Cell, Vol. 73, 585-596, May 7, 1993, Copyright 0 1993 by Cell Press

Target of Rapamycin in Yeast, TOR2, Is an Essential Phosphatidylinositol K inase Homolog Required for G1 Progression Jeannette Kuru, l Ruben Henriquez,‘t Ulrich Schneider,* Maja Deuter-Reinhard,’ N. Rao Movva,S and Michael N. Hall* *Department of Biochemistry Biocenter University of Base1 CH-4056 Base1 Switzerland tPreclinical Research Sandoz Pharma Limited CH-4002 Base1 Switzerland

Summary

The yeast TOP2 gene encodes an essential 282 kd phos- phatidylinositol (PI) Skinase homolog. TOP2 is related to the catalytic subunit of bovine PI Skinase and to yeast VPS34, a vacuolar sorting protein also shown to have PI Skinase activity. The immunosuppressant rapamycin most likely acts by inhibiting PI kinase activity because TOP2 mutations confer resistance to rapamycin and because a TO/?7 TOf?2 double disruption (TOR7 is a nonessential TOP2 homolog) confers G, arrest, as does rapamycin. Our results further suggest that 3-phosphor- ylated phosphoinosftides, whose physiological signifi- cance has not been determined, are an important signal in cell cycle activation. In yeast, this signal may act in a signal transduction pathway similar to the interleukin-2 signal transduction pathway in T cells.

Introduction

The cell division cycle is controlled primarily during the G, interval; once a ceil has progressed through G, into S, the decision has been made to divide and to ignore alternative choices. The decision to divide is controlled, depending on the ceil type, by nutrient availability, cell size, growth factors, mating pheromones, or immunosup- pressive drugs (reviewed in Pringle and Hartwell, 1981; Forsburg and Nurse, 1991; Reed, 1992; Norbury and Nurse, 1992; Schreiber, 1992). The critical point in G, at which the GJS transition is regulated and at which a cell is committed to a cell cycle has been termed START (Hart- well et al., 1974; Reed, 1980). in the budding yeast Sac- charomyces cerevisiae, START functions include the CDC28 kinase and the G1-specific cyclins CLNl, CLNP, and CLN3. The redundant cyclins, expressed only in G,, associate with and confer G, specificity on the constitu- tively expressed CDC28 kinase (Wittenberg et al., 1990). The input either to arrest at START or to proceed through G1 is received by the cyclins. Mutant strains lacking the G, cyclins or CDC28 arrest at START and strains producing

tPresent address: Preclinical Research, Sandoz Pharma Limited, CH-4002 Easel, Switzerland.

overactive cyclin enter S phase prematurely (Carter and Sudbery, 1980; Cross, 1988, 1990; Hadwiger et al., 1989; Nash et al., 1988; Richardson et al., 1989). The nature of the sensing pathway(s) or the signal(s) itself that may link the cyclins to the conditions or factors listed above that control progression through G, is not known. Components in such a signal transduction pathway may include FARl, FUS3, SWl4, and SW16. FAR1 and FUS3 inhibitthecyclins in the presence of mating pheromone (reviewed in Na- smyth, 1990). FAR1 is an inhibitor of CLN2, possibly as a transcriptional repressor of the CLN2 gene (Chang and Herskowitz, 1990). FUS3, aCDC28-related protein kinase, inhibits CLNl and CLNP transcriptionally and CLN3 post- transcriptionally (Elion et al., 1990,199l). SW14 and SW16 activate CLNl and CLN2 transcriptionally and CLN3 post- transcriptionally (Nasmyth and Dirick, 1991; Ogas et al., 1991; Dirick et al., 1992). The yeast RAS-CAMP pathway appears to regulate the cell cycle in response to nutrient availability. However, it has not yet been resolved whether this regulation is direct or indirect (reviewed in Broach, 1991; Thevelein, 1992).

The immunosuppressive drugs cyclosporin A (CsA), FK- 506, and rapamycin inhibit intermediate steps in signal transduction leading to cell cycle activation (reviewed in Schreiber, 1991; Heitman et al., 1992; Schreiber and Crab- tree, 1992; Sigal and Dumont, 1992). CsA and FK506 block the activation of quiescent Tcells (Go to G, transition) by interfering with the T cell receptor-mediated signal transduction pathway. Rapamycin inhibits subsequent G, to S progression by blocking response to the lymphokine interleukin-2 (IL-2). The action of these immunosuppres- sive drugs is dependent on the formation of a complex with intracellular receptors termed immunophil ins. CsA binds to cyclophilin; the related drugs FK506 and rapa- mycin bind to the FK506-binding protein (FKBP). The members of the structurally distinct cyclophilin and FKBP families are peptidyl-prolyl cis-trans isomerases that may be involved in protein folding (Fischer and Schmid, 1990). Drug binding inhibits proline isomerase activity, but is not sufficient for immunosuppression. Instead, recent evi- dencesuggests that an immunophil in-drug complex gains a new function that poisons signal transduction by acting on specific target proteins. The structurally unrelated, but functionally similar, cyclophilin-CsA and FKBP-FK506 complexes both bind and inhibit calcineurin, a Ca%almod- ulindependent serine/threonine phosphatase required for T cell activation (Liu et al., 1991a, 1992; Clipstone and Crabtree, 1992; Fruman et al., 1992; C’Keefe et al., 1992; Swanson et al., 1992). Binding of FKBP or cyclophilin to calcineurin is not detected in the absence of drug (Liu et al., 1991a). Calcineurin activity seems to be necessary for nuclear entry of a cytoplasmic subunit of NF-AT, a transcription factor required for lymphokine gene expres- sion (Flanagan et al., 1991). Thus, the immunophil in-drug complexes appear to block T cell activation by inhibiting nuclear import of an NF-AT subunit. The molecular target of FKBP-rapamycin has not been defined, but the com-

plex selectively inhibits IL-2-stimulated activation of p70 S6kinase(Calvoetal.,1992;Chungetal.,1992;Kuoetal., 1992; Price et al., 1992). The FKBP-rapamycin complex does not bind directly to p70 S6 kinase, but rather acts on an upstream component that controls activation of the p70 S6 kinase.

Rapamycin also arrests S. cerevisiae in the G, phase of the cell cycle by binding to a highly conserved FKBP (FKBP12, encoded by the FPR7 gene) (Heitman et al., 1991 a). Yeast strains lacking FKBPI 2 are viable and com- pletely resistant to rapamycin; this and other findings indi- cate that an FKBPlP-rapamycin complex mediates toxic- ity (Heitman et al., 1991a, 1991b; Koltin et al., 1991; Wiederrecht et al., 1991). The products of two novel genes, TOR7 and TOR2, are also involved in drug action; mutation of either gene confers rapamycin resistance (Heitman et al., 1991a). Nonallelic noncomplementation among FPRI, TO/V, and TOR2 alleles suggests that the gene products either interact physically, possibly as subunits of a protein complex, or are members of a common pathway.

