The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance. The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance. Rapamycin is an immunosuppressive and anti-proliferative antibiotic that targets the TOR 2The abbreviations used are: TOR, target of rapamycin; mTOR, mammalian TOR; SCD, synthetic complete dextrose; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; GEO, NCBI Gene Expression Omnibus; PHASI, phosphorylated heat and acid stable protein regulated by insulin. 2The abbreviations used are: TOR, target of rapamycin; mTOR, mammalian TOR; SCD, synthetic complete dextrose; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; GEO, NCBI Gene Expression Omnibus; PHASI, phosphorylated heat and acid stable protein regulated by insulin. kinase, a member of the phosphatidylinositol 3-kinase-like kinase family of protein kinases (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar). TOR is a large (∼280 kDa) protein and assembles into distinct membrane-associated protein complexes, termed TOR complex 1 (TORC1) and TORC2, in yeast as well as in higher eukaryotes (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar). In yeast, TORC1 contains Tor1p or Tor2p as well as several additional proteins, including Kog1p, Lst8p, and Tco89p (2Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar, 3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). TORC1 mediates a multitude of rapamycin-sensitive activities related to cell growth, including control of translation, gene expression, and protein trafficking and stability (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 2Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar). TORC2 contains Tor2p as well as Lst8p, Avo1p-Avo3p, and Bit61p (2Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar, 3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 4Wedaman K.P. Reinke A. Anderson S. Yates J.I. McCaffery J.M. Powers T. Mol. Biol. Cell. 2003; 14: 1204-1220Crossref PubMed Scopus (195) Google Scholar, 5Wullschleger S. Loewith R. Oppliger W. Hall M.N. J. Biol. Chem. 2005; 280: 30697-30704Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and its activity is required for polarized cell growth and cytoskeletal organization (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 2Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar). TORC2 does not interact with nor is inhibited by rapamycin (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 2Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar). In higher eukaryotes, TORC1 consists of mammalian TOR (mTOR), mLST8/GβL (the ortholog of Lst8p), and Raptor (the ortholog of Kog1p), whereas TORC2 consists of mTOR, mLST8/GβL, and mAVO3/Rictor (the ortholog of Avo3p) (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 6Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1441) Google Scholar, 7Kim D.-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2355) Google Scholar, 8Kim D.-H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V.P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 9Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2148) Google Scholar). Much of what we know about TORC1 has come from the use of rapamycin as a specific small molecule inhibitor of TOR activity (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar). Rapamycin, in conjunction with the highly conserved prolyl-isomerase FKBP, binds to the FRB domain of TOR located immediately N-terminal to its kinase domain (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 10Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar). A single amino acid change at a highly conserved serine residue within the FRB domain is sufficient to prevent binding of the rapamycin-FKBP complex to TOR and confer rapamycin resistance both in vivo and in vitro (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 10Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar, 11Chen J. Zheng X.F. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (446) Google Scholar, 12Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (618) Google Scholar, 13Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1543) Google Scholar, 14Lorenz M.C. Heitman J. J. Biol. Chem. 1995; 270: 27531-27537Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 15Stan R. McLaughlin M.M. Cafferkey R. Johnson R.K. Rosenberg M. Livi G.P. J. Biol. Chem. 1994; 269: 32027-32030Abstract Full Text PDF PubMed Google Scholar, 16Zheng X.