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Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast

2017; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês

10.1074/mcp.m116.066654

ISSN

1535-9484

Autores

Nadine Opitz, Kerstin Schmitt, Verena Hofer-Pretz, Bettina Neumann, Heike Krebber, Gerhard H. Braus, Oliver Valerius,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

The Asc1 protein of Saccharomyces cerevisiae is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent Biotin IDentification (BioID), an in vivo labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1R38D, K40Ep, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (e.g. of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes. The Asc1 protein of Saccharomyces cerevisiae is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent Biotin IDentification (BioID), an in vivo labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1R38D, K40Ep, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (e.g. of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes. As platforms or adaptors for protein-protein interactions, scaffold proteins dynamically organize protein proximities and thereby exert a major impact on cellular signaling (1.Pan C.Q. Sudol M. Sheetz M. Low B.C. Modularity and functional plasticity of scaffold proteins as p(l) acemakers in cell signaling.Cell Signal. 2012; 24: 2143-2165Crossref PubMed Scopus (60) Google Scholar, 2.Vondriska T.M. Pass J.M. Ping P. Scaffold proteins and assembly of multiprotein signaling complexes.J. Mol. Cell Cardiol. 2004; 37: 391-397Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The Gβ-like Asc1 protein from the unicellular budding yeast S. cerevisiae belongs to the WD40 family of scaffold proteins and folds into a seven-bladed β-propeller with a large surface for interactions (3.Chantrel Y. Gaisne M. Lions C. Verdière J. The transcriptional regulator hap1p (cyp1p) is essential for anaerobic or heme-deficient growth of saccharomyces cerevisiae: genetic and molecular characterization of an extragenic suppressor that encodes a wd repeat protein.Genetics. 1998; 148: 559-569PubMed Google Scholar, 4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar, 5.Tarnowski K. Fituch K. Szczepanowski R.H. Dadlez M. Kaus-Drobek M. Patterns of structural dynamics in rack1 protein retained throughout evolution: a hydrogen-deuterium exchange study of three orthologs.Protein Sci. Publ. Protein. Soc. 2014; 23: 639-651Crossref PubMed Scopus (9) Google Scholar). Asc1p is highly conserved throughout the eukaryotic kingdom and shares a high degree of structural similarity with its orthologs, e.g. the human receptor for activated C kinase 1 (RACK1 1The abbreviations used are: RACK1; Receptor for Activated C Kinase 1; BioID, proximity-dependent Biotin Identification; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; SILAC, stable isotope labeling with amino acids in cell culture; stage tips, stop and go extraction tips; YEPD, yeast extract peptone dextrose; YNB, yeast nitrogen base; LFQ, label-free quantification; tSIM, targeted single ion monitoring. 1The abbreviations used are: RACK1; Receptor for Activated C Kinase 1; BioID, proximity-dependent Biotin Identification; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; SILAC, stable isotope labeling with amino acids in cell culture; stage tips, stop and go extraction tips; YEPD, yeast extract peptone dextrose; YNB, yeast nitrogen base; LFQ, label-free quantification; tSIM, targeted single ion monitoring.; 4.Coyle S.M. 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Pedersen L.C. Structure of a signal transduction regulator, rack1, from arabidopsis thaliana.Protein Sci. Publ. Protein. Soc. 2008; 17: 1771-1780Crossref PubMed Scopus (93) Google Scholar). Asc1p is a core constituent of most eukaryotic ribosomes and crystallization of the S. cerevisiae 80S ribosome located Asc1p to the exposed head region of the 40S ribosome in direct proximity to the area of mRNA exit (10.Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 å resolution.Science. 