Several species of phosphatidylinositol (PI) kinase exist (reviewed in Berridge and Irvine, 1969; Carpenter and Cant- ley, 1990; Majerus, 1992). PI 4-kinase and PI-4-phosphate (PI+P) 5-kinase produce PI-4,5bisphosphate (PIP& which is cleaved by phospholipase C to generate the two canonical second messengers inositol-1,4,!5trisphos- phate and diacylglycerol. The status of the PI kinases and their products has recently become considerably more complex with the discovery of a novel PI kinase, PI 3-kinase (Whitman et al., 1966). PI 3-kinase phosphory- lates phosphoinositides at the 3 position of the inositol ring. The physiological significance of 3-phosphorylated phosphoinositides (PI-3-P, PI-3,4-P*, and PI-3,4,5-Pa) is not known. Strong circumstantial evidence suggests that at least one of them acts directly as a growth (division) signal. First, 3-phosphorylated phosphoinositides are not cleaved by phospholipase C (Lips et al., 1989; Serunian et al., 1989). Second, PI 3-kinase is associated with many pro- tein-tyrosine kinases, both Src-like and receptor-type, and this association often correlates with the mitogenic or on- cogenic action of the protein-tyrosine kinase (reviewed in Cantley et al., 1991). Third, the kinetics of appearance and degradation of 3-phosphorylated phosphoinositides upon activation of a protein-tyrosine kinase are consistent with their being second messengers (Auger et al., 1989b).

PI 3-kinase activity is conserved from yeast to human (Auger et al., 1989a; Hiles et al., 1992). The amount of PI 3-kinase activity found in the membrane fraction of yeast cells is significantly greater than that found in a similar fraction from mammalian cells; in S. cerevisiae, PI-3-P is as abundant as PI-4-P, whereas PI-3-P is only 3%-10% of the PIP content of mammalian cells (Auger et al., 1989a, 1989b; Whitman et al., 1988). The significance of the in- creased PI-3-P to PI-4-P ratio in yeast cells is not known.

Here we describe the cloning and characterization of an essential gene from S. cerevisiae, TOR2, whose product is argued, on genetic grounds, to be the target for G, arrest by FKBPl2-rapamycin. TOR2 exhibits significant homol- ogy with two previously identified PI 3-kinases.

Isolation of TO/?2 The TOR2-7 mutation was identified as a spontaneous mutation that confers resistance to the immunosuppres- sive drug rapamycin (Heitman et al., 1991 a). Genetic anal- ysis indicated that the mutation, corresponding to a single nuclear gene, confers a semidominant rapamycin-resis- tant phenotype at 30°C and 37OC and a recessive pheno- type at 24OC. Rapamycin normally arrests yeast cells in G, of the cell cycle (Heitman et al., 199la). To determine the role of the TOR2 gene product in G1 transition and in rapamycin drug action, we first cloned the mutant TOf?2-7 allele, making use of the semidominant phenotype ex- pressed at high temperature. A library was constructed from the TOR2-7 mutant strain R17 in the multicopy vector pSEY18 and transformed into JKS-3dala diploid cells. Ura+ transformants were selected at 37OC and subse- quently screened at 37°C for rapamycin resistance on selective platessupplemented with rapamycin (0.1 pglml). Only one plasmid, pJKl2, was able to confer rapamycin resistance at a level indistinguishable from the original TOR2-7 mutation. Restriction mapping demonstrated that the plasmid contained a genomic DNA insert of approxi- mately 11 kb. To determine whether the cloned gene was indeed identical to the TOR2-7 allele, we performed a tar- geted integration of the entire pJK12 plasmid into the ge- nome of strain JK9-3da (Rothstein, 1991). Integration at the chromosomal location homologous to the cloned insert was directed by linearizing pJK12 at a unique BamHl site within the insert. Stable Ura+ transformants, which were for unknown reasons sensitive to rapamycin at all tempera- tures, were crossed to the TOFf2-7 mutant strain JHl2- 17A, and resultant diploids were sporulated and dissected. Germinated spores were checked for Ura+ and rapamycin resistance. In all tetrads tested (24 of 24 from three dip- loids), Ura+ segregated 2:2, demonstrating integration of the plasmid at a single site in the genome. Moreover, in all cases, the Ura+ phenotype segregated opposite rapa- mycin resistance, indicating that the cloned insert had di- rected integration into the TOR2 locus and thus contained the TOR2-7 gene.

To clone the corresponding wild-type TOR2 gene, we performed a gap repair (Rothstein, 1991). Plasmid pJK12-8 (pJK12 lacking two adjacent internal Bglll frag- ments; see Experimental Procedures) was linearized with Bglll and transformed into strain JK9-3da. Plasmid DNA from Ura+ transformants was isolated and amplified in Escherichia coli. Restriction mapping demonstrated that the rescued plasmid, pJK3-3, had been gap repaired; Bglll fragments identical (by restriction analysis) to those de- leted in pJK12 had been restored using sequence from wild-type strain JK9-3d as a template. A restriction map of the gap-repaired DNA insert of pJK3-3 is shown in Fig- ure IA. The finding that plasmid pJK3-3 was able to com- plement the TOR2-7 mutation at room temperature, under conditions in which the mutation is recessive, indicated that we had indeed cloned the wild-type allele (data not shown). Furthermore, the gap-repaired DNA insert on a

;YFt Rapamycin Target Is PI 3-Kinase Homolog

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Figure 1. Restriction Map, Complementation Analysis, and TOR2 Disruption (A) Plasmid pJK3-3 and derivates containing the DNA indicated by a line were tested for complementation of a chromosomal TOP2 disruption. Complementation is indicated by a plus or minus (does complement or does not complement, respectively). The open arrow indicates the position and orientation of the TOR2 open reading frame as determined by DNA sequence analysis. The short bold line above the open reading frame indicates the position of the lipid kinase consensus sequence (see Figures 2 and 4). Restriction enzyme abbreviations are as follows: B, BamHI; G, Bglll; C, Clal; E, EcoRI; H, Hindlll; M, Sphl; SC, Sacl; S, Sall; Sm, Smal; X, Xbal (only one of several Xbal sites is indicated). (B) Diploid yeast strains were transformed with restriction fragments containing tor2.‘:ADE2-3 and tor2::LEU2-5 disruption alleles, and Ade+ or Leu’ transformants were selected (see Experimental Procedures). Sporulation and dissection of strains heterozygous for the TOR2 disruption yielded a 2:2 or a 1:3 segregation of viability to nonviability. Shown are 12 tetrads from the diploid strain MH346 (e/a tor2::ADf2-3/TOR2 ade2Nade2d). The four spores of each tetrad were positioned vertically. All viable spores were Ade-, indicating that TOP2 is an essential gene.

low copy vector (plasmid pJK4, GE/V4 LEUP) was able to complement a disruption in the chromosomal copy of the TOR2 gene (see below). Because the restriction map of the gap-repaired plasmid pJK3-3 was identical to that of plasmid pJK12, the TOR2-7 mutation is a point mutation or an otherwise small lesion.