-F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (246) Google Scholar). How rapamycin affects TORC1 activity remains unclear, although a number of in vitro studies have shown that the rapamycin-FKBP complex impairs TOR kinase activity (12Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (618) Google Scholar, 17Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (809) Google Scholar, 18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (411) Google Scholar, 19McMahon L.P. Choi K.M. Lin T.A. Abraham R.T. Lawrence Jr., J.C. Mol. Cell. Biol. 2002; 22: 7428-7438Crossref PubMed Scopus (85) Google Scholar, 20Crespo J.L. Hall M.N. Microbiol. Mol. Biol. Rev. 2003; 66: 579-591Crossref Scopus (282) Google Scholar, 21Alarcon C.M. Heitman J. Cardenas M.E. Mol. Biol. Cell. 1999; 10: 2531-2546Crossref PubMed Scopus (65) Google Scholar). Alternatively, it has been proposed that rapamycin prevents binding of a regulatory partner, for example Raptor, to mTOR that is essential for its activity (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar, 7Kim D.-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2355) Google Scholar, 22Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 23Yonezawa K. Tokunaga C. Oshiro N. Yoshino K. Biochem. Biophys. Res. Commun. 2004; 313: 437-441Crossref PubMed Scopus (69) Google Scholar, 24Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Interestingly, rapamycin-resistant mutant forms of mTOR have been shown to possess reduced kinase activity, prompting the suggestion that the FRB domain may somehow itself be involved in substrate recognition (19McMahon L.P. Choi K.M. Lin T.A. Abraham R.T. Lawrence Jr., J.C. Mol. Cell. Biol. 2002; 22: 7428-7438Crossref PubMed Scopus (85) Google Scholar). In addition to rapamycin, a number of other pharmacological agents have been shown to affect TOR, particularly in mammalian cells, including members of the methylxanthine family of compounds such as caffeine (25Scott P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; 273: 34496-34501Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar, 27McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (47) Google Scholar). Caffeine affects a diverse array of cellular processes related to cell growth, DNA metabolism, and cell cycle progression, most likely by acting as a low affinity ATP analog (28Kaufmann W.K. Heffernan T.P. Beaulieu L.M. Doherty S. Frank A.R. Zhou Y. Bryant M.F. Zhou T. Luche D.D. Nikolaishvili-Feinberg N. Simpson D.A. Cordeiro-Stone M. Mutat. Res. 2003; 532: 85-102Crossref PubMed Scopus (81) Google Scholar, 29Cortez D. J. Biol. Chem. 2003; 278: 37139-37145Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Both caffeine and the related compound theophylline have been shown to inhibit phosphorylation of mTOR-dependent substrates both in vitro as well as in vivo (25Scott P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; 273: 34496-34501Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar, 27McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (47) Google Scholar). Caffeine has not been used widely as a tool for probing mTOR function, however, potentially due to the pleiotropic behavior of this compound as well as the fact that it interacts with mTOR with relatively low affinity, i.e. in the submillimolar range (25Scott P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; 273: 34496-34501Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar, 27McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (47) Google Scholar). In Saccharomyces cerevisiae, increased sensitivity to caffeine has been correlated with defects in the “cell integrity pathway,” whereby cell well and/or plasma membrane stability is monitored in response to osmotic or thermal stress (30Levin D.E. Microbiol. Mol. Biol. Rev. 2005; 69: 262-291Crossref PubMed Scopus (882) Google Scholar, 31Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Several signaling pathways have been implicated in cell integrity maintenance, however, the relevant targets for caffeine have not yet been identified (30Levin D.E. Microbiol. Mol. Biol. Rev. 2005; 69: 262-291Crossref PubMed Scopus (882) Google Scholar, 31Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). In this regard, we as well as others have reported that mutation of distinct components of TORC1 lead to defects in cellular integrity, including increased