2011; 334: 1524-1529Crossref PubMed Scopus (804) Google Scholar, 11.Inada T. Winstall E. Tarun S.Z. Yates J.R. Schieltz D. Sachs A.B. One-step affinity purification of the yeast ribosome and its associated proteins and mrnas.RNA N Y N. 2002; 8: 948-958Crossref PubMed Scopus (97) Google Scholar, 12.Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2071) Google Scholar). The top surface of Asc1p is oriented to the ribosome interface, where it physically interacts with the ribosomal proteins Rps3p, Rps16p, and Rps17p (7.Rabl J. Leibundgut M. Ataide S.F. Haag A. Ban N. Crystal structure of the eukaryotic 40s ribosomal subunit in complex with initiation factor 1.Science. 2011; 331: 730-736Crossref PubMed Scopus (371) Google Scholar, 10.Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 å resolution.Science. 2011; 334: 1524-1529Crossref PubMed Scopus (804) Google Scholar, 13.Sengupta J. Nilsson J. Gursky R. Spahn C.M.T. Nissen P. Frank J. Identification of the versatile scaffold protein rack1 on the eukaryotic ribosome by cryo-em.Nat. Struct. Mol. Biol. 2004; 11: 957-962Crossref PubMed Scopus (200) Google Scholar). Furthermore, the ribosome facing side of Asc1p contacts helices 39 and 40 of the 18S rRNA with an area of positively charged amino acids (4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar, 7.Rabl J. Leibundgut M. Ataide S.F. Haag A. Ban N. Crystal structure of the eukaryotic 40s ribosomal subunit in complex with initiation factor 1.Science. 2011; 331: 730-736Crossref PubMed Scopus (371) Google Scholar, 10.Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 å resolution.Science. 2011; 334: 1524-1529Crossref PubMed Scopus (804) Google Scholar). The bottom side of Asc1p is not involved in ribosome association and exposed for further protein-protein interactions (10.Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 å resolution.Science. 2011; 334: 1524-1529Crossref PubMed Scopus (804) Google Scholar, 13.Sengupta J. Nilsson J. Gursky R. Spahn C.M.T. Nissen P. Frank J. Identification of the versatile scaffold protein rack1 on the eukaryotic ribosome by cryo-em.Nat. Struct. Mol. Biol. 2004; 11: 957-962Crossref PubMed Scopus (200) Google Scholar). Despite its ribosomal localization yeast Asc1p is not essential for viability and thus dispensable for general translation (3.Chantrel Y. Gaisne M. Lions C. Verdière J. The transcriptional regulator hap1p (cyp1p) is essential for anaerobic or heme-deficient growth of saccharomyces cerevisiae: genetic and molecular characterization of an extragenic suppressor that encodes a wd repeat protein.Genetics. 1998; 148: 559-569PubMed Google Scholar). Still, Asc1p affects the translational efficiency of transcripts in various ways: Asc1p physically interacts with the mRNA-binding protein Scp160p that locates specific mRNAs to polysomes and is required for its efficient ribosome association (4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar, 14.Baum S. Bittins M. Frey S. Seedorf M. Asc1p, a wd40-domain containing adaptor protein, is required for the interaction of the rna-binding protein scp160p with polysomes.Biochem. J. 2004; 380: 823-830Crossref PubMed Scopus (87) Google Scholar, 15.Hirschmann W.D. Westendorf H. Mayer A. Cannarozzi G. Cramer P. Jansen R.-P. Scp160p is required for translational efficiency of codon-optimized mrnas in yeast.Nucleic Acids Res. 2014; 42: 4043-4055Crossref PubMed Scopus (24) Google Scholar). Asc1p and Scp160p are both part of the SESA multiprotein complex (Smy2p, Eap1p, Scp160p, and Asc1p) which controls the translation of POM34 mRNA required for spindle pole body duplication (16.Sezen B. Seedorf M. Schiebel E. The sesa network links duplication of the yeast centrosome with the protein translation machinery.Genes Dev. 2009; 23: 1559-1570Crossref PubMed Scopus (63) Google Scholar). Furthermore, Asc1p influences the translation of specific mRNAs through their 5′UTRs (17.Rachfall N. Schmitt K. Bandau S. Smolinski N. Ehrenreich A. Valerius O. Braus G.H. RACK1/asc1p, a ribosomal node in cellular signaling.Mol. Cell. Proteomics MCP. 2013; 12: 87-105Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Asc1p/RACK1 was described as both, physical interaction partner and mediator for protein phosphorylation of various translation initiation factors (18.Ceci M. Gaviraghi C. Gorrini C. Sala L.A. Offenhäuser N. Marchisio P.C. Biffo S. Release of eif6 (p27bbp) from the 60s subunit allows 80s ribosome assembly.Nature. 