Physical and Genetic Mapping of TOR2 The internal 5.3 kb Bglll restriction fragment derived from plasmid pJK3-3 (see Figure 1A) was labeled and used to probe yeast chromosomes separated by pulsed-field

electrophoresis. This provided an unambiguous assign- ment of TOR2 to chromosome Xl. A more precise map position was determined by probing ordered h-based and cosmid-based libraries of S. cerevisiae. This placed TOR2 approximately 10 kb TRP3 distal to STEG. Linkage to STEG was confirmed by genetic mapping. Based on a parental ditype:nonparental ditype+tetratype ratio of 15:0:0, TOR2 is 93 CM from ST/Z.

TOR2 Is Related to PI 3-Kinase To locate the position of the TOR2 gene in the 11 kb DNA

Cell 500

$st Ftapamycin Target Is PI 3-Kinase Homolog

insert, deletion analyses were performed on plasmids con- taining the TOR2-7 allele and the wild-type TOR2 allele. Deletion variants of TOR2-7 were examined for their ability to confer rapamycin resistance; deletion variants of TOR2 were tested for complementation of a chromosomal TOR2 disruption (see below). A summary of the results from the TOR2 deletion analysis is given in Figure IA. The results from both deletion analyses were identical except for the Sacl deletion of plasmid pJKl2(TOR2-I), in which an inter- mediate level of rapamycin resistance compared with the original insert was observed, whereas no complementing activity was detected with the corresponding Sacl deletion of the wild-type allele (plasmid pJK3-10; Figure 1A). These results indicated that most, if not all, the DNA insert was required for TOR2 function. The sequence of the entire genomic DNA insert of plasmid pJK3-3 was therefore de- termined. The sequence revealed a long open reading frame of 7422 bp encoding a protein of 2474 amino acids (282 kd, calculated pl 7.5) (Figure 2). In accordance with an open reading frame of this size, Northern analysis using the internal 5.3 kb Bglll fragment as a probe detected a single message of approximately 8 kb that is overex- pressed in a strain containing TOR2 on a multicopy vector (data not shown).

A search of the EMBL (release 31) and the Swissprot (release 22) data bases using the FASTA program (Genet- ics Computer Group package; Devereux et al., 1984) re- vealed that TOR2 is a previously unidentified gene. Signifi- cant similarity was found between the C-terminal half of the TOR2 protein, the ~110 catalytic subunit of bovine PI 3-kinase (~110 of PI 3-kinase), and the yeast VPS34 PI 3-kinase (Hiles et al., 1992; Herman and Emr, 1990; Schu et al., 1993) (Figure 3); the N-terminal half of TOR2 did not show significant similarity to any protein on record. Amino acids 1107-2439 of TOR2 are 20% identical and 45% similar (accounting for conservative amino acid changes) to ~110 (1068 amino acids); amino acids 1329- 2417 are 22% identical and 48% similar to VPS34 (875 amino acids) (Bestfit program, Genetics Computer Group package; Devereux et al., 1984) (Figure 3). The regions of highest homology among the three proteins contain resi- dues conserved in the catalytic domain of protein kinases (Figure 4). Based on structural data from the catalytic sub- unit of CAMP-dependent protein kinase and earlier find- ings, these invariant residues are implicated in ATP bind- ing and phosphotransferase activity (Knighton et al., 1991; Hanks et al., 1988). Residues G-2176, G-2179, K-2198, D-2279, and N-2284 and the DFG-2300 triplet of TOR2 correspond to residues G-50, G-52, K-72, D-166, and N-171 and the DFG-186 triplet of CAMP-dependent protein kinase. Other residues that are part of the sequence motifs conserved among protein kinases are not found in TOR2 and are also missing in VPS34 and ~110. The protein kinase consensus sequence described above is part of a

Figure 2. Nucleotide Sequence of TOR2 and Deduced Amino Acid Sequence The nucleotide sequence of the TOR2 coding region and adjacent regions is shown. The deduced amino acid sequence in single-letter code is indicated below the nucleotide sequence. The lipid kinase consensus sequence (see Figure 4) is underlined.

larger consensus sequence found in TOR2, ~110, and VPS34 (Figure 4). Because this larger sequence motif may be a general feature of lipid kinases, we refer to it as the lipid kinase consensus sequence.

Also noteworthy is the finding that TOR2 is unusually rich in leucine; the TOR2 protein is 13% leucine. The con- figuration of the leucines in TOR2 is not characteristic of a leucine zipper (Busch and Sassone-Corsi, 1990).

TOR2 Is Essential for Growth To determine whether TOR2 is essential, we disrupted the chromosomal copy of the gene using the one-step gene disruption method (Rothstein, 1991). Two types of disrup- tion alleles were constructed (see Figure 1 B; Experimental Procedures). First, a 6.5 kb region of the 11 kb DNA insert in plasmid pJK3-3 was replaced with the S. cerevisiae LElJ2 gene. Second, the ADE2 gene was inserted at the unique BamHl site within the TOR2 coding region in plas- mid pJK3-3. The disrupted genes were excised and trans- formed into homozygous diploid strains selecting for leu- tine (tor2::LEU2-5) or adenine prototrophy (tor2::ADE2-3). Disruption of the TOR2 locus in Leu+ and Ade+ trans- formants was confirmed by Southern analysis (data not shown). Diploid strains heterozygous for each TOR2 dis- ruption were sporulated and dissected on nonselective (YPD) medium. From both strain MH343 (for2::LEU2-5/ TOR2) and MH346 (for2::ADE2-3flOR2), 16 tetrads (32 total) were dissected and yielded only two or fewer viable spores (see Figure 1 B). All viable progenies were auxotro- phic for the disruption marker, indicating that segregants containing the TOR2 disruption were not viable. Strain MH346 transformed with plasmid pJK4 (CEN4 LEU2) or plasmid pJK3-3 (2~ URA3) containing the wild-type TOR2 gene was also sporulated and dissected; progeny dis- rupted in the chromosomal copy of TOR2 were obtained, but in all cases they carried the plasmid-borne wild-type TOR2 gene. Neither pJK4 nor pJK3-3 was lost from these haploids, even after growth on nonselective medium for many generations. These results demonstrate that TOR2 is required for growth.

