Human Protein Phosphatase PP6 Regulatory Subunits Provide Sit4-Dependent and Rapamycin–Sensitive Sap Function in Saccharomyces cerevisiae
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{"title"=>"Human protein phosphatase PP6 regulatory subunits provide Sit4-dependent and rapamycin-sensitive sap function in Saccharomyces cerevisiae", "type"=>"journal", "authors"=>[{"first_name"=>"Helena", "last_name"=>"Morales-Johansson", "scopus_author_id"=>"6506203937"}, {"first_name"=>"Rekha", "last_name"=>"Puria", "scopus_author_id"=>"20735302500"}, {"first_name"=>"David L.", "last_name"=>"Brautigan", "scopus_author_id"=>"7006373533"}, {"first_name"=>"Maria E.", "last_name"=>"Cardenas", "scopus_author_id"=>"7101944474"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"354991745", "doi"=>"10.1371/journal.pone.0006331", "sgr"=>"67651246898", "scopus"=>"2-s2.0-67651246898", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"19621075"}, "id"=>"164d0f7a-d86b-3fe6-a831-37cc983e6043", "abstract"=>"In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G(1) to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G(1) gene expression and DNA synthesis, and partially abrogate the G(1) cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2alpha phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans.", "link"=>"http://www.mendeley.com/research/human-protein-phosphatase-pp6-regulatory-subunits-provide-sit4dependent-rapamycinsensitive-sap-funct", "reader_count"=>17, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>4, "Student > Master"=>1, "Student > Bachelor"=>1, "Professor"=>1, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>4, "Student > Master"=>1, "Student > Bachelor"=>1, "Professor"=>1, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>12, "Medicine and Dentistry"=>1, "Unspecified"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>12}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"China"=>1, "United Kingdom"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/890043"], "description"=>"<p>The protein sequences of the SAPS domain of <i>Saccharomyces cerevisiae</i> Saps and <i>Homo sapiens</i> PP6Rs were aligned using Jalview. Amino acid conservation is highlighted with grey boxes where the darkest grey indicates the highest level of similarity. Dashed line denotes non-homologous region.</p>", "links"=>[], "tags"=>["pp6r", "yeast", "saps", "proteins", "marked", "homology"], "article_id"=>560489, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_human_PP6R_and_the_yeast_Saps_proteins_share_marked_homology_within_the_SAPS_domain_/560489", "title"=>"The human PP6R and the yeast Saps proteins share marked homology within the SAPS domain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:12:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/890611"], "description"=>"<p>(A) Exponentially growing cultures of the quadruple <i>sap</i> mutant strain (JRY40) transformed with vector or its derivatives expressing <i>PP6R2</i> and <i>PP6R3</i> were treated with rapamycin over a 2 hr period. RNA was isolated at the indicated time points <i>GLN1</i>, <i>MEP2</i>, <i>GAP1</i>and <i>ACT1</i> mRNA expression levels were assayed by northern blot. (B) Northern blot signals from Panel A were quantified and normalized to the <i>ACT1</i> loading control signal. Results shown are the relative percentage of gene expression with maximal level of expression at 15 min as 100%. (C) PP6R proteins fail to restore normal levels of eIF2α dephosphorylation to the quadruple <i>sap</i> mutant strain (JRY40). Exponentially growing cultures of isogenic WT (MLY41) strain transformed with vector, and the quadruple <i>sap</i> mutant (JRY40) strain transformed with vector and derivatives expressing <i>PP6R1</i>, <i>PP6R2</i>, <i>PP6R3</i> and <i>SAP185</i> were treated with 100 nM rapamycin for 0 or 20 min. Whole-cell extracts were prepared and analyzed by sequencial western blotting with antibodies specific for the phosphorylated and non-phosphorylated form of eIF2α.</p>", "links"=>[], "tags"=>["pp6r3", "levels", "ncr", "cells", "lacking"], "article_id"=>561064, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g006", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R2_and_PP6R3_fail_to_restore_normal_levels_of_NCR_response_in_cells_lacking_all_SAP_genes_/561064", "title"=>"PP6R2 and PP6R3 fail to restore normal levels of NCR response in cells lacking all <i>SAP</i> genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:16:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/890319"], "description"=>"<p>Isogenic WT (MLY41) and <i>sit4</i> (SCY94) strains transformed with vector, and quadruple <i>sap</i> (JRY40) mutant strain transformed with vector and plasmids expressing <i>PP6R1</i>, <i>PP6R2</i> and <i>PP6R3</i> were grown to exponential phase and prepared for flow cytometry analysis. The plots show cell count (vertical axis) versus fluorescence intensity, which is proportional to DNA content (horizontal axis). The percentage of cells with 2N DNA content is shown in the upper right corner of each panel. (B) Table shows the percentage of budded cells, which was determined in cell cultures used for the flow cytometry. Results shown are representative of three independent experiments.</p>", "links"=>[], "tags"=>["proteins", "budding", "defects", "quadruple", "mutant"], "article_id"=>560774, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g004", "stats"=>{"downloads"=>1, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R_proteins_partially_overcome_the_G_1_delay_and_budding_defects_of_quadruple_sap_mutant_cells_/560774", "title"=>"PP6R proteins partially overcome the G<sub>1</sub> delay and budding defects of quadruple <i>sap</i> mutant cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:14:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/890441"], "description"=>"<p>Isogenic WT (MLY41) and quadruple <i>sap</i> (JRY40) mutant strains transformed with vector or plasmid expressing <i>PP6R3</i> were grown to exponential phase and elutriated to enrich for G<sub>1</sub> cells. Elutriated cells were collected, transferred to fresh SD-Ura media, and samples were taken over a 3 hours period for G<sub>1</sub> functions analysis. (A) Samples were analyzed for DNA content by flow cytometry as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006331#pone-0006331-g004\" target=\"_blank\">Figure 4A</a>. The percentage of cells with 2N DNA content is shown in the upper right corner of each panel). (B) Table shows the percentage of budded cells (ND, not detectable). (C) <i>CLN1</i> and <i>ACT1</i> mRNA expression was assessed by northern blot. (D) Northern blot signals from experiment shown in Panel C for <i>CLN1</i> were quantified and normalized to the <i>ACT1</i> loading control signal. Results shown are the relative percentage of gene expression with maximal level of expression at 30 min for WT as 100%.