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      Whi2 is a conserved negative regulator of TORC1 in response to low amino acids

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Yeast WHI2 was originally identified in a genetic screen for regulators of cell cycle arrest and later suggested to function in general stress responses. However, the function of Whi2 is unknown. Whi2 has predicted structure and sequence similarity to human KCTD family proteins, which have been implicated in several cancers and are causally associated with neurological disorders but are largely uncharacterized. The identification of conserved functions between these yeast and human proteins may provide insight into disease mechanisms. We report that yeast WHI2 is a new negative regulator of TORC1 required to suppress TORC1 activity and cell growth specifically in response to low amino acids. In contrast to current opinion, WHI2 is dispensable for TORC1 inhibition in low glucose. The only widely conserved mechanism that actively suppresses both yeast and mammalian TORC1 specifically in response to low amino acids is the conserved SEACIT/GATOR1 complex that inactivates the TORC1-activating RAG-like GTPases. Unexpectedly, Whi2 acts independently and simultaneously with these established GATOR1-like Npr2-Npr3-Iml1 and RAG-like Gtr1-Gtr2 complexes, and also acts independently of the PKA pathway. Instead, Whi2 inhibits TORC1 activity through its binding partners, protein phosphatases Psr1 and Psr2, which were previously thought to only regulate amino acid levels downstream of TORC1. Furthermore, the ability to suppress TORC1 is conserved in the SKP1/BTB/POZ domain-containing, Whi2-like human protein KCTD11 but not other KCTD family members tested.

          Author summary

          Yeast and human cells respond to declining levels of available nutrients to prepare ahead for leaner times. The detailed mechanisms of nutrient sensing are not well understood, but defects in these processes have key roles in diseases such as cancer. The evolutionarily conserved protein complex TORC1 is the control hub for responding to both high and low nutrients, particularly amino acids. We identified yeast Whi2 and the human tumor suppressor KCTD11 as novel suppressors of TORC1 activity in low amino acid conditions, and we investigated the detailed mechanisms for Whi2. Unexpectedly, Whi2 works differently from the usual mechanism where TORC1 is controlled by the SEACIT-Gtr complex (mammalian GATOR1-RAG complex). Furthermore, both the Whi2 and the SEACIT-Gtr pathways work independently and together in parallel to suppress TORC1. For this function, Whi2 requires its binding partners, the yeast protein phosphatases Psr1 and Psr2, which were previously thought to function downstream of TORC1 in amino acid signaling. These studies have important implications for human KCTD11 to help advance the understanding of its pathological role.

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          Most cited references 82

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          A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

          A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
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            Global analysis of protein expression in yeast.

            The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.
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              Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

              A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Validation
                Role: Formal analysisRole: Funding acquisitionRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                24 August 2018
                August 2018
                : 14
                : 8
                Affiliations
                [1 ] Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China
                [2 ] W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, United States of America
                [3 ] ISEM, Institut des Sciences de l’Evolution de Montpellier, Université de Montpellier, CNRS, EPHE, IRD, Montpellier, France
                [4 ] Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America
                Sheffield University, UNITED KINGDOM
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PGENETICS-D-17-01893
                10.1371/journal.pgen.1007592
                6126876
                30142151
                © 2018 Chen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 7, Tables: 2, Pages: 28
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31401197
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004608, Natural Science Foundation of Jiangsu Province;
                Award ID: BK20140318
                Award Recipient :
                Funded by: Jiangsu Key Laboratory of Translational Research for Neuropsychiatric Diseases
                Award ID: BM2013003
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: NS083373
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: NS37402
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: GM077875
                Award Recipient :
                This work is supported by the National Natural Science Foundation of China 31401197 (to XT), Natural Science Foundation of Jiangsu Province BK20140318 (to XT), Jiangsu Key Laboratory of Neuropsychiatric Diseases BM2013003 (to XT), National Institutes of Health USA [grant numbers R01NS083373 and R01GM077875 (to JMH)], Fondation ARC N° LS 172351 (to AA), and the Ligue Contre le Cancer Comité du Gard N° LS 176487 (to AA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Yeast
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Hydrolases
                Guanosine Triphosphatase
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Hydrolases
                Guanosine Triphosphatase
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Phosphorylation
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Amino Acid Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Amino Acid Analysis
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Model Organisms
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Yeast
                Saccharomyces
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Experimental Organism Systems
                Yeast and Fungal Models
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblotting
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblotting
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblot Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Immunoblot Analysis
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-09-06
                All relevant data are within the paper and its Supporting Information files.

                Genetics

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