TOR2 and Its Homolog TORI Are Required for Progression through G, Rapamycin arrests S. cerevisiae cells in the G, phase of the cell cycle. The current model for drug action is that rapamycin forms a toxic complex with its intracellular re- ceptor FKBP that binds to and inhibits an essential cellular component required for progression through the G, phase of the cell cycle. As shown in the preceeding section, TOR2 is essential. We therefore reasoned that if the TOR2 gene product is the target of, and is inhibited by, the immunophi- l in-drug complex, then disruption of the TOR2gene should cause the same phenotype as exposure to rapamycin. To test this hypothesis, we followed the fate of meiotic

Cdl 590

p110 165 Y”YPPN”EssPELPKHIYNKL.......DKGQII””IW”I”SPNNDKQKYTL.... KINHDC”eEo”IAEAIRKKTRsMLLssEQLK.....LC”LEY.Q III, ,I : :: : I : : :: 1: : : : I I ::/

TOR2 1306 . . . . . . ..5sSENPPEIYQ~LLNL"EFnEHDDKPLPIPI"T~KYAQKc~~F....AKALHYKE"EFLEEPKNSTIEALISINh(QL"*TDs~*G*LK~~Q :: I :,, I :: : :::,::, : II :://,

YPS34 68 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NRNLTLPIYTPYIPFRNSRTWDYWLTLPIRIKQLTFSSHLRIILWEYNGSKQIPFFNLETSIFNLKDCT

~110 248 GKYILKVCGCDEYFLEKY.. . ..PLsQYKYIRSC......IMLGRHPNL~LHAKESLYSQLp~DCFI~pSyS~~IsTA...................TpY I : I:,, :,/ :,:,:: I :: //:::: : I

TOR2 1394 Q"NELPL...KETWYEKLQRWEDALAAYNEKEAAGEDSVEVHMGKLRSLYyTs" ::: :,: ::: :: : :, / //: I I II1 I

"PS,4 137 LKR‘FES...LKFRYD"IDH..........CE""TDNKDQENLNK....YFPGEFTRLPWL..............................DEIT..ISK

~110 318 MNGETSTKSLWINSALR........ IKILCA.........TY"N"NIRDIDKIY"RTGIyNGG~pLCD.....N"NTQR"PCSNPRWNEWLNyDIyIpD I :: I :: 1: : I: I I I :I/ : I : 1: :: :/ :: /

TOR2 1491 HKSQSPDKEFYDAILCLHRNNFKKAEVHIFNARDLLVTELsAL"N~syN~AyN~~"~A~IIA~LE~IIKyKKLp~NsDK~LT~~~T~NT~LLGCQKNID" :: I :: : :: : I: : :/ ,::: I:: I I: : / I

VP534 188 LRKPRENRTWPQGTF"LNLEFp~L~Lp""FIEREIMNToM............................NIPTLKNNPGLSTDLREp..............

PIID 396 LPRAARLCLSICS"KGRKGAKEEHCPLAWGNINLFDyTDTL"SGK~ALNL~p"p"GL~DLLNpIG"TGSNpNK~TpCL~L~FD~FSs""KFpD~s"IE.. : I I: : I: : / : ,,:I!/ : 11: :1 : :\ :,,:: : : :

TOR2 1591 WQRILR"RSL"IKPKEDAQ"RIKFANLCRX..............SG~~A~KK"L~~TLL~~~DDpD"pNTAKASpp""yA~LKyL~ATGL~D~ALK~LINFT ::,:,::/ ::: :: ! : 1ll:II / I I I I

VP534 246 . . . . . . . . . . . ..NRNDPQIKISLGDKYHS............TLKFY..............DPDQPNNDPIEEK..YRRLERASKN~NLDKQ~KPDIKKR

p110 494 ..EHRNWS"SREAGFSYSHAGLsN~LA~DNE..........LRENDK....EQLRAICTRDPLSE....ITEQEKDFLWSHRXYC"TIp~ILpKLLLs"K . . . . : : : I I::, I I::: :::: I : : :: :::: /::,

TOR2 1679 SR~A"DLGLDPNNHIAQS"PQQSKRVPRHVEDYTKLLARCs"sK : : I ,I, 1: I :::/I / I /:: : I,: :: I

"PS34 305 DYLNKIINYPPG.....................TKLTAH......EKGSIWKYRYYLHNNKKALTKLLQSTNL~~Es.........ER"E"LELHDS...

p110 574 WASRDE"AQMYCL"KDWP.PIK~~QA~ELLDCN~~D~~"~GFA"~CL~K~.LTDDKL~Q~L~~L"Q"LK~E~~LDNLL"~F,.........LLKKALTNQ :, :::: ::::, ::: :::: I I : ::/ I ::i : ::

TOR2 1779 KKQEGSDASS"TDINEFDNG~IG"NTFDAKEVHYSSNLIHKGF.F"sISLS.......ESSSLQDALRLLTLWFTFGGIp~AT~A~~EGFNLI :, I: :: I:: I ::, : /:: : : I : ,I:: :

"PS34 366 . . . ..WAEIDIDDALELLGSTF.......KNLSVRSYAVNRLKKASDKELELYLLQLV.......EA"CFEN.......LsTFSDKSNSE..........

p110 662 RI‘HFF..FWHLKSEMH..NKT"SQ~FGLLLESYCRACGMyL.......... KHLNRO"EAM...EKL.INLTDILKQEKKDETQK"O~KFLVEonRRPD ,,::::,,:,III:II :I I / II I: ,,:I: ::I ::, / :: :

TOR2 1811 QICTWLEVLPQLISRIHQPNQI~SRSLLSLLSDLGKAHPQALVYPLMVAIKSESLSRQK~LSI~E~RIHSPVLVDQA~LVSHELIRMAVLWHEQW~EG : : :, I: I,, 1:: : : ::

"PS,4 430 . . . ..FTI"DA"SS.....QKLSGDSHL..... "ISPLAEF....LIRRAL"..NPRLGS

p110 744 F~DRLQGFLSPLNPAHQLGNLR..........L~~C~I"SSAK~pL....,.NLN~~NpDI~SEL......................... . : I, I:: I :: I II:, II ::,: :,:,

TOR2 1971 tDDASRQFFGEHNTEKMFRALEpLyENLKRGPETLREISFQNSFG~DLNDAy~~L~NyKKSKD"SNLNQA~DIyyN"F~KIGKQLpQLQ..TLELQH"SP : I II : I :: / ::/ I,,::: , :, : / /,:I : I I :