sensitivity to caffeine (3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 32Torres J. Di Como C.J. Herrero E. Angeles de la Torre-Ruiz M. J. Biol. Chem. 2002; 277: 43495-43502Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Independently, a large scale screen of yeast deletion mutants for altered drug sensitivities also identified TORC1 mutants as displaying increased caffeine sensitivity (33Lum P.Y. Armour C.D. Stepaniants S.B. Cavet G. Wolf M.K. Butler J.S. Hinshaw J.C. Garnier P. Prestwich G.D. Leonardson A. Garrett-Engele P. Rush C.M. Bard M. Schimmack G. Phillips J.W. Roberts C.J. Shoemaker D.D. Cell. 2004; 116: 121-137Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Here we have pursued these observations and present evidence that TORC1 is indeed a significant target for caffeine in yeast. As part of these studies, we have identified mutations within the FRB and kinase domains of Tor1p that together confer significant levels of caffeine resistance. Characterization of these mutants provides independent evidence that the FRB domain is an important regulator of TOR kinase function. Strains, Media, and General Methods—Strains and plasmids used in this study are listed in Tables 1 and 2, respectively. Cells were cultured in YPD (2% yeast extract, 1% peptone, and 2% dextrose) or synthetic complete dextrose (SCD) medium (0.8% yeast nitrogen base without amino acids, pH 5.5, 2% dextrose) supplemented with amino acids as described (34Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Rapamycin (Sigma) was dissolved in Me2SO, and caffeine (Sigma) was dissolved in H2O; both compounds were added to liquid cultures and agar plates at the final concentrations indicated in the text and figure legends. Yeast transformations were performed using a lithium acetate procedure (35Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2069) Google Scholar).TABLE 1Strains of S. cerevisiae used for this studyStrainDescriptionReference/sourceW303aMATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL+50W303αMATα ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL+50S288cMatα gal2 malBotstein laboratoryMY1384σ;2000 series, MataMicrobiaPLY254W303a, except tor1::His3MX63PLY332W303a, except tco89::His3MX63JK9-3daMata leu2-3,112 ura3-52 rmel trp1 his4 can1-100 GAL+ HMLa133H11-1cJK9-3da, except TOR1-113JH12-17bJK9-3da, except TOR2-113PLY297JK9-3da, except tor1::His3MX63PLY344JK9-3da, except tco89::His3MX63PLY365W303a/α, except tor1::TRP1 tco89:: His3MX6 [pNB100]3PLY366W303a/α, except TOR1/tor1:: His3MX63PLY369W303a/α, except TCO89/tco89::His3MX6This studyPLY475W303a/α, except AVO1/avo1::His3MX6This studyPLY477W303a/α, except AVO3/avo3::His3MX6This studyPLY479W303a/α, except KOG1/kog1::His3MX6This studyPLY315W303a/α, except LST8/lst8::His3MX6This studyPLY314W303a/α, except TOR2/tor2::His3MX6This studyPLY671W303a, except KOG1-MYC:TRP1This studyPLY673W303a, except LST8-MYC:TRP1This studyPLY675W303a, except TCO89-MYC:TRP1This study Open table in a new tab TABLE 2Plasmids used this studyPlasmidDescriptionReference/sourcepNB100TOR2 in Ycplac33 (URA3)51pJK4TOR2 in Ycplac111 (LEU2)51pRS315LEU2, CEN/ARS52pYDF23LEU2, CEN/ARS TOR1-116pPL132LEU2, CEN/ARS HA3-TOR1This studypPL155pPL132, HA3-TOR1 A1957V mutationThis studypPL156pPL132, HA3-TOR1 I1954V mutationThis studypPL157pPL132, HA3-TOR1 W2176R mutationThis studypPL158pPL132, HA3-TOR1 I1954V/W2176R mutationsThis studypPL159pPL132, HA3-TOR1 A1957V/W2176R mutationsThis studypPL164pPL132, HA3-TOR1 1a1 mutant from PCR mutagenesisThis studypPL165pPL132, HA3-TOR1 1a7 mutant from PCR mutagenesisThis study Open table in a new tab Construction of Yeast Strains—All gene deletions strains were constructed by replacing an entire open reading frame with a selectable maker following transformation of a linear fragment of DNA constructed by PCR. Specifically, we used His3MX6 as a selectable marker from plasmid pFA6a-His3MX6 (36Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4160) Google Scholar) and used forward and reverse primers that contained 50 bp corresponding to the 5′- or 3′-ends of the target open reading frame, followed by 20 bp corresponding to the 5′- or 3′-ends of the marker gene. All disruptions were confirmed by PCR using different primers that could detect specific integration events for each linear DNA fragment. Kog1p, Tco89p, and Lst8p were each tagged at their C termini with multiple copies of the Myc epitope using a PCR-based gene-tagging method as described previously (4Wedaman K.P. Reinke A. Anderson S. Yates J.I. McCaffery J.M. Powers T. Mol. Biol. Cell. 2003; 14: 1204-1220Crossref PubMed Scopus (195) Google Scholar). Plasmid Construction—Plasmid pPL132 was constructed in multiple steps. The starting plasmid was pYDF23, which carries the TOR1-1 allele under control of its native promoter (16Zheng X.