2003; 426: 579-584Crossref PubMed Scopus (325) Google Scholar, 19.Gavin A.-C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.-M. Cruciat C.-M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.-A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3993) Google Scholar, 20.Guo J. Wang S. Valerius O. Hall H. Zeng Q. Li J.-F. Weston D.J. Ellis B.E. Chen J.-G. Involvement of arabidopsis rack1 in protein translation and its regulation by abscisic acid.Plant Physiol. 2011; 155: 370-383Crossref PubMed Scopus (93) Google Scholar, 21.Kouba T. Rutkai E. Karásková M. Valášek L.S. The eif3c/nip1 pci domain interacts with rna and rack1/asc1 and promotes assembly of translation preinitiation complexes.Nucleic Acids Res. 2012; 40: 2683-2699Crossref PubMed Scopus (49) Google Scholar, 22.Ruan Y. Sun L. Hao Y. Wang L. Xu J. Zhang W. Xie J. Guo L. Zhou L. Yun X. Zhu H. Shen A. Gu J. Ribosomal rack1 promotes chemoresistance and growth in human hepatocellular carcinoma.J. Clin. Invest. 2012; 122: 2554-2566Crossref PubMed Scopus (104) Google Scholar, 23.Schmitt K. Smolinski N. Neumann P. Schmaul S. Hofer-Pretz V. Braus G.H. Valerius O. Asc1p/rack1 connects ribosomes to eukaryotic phospho-signaling.Mol. Cell. Biol. 2017; 37: e00279Crossref PubMed Scopus (24) Google Scholar, 24.Wang Y. Shen G. Gong J. Shen D. Whittington A. Qing J. Treloar J. Boisvert S. Zhang Z. Yang C. Wang P. Noncanonical gβ gib2 is a scaffolding protein promoting camp signaling through functions of ras1 and cac1 proteins in cryptococcus neoformans.J. Biol. Chem. 2014; 289: 12202-12216Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and Asc1p-depletion accordingly results in a decreased 40S binding affinity of eIF3 (21.Kouba T. Rutkai E. Karásková M. Valášek L.S. The eif3c/nip1 pci domain interacts with rna and rack1/asc1 and promotes assembly of translation preinitiation complexes.Nucleic Acids Res. 2012; 40: 2683-2699Crossref PubMed Scopus (49) Google Scholar). Besides translation, Asc1p/RACK1 has a major impact on signal transduction and interacts with multiple players of signal transduction pathways including components of the cAMP/PKA and different MAPK pathways in S. cerevisiae (25.Breitkreutz A. Choi H. Sharom J.R. Boucher L. Neduva V. Larsen B. Lin Z.-Y. Breitkreutz B.-J. Stark C. Liu G. Ahn J. Dewar-Darch D. Reguly T. Tang X. Almeida R. Qin Z.S. Pawson T. Gingras A.-C. Nesvizhskii A.I. Tyers M. A global protein kinase and phosphatase interaction network in yeast.Science. 2010; 328: 1043-1046Crossref PubMed Scopus (524) Google Scholar, 26.Zeller C.E. Parnell S.C. Dohlman H.G. The rack1 ortholog asc1 functions as a g-protein beta subunit coupled to glucose responsiveness in yeast.J. Biol. Chem. 2007; 282: 25168-25176Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and PKC, Src, and c-Jun kinases in mammals (18.Ceci M. Gaviraghi C. Gorrini C. Sala L.A. Offenhäuser N. Marchisio P.C. Biffo S. Release of eif6 (p27bbp) from the 60s subunit allows 80s ribosome assembly.Nature. 2003; 426: 579-584Crossref PubMed Scopus (325) Google Scholar, 22.Ruan Y. Sun L. Hao Y. Wang L. Xu J. Zhang W. Xie J. Guo L. Zhou L. Yun X. Zhu H. Shen A. Gu J. Ribosomal rack1 promotes chemoresistance and growth in human hepatocellular carcinoma.J. Clin. Invest. 