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Title: A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.

Authors:
ORCiD logo [1];  [2];  [3];  [3];  [4];  [5]
  1. California Inst. of Technology (CalTech), Pasadena, CA (United States); Harvard Univ., Cambridge, MA (United States)
  2. Harris County Public Health, Houston, TX (United States); Harvard Univ., Cambridge, MA (United States)
  3. Princeton Univ., Princeton, NJ (United States)
  4. Howard Hughes Medical Inst., Chevy Chase, MD (United States); Harvard Univ., Cambridge, MA (United States)
  5. Texas A & M Univ., College Station, TX (United States)
Publication Date:
Research Org.:
Princeton Univ., NJ (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1357715
Alternate Identifier(s):
OSTI ID: 1389780
Grant/Contract Number:
SC0012461
Resource Type:
Journal Article: Published Article
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 12; Journal Issue: 5; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; adenine; metabolic pathways; epistasis; genetic screens; phenotypes; gene regulation; genomic signal processing; Saccharomyces cerevisiae

Citation Formats

Choi, Joonhyuk, Rajagopal, Abbhirami, Xu, Yi -Fan, Rabinowitz, Joshua D., O’Shea, Erin K., and Polymenis, Michael. A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae. United States: N. p., 2017. Web. doi:10.1371/journal.pone.0176085.
Choi, Joonhyuk, Rajagopal, Abbhirami, Xu, Yi -Fan, Rabinowitz, Joshua D., O’Shea, Erin K., & Polymenis, Michael. A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae. United States. doi:10.1371/journal.pone.0176085.
Choi, Joonhyuk, Rajagopal, Abbhirami, Xu, Yi -Fan, Rabinowitz, Joshua D., O’Shea, Erin K., and Polymenis, Michael. Wed . "A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae". United States. doi:10.1371/journal.pone.0176085.
@article{osti_1357715,
title = {A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae},
author = {Choi, Joonhyuk and Rajagopal, Abbhirami and Xu, Yi -Fan and Rabinowitz, Joshua D. and O’Shea, Erin K. and Polymenis, Michael},
abstractNote = {Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient Pi. Pi-limitation induces upregulation of inositol heptakisphosphate (IP7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of Pi starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.},
doi = {10.1371/journal.pone.0176085},
journal = {PLoS ONE},
number = 5,
volume = 12,
place = {United States},
year = {Wed May 17 00:00:00 EDT 2017},
month = {Wed May 17 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1371/journal.pone.0176085

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  • Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less
  • In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts-) lethal mutations that had been induced by ethyl methane-sulfonate or nitrosoguanidine were analyzed. Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously. In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes. In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts- lethal mutations, we isolated a newmore » set of such mutations after mutagenesis with UV or nitrogen mustard. Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis. Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped. These results suggest that only a minority of the essential genes in yeast can be identified using Ts- lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.« less
  • To study the functions of DNA topoisomerase I and Hpr1 protein, a suppressor mutant of the temperature-sensitive growth of an hpr1 top1-5{sup ts} double mutant was isolated. The isolated triple mutant showed cold-sensitive growth. By complementation of this phenotype, the suppressor gene was cloned. DNA sequencing showed it to be GCR3, a gene involved in the expression of glycol genes. Further analysis showed that gcr3 mutations also suppressed the temperature-sensitive growth of hpr1 single mutants. Experiments with gcr3 truncation mutants also suggested a genetic interaction between GCR3 and HPR1. The fact that top1 suppressed the growth defect of gcr3 suggestedmore » an interaction between those two genes also. Plasmid DNA isolated from gcr3 mutants was significantly more negatively supercoiled than normal, suggesting that Gcr3 protein, like topoisomerase I and Hpr1p, affects chromatin structure, perhaps during transcription. 43 refs., 2 figs., 6 tabs.« less
  • Gtr1p and Gtr2p of Saccharomyces cerevisiae are members of the Ras-like GTP binding family and interact genetically with Prp20p (yeast RCC1), which is a guanine nucleotide exchange factor for Gsp1p (yeast homolog of Ran, involved in nuclear export). Recently, Gtr1p and Gtr2p were suggested to be molecular switches in the rapamycin-sensitive TOR signaling pathway. Here, we show that Gtr1p and Gtr2p genetically interact with the chromatin remodeling factor Ino80p. Gtr2p interacted physically with both Rvb1p and Rvb2p. Consistent with these results, Gtr2p localized to chromatin and could activate transcription. Gtr1p and Gtr2p were found to be involved in chromatin silencingmore » in the vicinity of telomeres. Gtr1p and Gtr2p were required to repress nitrogen catabolite-repressed genes, which are repressed by the TOR signaling pathway. We propose that Gtr1p and Gtr2p are involved in epigenetic control of gene expression in the TOR signaling pathway.« less