"PS34 497 F..............,.....,.FYWYLKSESED...... K~YLDQILS~FWSRLD..KKSRNILN.DQ~~L~..N~L~ECC~TIKKLKDTTAKKMELLV

p110 793 LFQNNEI.......,..........,,.......,...................... IFKNGDDL8QDMLTMIIRIMENIWQNQGLDLR....MLPYG I: ::: ::, :,: I,,, :/:::::::,,:: :, I :

TOR2 2069 KLLSAHDLELAVPGTRASGGKPIVKISKFEPVFS.VISSKQRPRKFCIKGSDGKDYKYVLKGHEDIRQDSLVMQLFGL~TLLQNDAECFRRHLDI~yp I I I I III: I I I: / I /: :, ::, :,:/,I I,:,:::,:, /I I: I: I

"~534 564 "LLETKVRPLVKVRPIRLPLDPDVLICDVCPETSKVFKSSLSPLKITFKTTLNQPYHLMFKVGDDLRQDQLVVQIISL~ELLKNENV....DLKLTPYK

PllO 838 CLSIGDC"GLIE""RNSXTI......,....... MQIQCKGGLKGRLQFNSHTLNQ,,................... WLKDKNKGEIYDAAIDLFTRSCA I,:: , ,, 1: :,: I I::: II I ,/I : :/ : :/II I

TOR2 2168 A~p~~pKSGLU;W"pNSDTFN"LI~~~~EAKKIPLNIEHW"MLQMApDyDNLTLL~K"~"FTyALNNT~GQDLYK"L~LKs~S.SET~L~~~TTyT~sLA :: :, / ::::,, ,I: :: :: I ::,::: I :/ : /:I : :, ,

VP534 660 ILATGPQEGAIEFIPN.DTLASILSKYHG.......ILGYLKLHYPDENATL...... G"QGW"LDN........................... FVKSCA

PIlO 909 GyC"ATFILGIGDRHNSNIH",KDDGQLFHIDFG"FLDHKKKKFGYKRERVPF"LTQDFLI"ISKGA~~......CTKTREFERFQE~CY~YLAI~QNA : : :/,,:,,II I,:,: : I ::I,,!, :: : : I:,,, /, : : :: 1 I : /I

TORZ 2267 "~SMTG~ILGU;D~"PSNLULDRITGKVIHIDFCDCFEAAILREKF.PEKVPFRLTRMLTYAHEVSG~EGSFRITCEN~KVLRDNKGSL~ILEAFAFD : : ,/Il:,III I,:: I :I HI :: I I : : : :: I: : I, / :,, I I :: ::,

VP534 719 GYC"IT~ILG"GD~"LDNLL"T~.DGHFF"A~FGYILGQD..PKPF.PPLMKLPPQIIEAFGGAESSNYDKFRS~CFVAYSILRRNAGLILNL~E.. .

PI10 996 NLFINLFSHMLI;SGHPELQSFDDIAyI~KTLRLDKTEQEALEYF~KQ~DA""GG~TT.~DNIF"TIKQNALN 1068 I: I: ,: :,:: : :,::: ::, : ::

TOR2 2366 PLINWGFDLPTKKIEEETGIQLP~~NANELLSNGAITEEEI~NA~A~L~LK~ITDKLTGNDI~ 2439

VP534 810

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136

317

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187

395

1590

245

493

1678

304

573

1778

365

661

1870

429

743

1970

496

792

2068

563

837

2167

659

908

2266

718

995

2365

809

Yeast Rapamycin Target Is PI 3-Kinase Homolog 591

VX2: w;HEDI~~~GL~~--(17)---LsP~ Gw ltXl: rsoC~~~~GL~~--(17)---LSP~L c;w

vPs34: FKVGtBrqp;;awPIISLMEUKNE--(l l)---LA ' IWQEGn pno: FWNXD~TL@IRIMCN~-(11)---U ImGL

-: mm~~v~IIx-(ll-l7)-LS xc%xxGx protkin: LG XGXXGX

V--(14)---EAK--(I%---RSLA-(6)- ~SNIMLDRITIEIEG V--(14) ---DAK--(64)---RSIA-(6)- YIII;LCEIRBPsNIsvDRITQNIIIIIEG I--(ll)---LSK--(30)---KSCA-(6)--V TP- I--(CA)--m--(35)---RX?+(6)-FIIGIcEAw%IW KDcGQLFglIR; 1-(11-14)-xxK-(30-64)-RscA-(6)-~SiLW XXDGXXFBIIEG "-- (+1*)-~--------(go-g5)- ------- -e ---- (11) ----m

Figure 4. Lipid Kinase Consensus Sequence

Shown is a conserved sequence motif found in TOR2, TORl, ~110, and VPS34. The first amino acid in each line corresponds to amino acid position 2127,623, and 901 of TOR2, VPS34, and ~110, respectively. A proposed lipid kinase consensus sequence derived from the TOR2, VPS34, and ~110 sequences is given. The last line shows residues conserved in the catalytic domain of protein kinases. Bold letters indi- cate residues conserved in all sequences; x is any amino acid.

segregants carrying a TOR2 disruption. Germinating spores of strain MH346 heterozygous for the tor2::ADE2-3 disruption were monitored microscopically; spores were incubated at 30°C and monitored for 36 hr. In all tetrads assayed, the two nonviable segregants germinated and underwent three to four rounds of cell division before ar- resting growth randomly in the cell cycle. Loss of TOR2 activity, therefore, did not cause G, arrest. However, TOR2 is strikingly homologous to TOR7, a second gene involved in rapamycin toxicity that maps 14 CM centromere- proximal to ST,58 on chromosome X (Heitman et al., 1991 a; P. Wagner, M. D.-R., S. B. Helliwell, Fi. H., and M. N. H., unpublished data). The two genes and their prod- ucts are approximately 65% identical by sequence com- parison. Interestingly, TORl also contains a lipid kinase consensus sequence that is highly conserved (96% amino acid identity) between TORl and TOR2; all residues in the lipid kinase consensus sequence that are conserved among TOR2, VPS34, and ~110 are also conserved in TORl (Figure 4). Despite the sequence similarity, a TON disruption confers no evident growth defect (M. D.-R. and M. N. H., unpublished data). Moreover, the TOW gene does not complement a TOR2 disruption, even when over- expressed. It seemed possible that TORl and TOR2 have partly overlapping function and that either one can medi- ate G1 to S phase progression. To test this possibility, a diploid, JK350, was constructed heterozygous for a TOR7 TOR2doubledisruption (tor7::LEU2-4/TOR7 tor2::ADE2-3/ TOR2). This diploid was sporulated and dissected, and spores were again monitored microscopically. Two or fewer viable progeny were recovered from each tetrad. Because TOR7 and TOR2 are unlinked, the two genes segregate independently in meiosis and frequently give

rise to recombinant spores (Leu+ Ade+ and Leu- Ade-). In those tetrads where the TOR7 disruption was inferred to segregate with the TOR2 disruption (that is, two viable Leu- Ade- spores were produced), the two spores con- taining the double disruption enlarged (germinated) but did not form buds. Occasionally, germinated spores did go through one round of cell division, but the two resulting cells also always stopped growth as unbudded cells. Of 18 segregants containing the double disruption, 16 ar- rested as a single cell and 2 arrested after one division. Thus, cells lacking TOR function germinate but fail to prog- ress through G,.

