt1 had a higher occupancy on the <i>HXT2</i> promoter in the <i>rpc128-1007</i> strain with low RNAP III activity. (D) Tup1 shows enhances association with the <i>HXT2</i> promoter in <i>rpc128-1007</i> despite the carbon source.</p
Diagram illustrating the crosstalk between the Snf1-Mig1 and Snf3/Rgt2-Rgt1 (SRR) pathways and glycolysis in yeast resulting in <i>HXT2</i> gene repression, under fermentable and non-fermentative growth conditions.
<p>Transcription of the <i>HXT2</i> gene is repressed by the Rgt1/Ssn6-Tup1 complex in the absence of glucose and under non-fermentable growth conditions (A). Rgt1 recruits Ssn6-Tup1 in a Mth1-dependent manner to form the repressor complex. Mth1 protein prevents Rgt1 phosphorylation by PKA kinase. Snf1 kinase is activated under these conditions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185516#pone.0185516.ref033" target="_blank">33</a>]. Snf1 kinase phosphorylates and regulates the Mig1 interaction with DNA and Ssn6-Tup1. See the text for further details. LEGEND: Fps1- glycerol transporter/exporter, Stl1 –glycerol transporter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185516#pone.0185516.ref057" target="_blank">57</a>], Gut1- glycerol kinase converts glycerol to glycerol-3-phophate. (B) Under high-glucose conditions, the <i>HXT2</i> gene is repressed by the Snf1-Mig1 pathway. Mig1 is dephosphorylated and recruits Ssn6-Tup1 to mediate the repression. Snf1 induces the repression of <i>MTH1</i> gene expression. Glucose binding generates a signal that leads to proteasomal degradation of Mth1 <i>via</i> the Snf3/Rgt2-Rgt1 (SRR) pathway, which causes Rgt1 phosphorylation by PKA. See the text for further details. LEGEND: Yck1, 2 casein kinases, Std1- SCF<sup>Grr1</sup> complex, Hxk2- glucose hexokinase, Reg1-Glc7- phosphatase complex. The diagrams are presented in SBGN format using CellDesigner<sup>™</sup> 4.4 software.</p
RNAP III changed activity does not affect glucose repression.
<p>The WT strain (MB159-4D) and the <i>rpc128-1007</i>, <i>reg1-Δ</i> and <i>maf1-Δ</i> mutant strains were grown under repressing (2% glucose) and activating (2% sucrose) conditions. The experiment was performed in cell-free extracts isolated from the aforementioned strains. Invertase activity is expressed in μmol of glucose·min<sup>-1</sup>·mg<sup>-1</sup> protein. Data are expressed as the mean obtained from at least three independent experiments measured in triplicate. The ± standard deviations are shown.</p
Increased stability of Mth1 in the <i>rpc128-1007</i> strain under non-fermentative growth conditions.
<p>The wild-type (MB159-4D) and <i>rpc128-1007</i> mutant strain expressing HA-tagged Mth1 were grown to A<sub>600</sub> ≈ 1.0 in 2% glucose or 2% glycerol-rich medium. Proteins were extracted and immunoprecipitated with magnetic beads conjugated to mouse anti-HA antibodies. Mth1-HA was visualized with rabbit anti-HA tag antibodies. The relative level of Mth1 (B) protein in comparison to yeast Vma2 protein from four independent biological replicates. The molecular weight (MW) of Mth1 with the HA tag was ~56.28 kDa, and the MW Vma2 was ~57.7 kDa.</p
Low RNAP III activity caused by Gly1007Ala point mutation in C128 RNAP III subunit, correlates with transcription of <i>HXT2</i> gene by RNAP II, despite repressing conditions.
<p>Activation of the Snf1-dependent glucose repression and Snf3/Rgt2-Rgt1 (SRR) signaling pathways do not repress the <i>HXT2</i> transcription by RNAP II. We propose Ssn6-Tup1 complex as a <i>HXT2</i> transcription coactivator in <i>rpc128-1007</i>. Under high-glucose conditions, Mth1 degradation occurs. Rgt1, which is phosphorylated by PKA, dissociates from the <i>HXT2</i> promoter. Mig1, which is bound to the regulatory region, recruits Ssn6-Tup1. The complex transforms into a coactivator complex due to an unidentified intracellular signal and the expression of <i>HXT2</i> is induced. Under non-fermentable growth conditions in the strain with low RNAP III activity, the Snf3/Rgt2-Rgt1 (SRR) pathway transduces the signal for unfavorable external conditions to Mth1, preventing its degradation. The Rgt1 and Tup1 corepressor complex transforms into an activator complex and strongly induces <i>HXT2</i> expression.</p
Glucose flux is present in all tested strains.
<p>High-affinity glucose transport was measured in the WT (MB159-4D), <i>rpc128-1007</i> and <i>maf1</i>-Δ strains. The assay was performed using fluorescently labeled glucose 2-NDBG. The cells were grown to exponential phase of <i>A</i><sub><i>600</i></sub> ≈ 1.0 in 2% glucose-rich medium (YPD), transferred to SC medium supplemented with amino acids without a carbon source and incubated for 10 min at 30°C. The uptake of 2-NDBG (1 mM concentration) was performed over time. As a control, cell suspensions without fluorescently labeled glucose were assessed. The results are expressed as the mean fluorescence intensity (MFU) of four independent biological replicates with standard deviations.</p
Mig1 cellular concentration and phosphorylation pattern under different growth conditions.
<p>The WT strain (MB159-4D) and <i>rpc128-1007</i> mutant strain expressing HA-tagged Mig1 proteins were grown to A<sub>600</sub> ≈ 1.0 in 2% glucose or 2% glycerol-rich medium. Total cell protein extracts were subjected to SDS-PAGE and examined by Western blotting using anti-HA antibodies (A) P-Mig1-HA: phosphorylated Mig1-HA. The quantified relative level of Mig1–HA (B) protein compared to yeast Vma2 protein was calculated for at least three independent experiments conducted in triplicates with standard deviations. The molecular weight (MW) of Mig1-HA was ~ 64.8 kDa the MW of Vma2 was ~ 57.7 kDa.</p