2012; 122: 2554-2566Crossref PubMed Scopus (104) Google Scholar, 27.Chang B.Y. Chiang M. Cartwright C.A. The interaction of src and rack1 is enhanced by activation of protein kinase c and tyrosine phosphorylation of rack1.J. Biol. Chem. 2001; 276: 20346-20356Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 28.Gandin V. Gutierrez G.J. Brill L.M. Varsano T. Feng Y. Aza-Blanc P. Au Q. McLaughlan S. Ferreira T.A. Alain T. Sonenberg N. Topisirovic I. Ronai Z.A. Degradation of newly synthesized polypeptides by ribosome-associated rack1/c-jun n-terminal kinase/eukaryotic elongation factor 1a2 complex.Mol. Cell. Biol. 2013; 33: 2510-2526Crossref PubMed Scopus (41) Google Scholar). A functional requirement for ribosome association of Asc1p in MAPK signaling, however, remains elusive. An artificial asc1R38D, K40E mutant, hereafter referred to as asc1DE, has been constructed to diminish the binding affinity of Asc1DEp to the ribosome and to study possible ribosome-independent functions (4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar). The exchange of the two residues within the rRNA contact region to negatively charged amino acids results in repelling forces between the protein and the rRNA and to a substantial shift of the Asc1DE protein into nonribosomal fractions during sucrose density ultracentrifugation (4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar). However, only a minor impact on Asc1p-dependent phenotypes was observed (4.Coyle S.M. Gilbert W.V. Doudna J.A. Direct link between rack1 function and localization at the ribosome in vivo.Mol. Cell. Biol. 2009; 29: 1626-1634Crossref PubMed Scopus (115) Google Scholar). Just recently, Thompson et al. (29.Thompson M.K. Rojas-Duran M.F. Gangaramani P. Gilbert W.V. The ribosomal protein asc1/rack1 is required for efficient translation of short mrnas.eLife. 2016; 5Crossref Scopus (73) Google Scholar) revealed that the mutant is still ribosome-associated in vivo. The degree of ribosome binding of the Asc1DE protein and its exact position at the ribosome, however, remained uncertain. Proximity-dependent Biotin IDentification, short BioID, was first described by Roux, Burke and coworkers and was developed as an in vivo screen for interacting and proximal proteins of a bait in mammalian cells (30.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar). The emerging in vivo labeling technique uses the E. coli-derived promiscuous biotin protein ligase BirAR118Gp (hereafter referred to as BirA*p), which is fused to a bait protein, and covalently biotinylates interacting and proximal proteins of the bait if biotin is available. Thereafter, cells are lysed under denaturing conditions, and the modified proteins are purified by biotin affinity capture and finally identified by liquid chromatography mass spectrometry (LC-MS). The method was meanwhile adapted for other organisms including Trypanosoma brucei, Toxoplasma gondii, or Dictyostelium amoebae (31.Batsios P. Meyer I. Gräf R. Proximity-dependent biotin identification (bioid) in dictyostelium amoebae.Methods Enzymol. 2016; 569: 23-42Crossref PubMed Scopus (21) Google Scholar, 32.Chen A.L. Kim E.W. Toh J.Y. Vashisht A.A. Rashoff A.Q. Van C. Huang A.S. Moon A.S. Bell H.N. Bentolila L.A. Wohlschlegel J.A. Bradley P.J. Novel components of the toxoplasma inner membrane complex revealed by bioid.mBio. 2015; 6: e02357Crossref PubMed Scopus (111) Google Scholar, 33.Morriswood B. Havlicek K. Demmel L. Yavuz S. Sealey-Cardona M. Vidilaseris K. Anrather D. Kostan J. Djinovic-Carugo K. Roux K.J. Warren G. Novel bilobe components in trypanosoma brucei identified using proximity-dependent biotinylation.Eukaryot. Cell. 2013; 12: 356-367Crossref PubMed Scopus (99) Google Scholar). In this study, we used BioID in combination with stable isotope labeling with amino acids in cell culture (SILAC) to quantitatively monitor the proteinaceous Asc1p-neighborhood in exponentially growing S. cerevisiae cells, but also to record alterations in response to glucose deprivation and heat stress. Furthermore, we analyzed the molecular microenvironment of the "ribosome-binding deficient" Asc1DE protein, which indicates a ribosome-associated population of Asc1DEp in vivo and suggests a distorted position of the variant at the ribosome. In direct proximity to wt-Asc1p, we identified important regulators of translation, including mRNA-binding proteins