To examine further the cell cycle arrest caused by TOR depletion, the TOR2 gene was placed under control of the regulatable GAL7 promoter (plasmid pJK5) and introduced into TOR7 TOR2, for7 TOR2, TOR7 tor2, and tori tor2 strains (see Experimental Procedures). These strains con- taining pJK5 all grew normally in the presence of galac- tose, conditions under which the pJK5-borne copy of TOR2 is expressed. After shifting to glucose media, conditions under which the pJK5-borne TOR2 gene is repressed, the strains lacking a wild-type chromosomal TOR2 gene (TOR7 tor2 and for7 for2) stopped growing, and strains containing a wild-type TOR2 gene (TO/?7 TOR2 and rorl TOR2) continued to grow normally. The arrested, TOR2- depleted cells gave rise only to faintly visible microcolonies on solid media, whereas the cells that continued to grow gave rise to normal colonies. Microscopic examination of the TOR2-depleted cells revealed that the TOR7 tor2 strain arrested growth randomly in the cell cycle and the tori for2 strain arrested as large unbudded cells (Figure 5), confirming our previous observations that TOR2 is essen- tial and that TOR function (TORl and TOR2) is necessary for G, progression.

Discussion

We have cloned and characterized agene from S. cerevis- iae, TOR2, previously identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 disruption is lethal but does not cause a cell cycle-specific arrest. TOR2 disruption in combination with disruption of TOR7, a nonessential homolog of TOR2, causes a G, ar- rest, indicating that TOR function is necessary for progres- sion through G,. TOR2 encodes a 2474 amino acid protein with homology to the ~110 catalytic subunit of bovine PI 3-kinase and the yeast VPS34 PI 3-kinase (Hiles et al., 1992; Herman and Emr, 1990; Schu et al., 1993). The homology among ~110, VPS34, and TOR2 is particularly striking in a sequence we term the lipid kinase consensus; this consensus includes a conserved sequence implicated in the ATP binding and phosphotransferase activities of protein kinases (Figure 4) (Hanks et al., 1988).

Figure 3. TOR2 Is a PI 3-Kinase Homolog

Comparison of amino acids 1107-2439 of TOR2 with the amino acid sequences of the pIlO catalytic subunit of bovine PI 3-kinase (Hiles et al., 1992) and the S. cerevisiae VPS34 PI 3kinase (Herman and Emr, 1990; Schu et al., 1993) using the Bestfit program (Genetics Computer Group package; Devereux et al., 1994). A line between two amino acids indicates identity, and two dots represent a conservative difference.

Cdl 592

GLUCOSE GALACTOSE

TORT rod

TORl TOR2

Figure 5. TOR Depletion Causes G, Arrest

Ceils containing a plasmid-borne TORPgene under control of the GAL1 promoter (pJK5) were transferred from galactoselglycerol medium to glucose (A, C, and E) and galactose/glycerol (8, D, and F) media and grown overnight at 30% (see Experimental Procedures). The strains are torl::LEU2-4 torZ::ADE2-3/pJK5 (A and B), TOR7 tor2::ADE2-3/ pJK5 (C and D), and wild-type TOM TOR2/pJK5 (E and F). The TOR2 gene under control of the GAL1 promoter is induced by galactose and repressed by glucose. Depletion of TOR2 by glucose repression of the plasmid-borne TOR2 gene in a tori tor2 double mutant causes cells to arrest as large unbudded cells (A).

Based on its homology with PI 3-kinases, TOR2 (and TORl) is likely a PI kinase. Less certain is what species of PI kinase TOR2 might be. The various types of PI kinase could have structural motifs in common, though, as of yet, no sequence information is available on any defined PI kinase other than PI 3-kinase. A PI 4-kinase has been implicated in cell cycle (G,) regulation of S. cerevisiae (Uno et al., 1988; Kato et al., 1989). However, these early stud- ies predated the discovery of PI 3-kinase in yeast and may not have distinguished between the different PI kinases (Auger et al., 1989a). Two PI 4-kinases purified from yeast have apparent molecular masses, 45 kd and 125 kd, much smaller than that predicted for TOR2, 282 kd (Buxeda et al., 1991; Flanagan and Thorner, 1992). Consideration of the IL-2 signal transduction pathway and similarities of rapamycin action in yeast and T cells (see below) argue that TOR2 is indeed a PI 3kinase. The identity of the puta- tive TOR2 PI kinase remains to be determined.

Our results indicate that TOR2 is a target of FKBPl2- rapamycin and, furthermore, that FKBP12-rapamycin in- hibits TOR2 activity. Three observations support the for- mer conclusion (Heitman et al., 1991a). First, the TOR2 gene was originally identified by mutations that confer re- sistance to rapamycin. Second, FKBPlP is required for rapamycin toxicity; strains lacking FKBP12 (fprl mutants)

are viable and completely resistant to rapamycin. Third, rapamycin resistance in a diploid heterozygous for two recessive rapamycin-resistant FPR7 and TOR2 alleles (nonallelic noncomplementation) indicates that FKBP12 and TOR2 interact in the presence of rapamycin. The ob- servation that both rapamycin and loss of TOR function by gene disruption confer similar phenotypes, G, arrest and lethality, suggests that the interaction of FKBP12- rapamycin with TOR2 causes TOR2 inhibition. Thus, mu- tations in TOR2 that confer resistance to rapamycin pre- sumably prevent FKBP-rapamycin from interacting with TOR2 and do not otherwise affect TOR2 function.