-F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (246) Google Scholar). We first introduced sequences corresponding to three copies of the HA epitope (HA3) at the N terminus of the coding region of TOR1-1. This was accomplished by PCR amplification using genomic DNA from strain PLY298 (3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) that expresses HA3-TOR1 to generate a linear fragment of DNA that encodes the promoter region of TOR1 followed by the N terminus of TOR1 fused to HA3. This fragment was used in a co-transformation along with gapped pYDF23 to generate an intact HA3-tagged TOR1-1 gene. Next, the FRB region of this plasmid was removed by digestion with BglII and NdeI followed by transformation of strain JK9-3da to recover the wild-type FRB sequences by gap repair. The success of each step was verified by restriction digestion, PCR, and DNA sequence analysis. The functionality of pPL132 was demonstrated by its ability to rescue cell integrity defects of strain PLY254 carrying a tor1 deletion in the W303a background (3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Mutagenesis of pPL132—Hydroxylamine mutagenesis of pPL132 was carried out essentially as described (37Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (493) Google Scholar). Briefly, 10 μg of plasmid was incubated at 75 °C in 1 m hydroxylamine for ∼15 min, followed by transformation of W303a cells. Cells were allowed to recover for 6 h in SCD minus leucine media and were then plated on SCD minus leucine agar plates containing 9 mm caffeine and then incubated for several days at 30 °C. Control transformations onto plates lacking caffeine demonstrated that ∼3 × 105 cells were transformed using this procedure. Plasmids were recovered from several colonies that appeared on caffeine-containing plates and were used to transform fresh W303a cells, followed by re-plating on 9 mm caffeine plates. Three plasmids that passed this criteria where subjected to a combination of Gap-repair and fragment exchange reactions, where in each case the caffeine-resistant phenotype was localized to an NcoI-BsiWI fragment that spanned the FRB and kinase domains of TOR1. This region was sequenced, and all three plasmids were found to possess nucleotide changes predicted to create an A1957V mutation in TOR1. Error-prone PCR mutagenesis of pPL132 was carried out essentially as described (38Collins C.A. Guthrie C. Genes Dev. 1999; 13: 1970-1982Crossref PubMed Scopus (103) Google Scholar) with several modifications. A 1583-bp fragment spanning the NcoI and BsiWI sites in TOR1 was amplified using pPL132 DNA as template. Mutagenic PCR conditions used 5 mm MnCl2, 2 mm each dCTP, dGTP, and dTTP, and 1 mm dATP. A 1383-bp fragment was excised from pPL132 by digestion with NcoI and BsiWI, and the resulting linearized plasmid vector was mixed with the PCR product in a 1:30 ratio and used to co-transform W303a cells. Following recovery in SCD minus leucine media, cells were plated directly onto SCD minus leucine agar plates containing 9 mm caffeine. Control transformations onto plates lacking caffeine demonstrated that ∼3.5 × 105 cells were transformed using this procedure. Following several days of growth at 30 °C, plasmids were rescued from colonies that appeared on caffeine-containing plates, re-tested by transformation of fresh W303a cells, and then ultimately sequenced. Site-directed mutagenesis was carried out using a QuikChange XL II kit (Stratagene) and pPL132 DNA as a template, following instructions provided by the manufacturer. Gene Expression Studies—Northern blot analysis was performed as described (39Powers T. Walter P. Mol. Biol. Cell. 1999; 10: 987-1000Crossref PubMed Scopus (324) Google Scholar). A 10-ml culture was grown to mid log phase (A600 = 0.5) and harvested by centrifugation, and total RNA was extracted by performing a hot phenol method. 