and initiation factors, as well as transcription-related proteins. Additionally, we observed major alterations in response to glucose withdrawal. In contrast, mild heat stress caused only minor effects on the Asc1p-neighborhood. Altogether, Asc1p seems to connect signal transduction not only with the translational machinery, but also with fundamental nuclear processes. The S. cerevisiae strains used in this work are of the Σ1278b, S288c (BY strains), and W303 background and are listed in Table I. The construction of RH3494 was performed as described in Schmitt et al. (23.Schmitt K. Smolinski N. Neumann P. Schmaul S. Hofer-Pretz V. Braus G.H. Valerius O. Asc1p/rack1 connects ribosomes to eukaryotic phospho-signaling.Mol. Cell. Biol. 2017; 37: e00279Crossref PubMed Scopus (24) Google Scholar) using RH3263 as background strain. Yeast strains were cultivated at 30 °C in yeast extract peptone dextrose medium (YEPD; 1% yeast extract, 2% peptone, 2% glucose) or yeast nitrogen base minimal medium (YNB; 0.15% YNB without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) with the respective supplements or in Synthetic Complete medium (SC, 0.15% YNB without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose, 0.2 mm myo-inositol, 2 g amino acid/nitrogenous base mix). 2% agar was supplied to obtain solid medium. l-arginine (20 mg/l), l-lysine HCl (30 mg/l), l-tryptophan (20 mg/l) and uracil (20 mg/l) were supplemented if required. For differential protein labeling according to the SILAC approach 50 mg/l differentially labeled l-arginine and l-lysine were added to the growth medium (13C6-l-arginine HCl (#201203902), 13C6 15N4-l-arginine HCl (#201603902), 4,4,5,5-D4-l-lysine HCl (#211103912), 13C6-l-lysine HCl (#211203902); Silantes GmbH, München, Germany).Table IS. cerevisiae strains used in this workStrainGenotypeReferenceRH2817MATα, ura3-52, trp1::hisG40.Melamed D. Bar-Ziv L. Truzman Y. Arava Y. Asc1 supports cell-wall integrity near bud sites by a pkc1 independent mechanism.PloS One. 2010; 5: e11389Crossref PubMed Scopus (14) Google ScholarRH3263MATα, ura3-52, trp1::hisG, leu2::hisG, Δasc1::LEU240.Melamed D. Bar-Ziv L. Truzman Y. Arava Y. Asc1 supports cell-wall integrity near bud sites by a pkc1 independent mechanism.PloS One. 2010; 5: e11389Crossref PubMed Scopus (14) Google ScholarRH3493MATα, ura3-52, trp1::hisG, Δarg4::loxP, Δlys1::loxP23.Schmitt K. Smolinski N. Neumann P. Schmaul S. Hofer-Pretz V. Braus G.H. Valerius O. Asc1p/rack1 connects ribosomes to eukaryotic phospho-signaling.Mol. Cell. Biol. 2017; 37: e00279Crossref PubMed Scopus (24) Google ScholarRH3494MATα, ura3-52, trp1::hisG, leu2::hisG, Δasc1::LEU2, Δarg4::loxP, Δlys1::loxPThis workBY4741MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0EuroscarfY03444MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, Δsro9::kanMXEuroscarfY06556MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, Δasc1::kanMXEuroscarfRH3694 RH3695MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, Δsro9::kanMX, Δasc1::URA3This workRH3686MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, SRO9::GFP::HIS3MXInvitrogen, 75.Huh W.-K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3291) Google ScholarRH3687MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, UBP3::GFP::HIS3MXInvitrogen, 75.Huh W.-K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3291) Google ScholarRH3696 RH3697MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, UBP3::GFP::HIS3MX, Δasc1::URA3This workW303-1aW303, MATa, ura3, leu2-3 112, his3-11 15, trp1-1, ade2-1, can1-10034.Wilson M.D. Harreman M. Taschner M. Reid J. Walker J. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Proteasome-mediated processing of def1, a critical step in the cellular response to transcription stress.Cell. 2013; 154: 983-995Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarJSY1194W303, MATa, ura3, leu2-3 112, his3-11 15, trp1-1, ade2-1, can1-100, eGFP-DEF134.Wilson M.D. Harreman M. Taschner M. Reid J. Walker J. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Proteasome-mediated processing of def1, a critical step in the cellular response to transcription stress.Cell. 2013; 154: 983-995Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarRH3698W303, MATa, ura3, leu2-3 112, his3-11 15, trp1-1, ade2-1, can1-100, Δasc1::URA3This workJSY1198W303, MATa, ura3, leu2-3 112, his3-11 15, trp1-1, ade2-1, can1-100, 9xMyc-2xTEV-6xHis-DEF134.Wilson M.D. Harreman M. Taschner M. Reid J. Walker J. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Proteasome-mediated processing of def1, a critical step in the cellular response to transcription stress.Cell. 2013; 154: 983-995Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarRH3699W303, MATa, ura3, leu2-3 112, his3-11 15, trp1-1, ade2-1, can1-100, 9xMyc-2xTEV-6xHis-DEF1, Δasc1::URA3This work Open table in a new tab Plasmids used in this study are listed in Table II. Their construction is described in detail in Supplementary Experimental Procedures. A plasmid encoding an ASC1-birA* fusion gene was generated by linearizing plasmid pME2624 by PCR with a primer pair resulting in the removal of the ASC1 stop codon and the addition of a large overhang containing the linker sequence and a sequence complementary to the birA* gene. The birA* allele containing the point mutation R118G was amplified from plasmid pRS313 and supplied with a sequence complementary to the plasmid backbone by PCR. The linearized plasmid backbone and the birA* fragment were fused by homologous recombination using the In-Fusion® HD Cloning Kit (#639650, Clontech, Mountain View, CA). For the generation of a plasmid expressing the mere birA*, plasmid pME2624 was linearized by PCR with primers resulting in the removal of the ASC1 ORF and the addition of a 20 bp overhang complementary to the birA* gene. The birA* allele was amplified by PCR and fused with the linearized plasmid backbone by homologous recombination. A plasmid encoding an asc1DE-birA* fusion was generated by site directed mutagenesis using pME4478 as template. To insert the R38D and K40E substitutions within the ASC1-birA* allele, the plasmid was fully amplified by PCR with a complementary primer pair carrying the two mutations resulting in the asc1DE-birA* vector. The template DNA was removed by DpnI treatment. Plasmid pME4481 with ASC1 under control of its native promoter was constructed as described by Schmitt et al. (23.Schmitt K. Smolinski N. Neumann P. Schmaul S. Hofer-Pretz V. Braus G.H. Valerius O. Asc1p/rack1 connects ribosomes to eukaryotic phospho-signaling.Mol. Cell. Biol. 2017; 37: e00279Crossref PubMed Scopus (24) Google Scholar) for the same plasmid with a URA3 marker for selection.Table IIPlasmids used in this workPlasmidDescriptionReferencepUG72AmpR, pUCori, loxP::URA3::loxP76.Gueldener U. Heinisch J. Koehler G.J. Voss D. Hegemann J.H. A second set of loxp marker cassettes for cre-mediated multiple gene knockouts in budding yeast.Nucleic Acids Res. 2002; 30: e23Crossref PubMed Scopus (750) Google ScholarpME2787MET25Prom, CYC1Term, URA3, 2 μm77.Mumberg D. Müller R. Funk M. Regulatable promoters of saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression.Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (801) Google Scholar, 78.van Werven F.J. Timmers H.T.M. The use of biotin tagging in saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation.Nucleic Acids Res. 2006; 34: e33Crossref PubMed Scopus (45) Google ScholarpME2624pME2787 with ASC1Our collectionpRS313PGK1Prom, CYC1Term, HIS3, CEN/ARS, birAR118G (based on van Werven and Timmers (78))provided by H. D. SchmittpME4478MET25Prom, CYC1Term, URA3, 2 μm, ASC1-birA*This workpME4479MET25Prom, CYC1Term, URA3, 2 μm, asc1DE-birA*This workpME4480MET25Prom, CYC1Term, URA3, 2 μm, birA*This workpME2781MET25Prom, CYC1Term, TRP1, CEN/ARS77.Mumberg D. Müller R. Funk M. Regulatable promoters of saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression.Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (801) Google ScholarpME4481native promoter, CYC1Ter

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