What is the role of TOR2? The ~110 subunit of bovine PI 3-kinase, in complex with the p85a subunit, binds to activated colony-stimulating factor 1 receptor and is likely required for signaling cell proliferation in response to col- ony-stimulating factor 1 receptor activation (Hiles et al., 1992; Varticovski et al., 1989). VPS34 is required for the efficient sorting of proteins to the lysosome-like vacuole and for vacuole segregation in S. cerevisiae (Herman and Emr, 1990); strains lacking VPS34 are nonviable only at high temperature and do not exhibit a cell cycle arrest phenotype (S. Emr, personal communication). Because strains lacking TOR function (tor7 tor2 double disruption mutants) arrest in G1, we favor a model in which TOR2 performs a function similar to that proposed for bovine ~110 and for PI 3-kinases in general. TOR2 (and TORl) could be a component of a signal transduction pathway in yeast that acts on G1 cyclin and is required for cell cycle activation.

The finding that a TOR2 disruption is lethal but causes a G1 arrest only when combined with an otherwise nonlethal TOR7 disruption suggests that TOR2 has more than one function. As discussed above, one function is presumably a TORl-like, essential cell cycle function. Accordingly, a TOR7 disruption alone is not lethal because it is comple- mented by TOR2, a TOR2 disruption alone does not cause a GI arrest because it is partly complemented by TOR7, and rapamycin arrests cells in G, because it inhibits both TORI and TOR2. Because a TOR2 disruption alone is lethal but does not confer a cell cycle arrest, the second TOR2 function is unique to TOR2, also essential, and not necessarily related to the cell cycle. TOR2 may also have a role in membrane traffic similar to that of VPS34. Alterna- tively, the second function of the large TOR2 protein could be independent of PI kinase activity.

We anticipate, based on five observations, that the mechanism of action of rapamycin in yeast and T cells is similar and that the target of FKBPlP-rapamcyin in the IL-2 signal transduction pathway of T cells is a TOR homo- log. First, rapamycin causes a similar G, arrest in both cell types (Dumont et al., 1990b; Heitman et al., 1991a). Second, FK506 antagonizes rapamycin toxicity in T cells and yeast (Bierer et al., 1990; Dumont et al., 1990a; Heit- man et al., 1991a). Third, the target protein FKBPl2 is

highly conserved between yeast and human (Heitman et al., 1991b; Koltin et al., 1991; Wiederrecht et al., 1991). Fourth, the target of the FKBPl2-FK506 and cyclophilin- CsA complexes, calcineurin, and the action of these com- plexes on calcineurin are also highly conserved between

;Yr’ Rapamycin Target is PI 3-Kinase Homoiog

yeast and human(Cyettet al., 1991; Liuet al., 1991 b; Foor et al., 1992). Fifth, human FKPBlP restores rapamycin sensitivity to yeast strains missing endogenous FKBP12, and a host rapamycin-resistant TOR mutation protects the cell from rapamycin in the presence of human FKBP12, suggesting that protein-protein interactions between FKBP12 and TOR are conserved (Koltin et al., 1991; A. Koller and M. N. H, unpublished data). Interestingly, an early event in IL-2 signaling is a rapid increase in PI 3-kinase activity associated with the Src-like tyrosine ki- nase p5W (Augustine et al., 1991). The IL-2 signal trans- duction pathway(s) of T cells is poorly defined, but recent evidence suggests that it includes, in addition to the recep- tor-proximal p5W” and PI 3-kinase, glycosylphosphatidyli- nositol, tyrosine kinases ~56~” and p7Vk, serine/threonine kinase Raf-1 , Ras-p21 , p70 S6 kinase, and transcription factor NF-KB (Merida et al., 1990; Eardley and Koshland, 1991; Hatakeyamaet al., 1991; Horaket al., 1991; Satoh etal., 1991;Turneretal., 1991;Silicianoetal., 1992;Arima et al., 1992; Calvo et al., 1992; Kuo et al., 1992; Maslin- ski et al., 1992). How these components might be related in a pathway is obscure. However, the pathway appears ultimately to control the D2 and D3 cyclins, cyclins analo- gous to the G, cyclins of yeast (Matsushime et al., 1992).

Experimental Procedures

Strains, Plasmids, and Media The S. cerevisiae strains used in this work were JK9-3da, a, or ala /eo2-3,772 ura3-52 trpl his4 me7 HMLa or isogenic derivatives of JK9-3d. The dipioid strain JKS-Sdala is homozygous for ail markers except mating type. isogenic derivatives with only the changes indi- cated were as follows: JH12-17A, a TOR2-7; JK349-2A, a torl::LEU2-4 ade2A; JK350, a/a torl::LEU2-4/lOR7 tor2::ADE2-3/rOR2 ade2A/ ade2A; MH343, ala tor2::LEU2-5/TOR2; MH346, a/a tor2::ADE2-3/ TOR2 ade2A/ade2A; MH346-1 A, a tor2::ADE2-3 ade2AlpJK3-3; R17, a TOR2-7 HIS4. Strain JK350 was constructed by crossing strain JK349-2A with strain MH346-1 A and selecting against piasmid pJK3-3 on medium containing 5-fiuoroorotic acid. Strain K61-2c, a s&6-/acZ /eu2-3.7 7 2 ura3-52 trp7 his3 his4, was used for meiotic mapping. Pias- mid pJKl2 is pSEY16, amp’2p URAI (S. Emr, University of California, San Diego), carrying a 11 kb genomic DNA insert containing the TOR2-7 allele (see Construction of a Genomic DNA Library); piasmid pJK12-6 is pJK12 lacking the two Bgili fragments of the genomic DNA insert; piasmid pJK3-3 is a gap-repaired derivative of pJK12-6 con- taining the wild-type TOR2 allele (see Cloning of TOR2). Piasmid pJK5 is pSEYCllgaip, amp GAL7-GAL70 promoter CEN4 lJRA3 (S. Emr, University of California, San Diego), containing the TOR2 coding se- quence under control of the GAL7 promoter. Other piasmids are de- scribed below where relevant. The composition of rich medium (YPD), synthetic minimal medium (SD) complemented with the appropriate nutrients for plasmid maintenance, and sporuiation medium was as described (Sherman, 1991). YPGaiGiy and SGaiGiy were prepared as described for YPD and SD, respectively, with 2% galactose and 2% glycerol substituted for the glucose. Rapamycin was added to the medium to a final concentration of 0.1 pg/mi. Prior to addition to media, the drug was dissolved in 10% Tween 20 in ethanol (1 mglmi). The medium for Sfiuoroorotic acid selection was as described (Boeke et al., 1967).

General Genetic Manipulations Standard genetic techniquesof crossing, sporuiation, and tetrad analy- sis were as described (Guthrie and Fink, 1991). S. cerevisiae transfor- mation was performed by the lithium acetate procedure (ito et al., 1963). E. coli strains MHl or DH5a were used for propagation and iSolation of plasmids as described (Sambrook et al., 1969).