20 μg of total RNA was loaded onto a 1.5% agarose, 6.9% formaldehyde gel and run in 1× E buffer (20 mm MOPS, pH 7.0, 5 mm sodium acetate, 0.5 mm EDTA). The RNA was transferred overnight onto a Duralon-UV membrane (Stratagene, La Jolla, CA) and probed overnight at 65 °C in Church hybridization buffer (0.5 m NaPO4, pH 7.2, 7% SDS, 1 mm EDTA). Membranes were washed in 2× sodium saline citrate (1 × SSC = 0.15 m NaCl and 0.015 m sodium citrate) buffer, exposed to a phosphorimaging screen, and analyzed using a Storm 860 imaging system and software provided by the manufacturer (Amersham Biosciences). DNA templates for the Northern probes were made as described previously (39Powers T. Walter P. Mol. Biol. Cell. 1999; 10: 987-1000Crossref PubMed Scopus (324) Google Scholar). Relative levels of specified mRNAs were normalized to actin (ACT1) mRNA. For gene expression analysis using DNA microarrays, 250-ml cultures were harvested for each sample, poly(A) mRNA was isolated, and fluorescently labeled cDNAs were prepared essentially as described (40Chen J.C. Powers T. Curr. Genet. 2006; 49: 1-13Crossref PubMed Scopus (38) Google Scholar). Arrays were scanned using a GenePix 4000a microarray scanner (Axon Instruments, Inc.) and analyzed using GenePix Pro 3.0 provided by the manufacturer. Data were uploaded to an AMAD microarray data base using a Linux operating system, and analyzed using software Cluster 3.0 and Java Treeview (all publicly available at derisilab.ucsf.edu/microarray/index.html). All microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo) and are available through GEO Series accession numbers GSE4584 and GSE4586. Immune Complex Kinase Assay and Co-immunoprecipitations—The protocol for kinase assays using PHAS-I as a substrate was developed based on previous reports (5Wullschleger S. Loewith R. Oppliger W. Hall M.N. J. Biol. Chem. 2005; 280: 30697-30704Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19McMahon L.P. Choi K.M. Lin T.A. Abraham R.T. Lawrence Jr., J.C. Mol. Cell. Biol. 2002; 22: 7428-7438Crossref PubMed Scopus (85) Google Scholar, 21Alarcon C.M. Heitman J. Cardenas M.E. Mol. Biol. Cell. 1999; 10: 2531-2546Crossref PubMed Scopus (65) Google Scholar) with several modifications based on our immunoprecipitation conditions for TORC1 (3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Two liters of each strain was grown in SCD minus leucine media to mid log phase (0.5 A600/ml) at 30 °C. Cells were washed with sterile H2O, pelleted again, and resuspended in yeast extract buffer (YEB, 50 mm HEPES-KOH, pH 7.1, 100 mm β-glycerol phosphate, 50 mm NaF, 5 mm EGTA, 5 mm EDTA, 10% glycerol, 0.25% Tween 20, and 150 mm KCl). Cells were again pelleted, resuspended in 2 ml of YEB containing protease inhibitors (mixture tablet, Roche Applied Science), 2 mm dithiothreitol, and 2 mm phenylmethylsulfonyl fluoride and frozen dropwise by transfer pipette into liquid nitrogen. The cell pellet was then transferred to a pre-chilled mortar and pestle containing liquid nitrogen and ground into a fine powder (∼150 strokes followed by the addition of liquid nitrogen, repeated three times). After the liquid nitrogen boiled off, the powder that remained was transferred to 1.5-ml microcentrifuge tubes and thawed. All of the following steps were performed at 4 °C unless stated otherwise. Extracts were then centrifuged twice at 20,000 × g for 20 min, transferring the supernatant to a new tube each time. 500 μl of each extract was then transferred into new tubes and pre-cleared by binding to 25 μl of protein A-coupled beads (Amersham Biosciences) for 45 min. 5 μl of affinity-purified anti-HA polyclonal antibody (3Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. J. Biol. Chem. 2004; 279: 14752-14762Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and 25 μl of fresh protein A-coupled beads were incubated together at room temperature for 1 h and were added to each tube of cleared extract. Extract-bead mixtures were then incubated for 3 h at 4 °C with end-over-end rotation. Following this incubation, beads were washed five times with 1 ml of YEB. To each tube of beads, 56 μl of kinase buffer (YEB containing protease inhibitors (mixture tablet, Roche Applied Science)), 2 mm dithiothreitol, 4 mm MnCl2, and 1.5 μg of PHAS-I (Stratagene) substrate was added. Reactions were started by adding 4 μl of ATP mix (1.5 mm ATP, 2.5 μCi/μl[γ-32P]ATP, 3000 Ci/mmol, Amersham Biosciences). Where indicated, caffeine was added at final concentrations listed in the text and figure legends prior