</p>", "links"=>[], "tags"=>["enhances"], "article_id"=>560898, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g005", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R3_partially_enhances_CLN1_expression_and_cell_cycle_progression_/560898", "title"=>"PP6R3 partially enhances <i>CLN1</i> expression and cell cycle progression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:15:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/890733"], "description"=>"<p>Isogenic WT (MLY41) transformed with vector, the quadruple <i>sap</i> mutant (JRY40), and only <i>SAP4</i> (<i>sap155 sap185 sap190</i>; JRY45) strains transformed with vector or constructs expressing <i>PP6R1</i>, <i>PP6R2</i>, <i>PP6R3</i>, or <i>SAP185</i> were grown overnight in SD-Ura medium. Equivalent numbers of cells were serially diluted, and aliquots were spotted onto plates of YPD and YPD containing zymocin. After 3 days of incubation at 30°C, the plates were photographed.</p>", "links"=>[], "tags"=>["proteins", "zymocin", "quadruple", "mutant"], "article_id"=>561186, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g007", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R_proteins_partially_restore_zymocin_sensitivity_to_the_quadruple_sap_mutant_strain_/561186", "title"=>"PP6R proteins partially restore zymocin sensitivity to the quadruple <i>sap</i> mutant strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:16:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/890143"], "description"=>"<p>The quadruple <i>sap</i> mutant (JRY40) and <i>sap185 sap190</i> double mutant (JRY29) strains were transformed with vector and derivatives expressing N-terminal FLAG tagged versions of <i>PP6R1</i>, <i>PP6R2</i> and <i>PP6R3</i>. Proteins were immunoprecipitated with anti-FLAG antibody and analyzed by western blotting. The membrane was stripped and interaction with Sit4 was detected by reprobing with a Sit4 specific antiserum. Sit4 was also detected in equivalent amounts of protein extracts and Pgk1 served as a loading control.</p>", "links"=>[], "tags"=>["proteins", "pp2a-like", "phosphatase", "catalytic", "subunit"], "article_id"=>560595, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R_proteins_associate_with_the_PP2A_like_phosphatase_catalytic_subunit_Sit4_/560595", "title"=>"PP6R proteins associate with the PP2A-like phosphatase catalytic subunit Sit4.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:13:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/890827"], "description"=>"<p>Yeast strains.</p>", "links"=>[], "tags"=>["microbiology", "biochemistry/cell signaling and trafficking structures", "biochemistry/drug discovery", "genetics and genomics/functional genomics"], "article_id"=>561286, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.t001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Yeast_strains_/561286", "title"=>"Yeast strains.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-21 05:17:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/890211"], "description"=>"<p>Isogenic WT (MLY41) strain transformed with vector, quadruple <i>sap</i> (JRY40), <i>sit4</i> (SCY94) and <i>sap185 sap190</i> (JRY29) mutant strains transformed with vector and plasmids expressing <i>SAP185</i>, <i>SAP155</i>, <i>PP6R1</i>, <i>PP6R2</i>, and <i>PP6R3</i> were grown overnight in SD-Ura medium. Equivalent numbers of cells were serially diluted, and aliquots were spotted onto SD-Ura plates containing drug vehicle or 50 nM and 100 nM rapamycin. After 3 days of incubation at 30°C, the plates were photographed.</p>", "links"=>[], "tags"=>["pp6r3", "defect", "rapamycin", "hypersensitivity", "yeast", "cells", "lacking"], "article_id"=>560658, "categories"=>["Microbiology", "Biochemistry", "Genetics"], "users"=>["Helena Morales-Johansson", "Rekha Puria", "David L. Brautigan", "Maria E. Cardenas"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006331.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PP6R2_and_PP6R3_rescue_the_growth_defect_and_rapamycin_hypersensitivity_of_yeast_cells_lacking_all_SAP_s_/560658", "title"=>"PP6R2 and PP6R3 rescue the growth defect and rapamycin hypersensitivity of yeast cells lacking all <i>SAP</i>s.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:13:53"}

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  • {"unique-ip"=>"2", "full-text"=>"1", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"2"}
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  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"2", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
  • {"unique-ip"=>"4", "full-text"=>"4", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"4", "full-text"=>"2", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2017", "month"=>"1"}
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  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
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  • {"unique-ip"=>"3", "full-text"=>"2", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
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  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
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  • {"unique-ip"=>"8", "full-text"=>"8", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"6", "full-text"=>"7", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
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  • {"unique-ip"=>"8", "full-text"=>"6", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"9", "full-text"=>"6", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"13", "full-text"=>"14", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"8", "full-text"=>"6", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
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Relative Metric

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