Construction of a Genomic DNA Library Total genomic DNA from the S. cerevisiae strain R17 was isolated as described (Phiiippsen et al., 1991) and subjected to partial SauiiiA digestion. Fragmentswerefractionatedon a0.6% low melting agarose gel, and a region corresponding lo a size of 6-12 kb was cut from the gel and treated with agarose. The DNA was ligated into the BarnHI site of vector pSEY16. The ligation mixture was transformed into E. coii strain MHl, 40,000 independent transformants were pooled, and plasmid DNA was isolated. The mean insert size was 5-7 kb, and insert frequency was 60%. The single positive clone isolated from this library was named pJK12.

Cloning of TOR2 The TOR2-7 gene was cloned by using the semidominant rapamycin- resistant phenotype conferred by this mutant allele at 30°C and 37V. JKQSda/a dipioid wild-typeceils were transformed with the yeast gene- mic library constructed from the TOR2-7 mutant strain R17 in thevector pSEY16. Approximately 25,000 Ura+ transformants were selected at 37OC on minimal plates lacking uracii (SD minus Ura) and subse- quently replica plated to prewarmed 37°C SD minus Ura plates con- taining rapamycin (0.1 rg/mi). Rapamycin-resistant colonies were picked, and the piasmid DNA was isolated (Hoffman and Winston, 1967). After amplification in E. coii, the piasmid DNA was reintroduced into the original yeast strain JKS-3da/a, and piasmid-dependent resis- tance to rapamycin at 37OC was confirmed on selective plates con- taining rapamycin (0.1 pg/mi). To clone the wild-type TOR2 gene by gap repair, piasmid pJK12-6 was linearized with Bgiii. This plasmid contained a 6.5 kb internal deletion spanning two adjacent Bglii restric- tion fragments. The linearized piasmid was transformed into the wiid- type strain JK9-3da. Piasmids from several independent Ura+ trans- formants were reisolated and mapped by restriction analysis to confirm the gap repair. For integrative mapping, piasmid pJK12 was linearized at the unique BamHi site within the genomic DNA insert and trans- formed into strain JK9-3da. Ura+ transformants were selected on mini- mal plates and tested for sensitivity to 5fiuoroorotic acid, indicative of stable integration of the piasmid. integrants were subsequently crossed to strain JH17-12A. Sporuiation of the resultant diploids was induced, and meiotic segregants were tested for the segregation pat- tern of the URAB and TOR2-7 genes. Rapamycin resistance as a marker for the TOR2-7 allele and uracii prototrophy as a marker for the integrated piasmid were tested. Eight tetrads each from three inde- pendent dipioids were analyzed.

Mapplng the Chromosomal Site of TOR2 The internal 5.3 kb Bgili fragment of piasmid pJK3-3 (see Figure IA) was used as a probe for hybridization to yeast chromosomes separated by pulsed-field gel eiectrophoresis and for hybridization to a set of overlapping 5 and cosmid clones representing - 99% of the S. cerevis- iae genome (gift of L. Riles and M. Olson, Washington University, St. Louis, Missouri). Hybridization was done according to the recommen- dations of L. Riles. Overlapping cosmid clones of chromosome Xi were also probed (performed by 8. Dujon, institut Pasteur) with results similar to those obtained with the Riles and Olson clones. For meiotic mapping, strain JK12-17A was crossed to strain K61-2c, resultant dip- ioids were sporuiated and dissected, and the segregation pattern of ste6 (a-specific sterility) and TOR2-7 (rapamycin resistance) was foi- lowed. Because both sfe6 segregants were detectable only in those tetrads in which sfe6 segregated with MATa, only a subset of the total number of tetrads dissected were used to calculate the steG-TORZ-7 genetic distance.

Plasmids for Deletion Analysis Piasmidsfor deletion analysis were obtained by cutting piasmid pJK3-3 (TOR2) or plasmid pJK12 (TOR2-7) at sites that were present both in the polylinker and in the insert (or multiple times within the insert) and by reiigating. Alternatively, restriction fragments were subcloned into the multicopy vector YEpiacl61, amp’ 2~ LEU2 (Gietz and Sugino, 1966). Piasmids listed in Figure 1A were ail derived from piasmid pJK3-3 and represent the following deletions and subclones (plasmids indicated in parentheses are the equivalent deletions in piasmid pJK12): pJK3-4 (pJK12-4). Ciai deletion; pJK3-6 (pJK12-6), Smal dele- tion; pJK3-7 (pJK12-7), Hindili deletion; pJK3-6 (pJK12-6), Bglll dele- tion; pJK59 (pJK12-9), EcoRl deletion; pJK3-10 (pJKlP-lo), Sacl de-

Cdl 594

letion. Plasmid pJK3-5 was constructed by subcloning the 8.0 kb Smal fragment into the vector YEplacl81 and replacing the 1.4 kb Hindlll- Smal fragment with the 1.95 kb Hindlll fragment from plasmid pJK3-3 in the correct orientation. Plasmid pJK3-11 was derived from plasmid pJK38 by subcloning a 3.0 kb Xbal-Pstl fragment (the Pstl site is part of the vector polylinker on the right side of the insertion, as shown in Figure 1) into the vector YCplacl II, amp CEN4 LEU2 (Gietz and Sug- ino, 1988); the 5.3 kb Bglll fragment from plasmid pJK3-3 was then cloned into the unique Bglll site of the resultant plasmid. For generating plasmid pJK3-12, the 5.3 kb Bglll fragment from plasmid pJK3-3 was cloned into the vector YEplacl81. Plasmids were tested for their ability to complement a chromosomal TOR2 disruption by transforming strain MH348, inducing transformants to sporulate and dissecting individual spores. Complementing activity was assigned if viable Ade+ Ura’ or, depending on the deletion construct used, viable Ade+ Leu’ haploids were recovered.

TOFf2 Dlsruption The TOR2 disruption vector pMD7A was constructed by replacing the 5.3 kb and 1.3 kb Bglll fragments of plasmid pJK3-3 with a 2.5 kb Bglll cassette containing the LEU2 gene. Plasmid pMD12B was con- structed by inserting a 2.3 kb Bglll cassette containing the ADE2 gene into the unique BamHl site located within the TOR2 coding region of plasmid pJK3-3. The disruption was excised from plasmid pMD7A by Sacl and Xbal digestion and from plasmid pMDl2B by Bglll digestion and was transformed into the diploid strains JK9-3da/a and JK9- &Q/a ade2A/ade2A, selecting for Leu+ and Ade+ transformants. re- spectively. The successful disruption of the chromosomal 7OR2 locus was verified in transformants by Southern analysis. Diploid strains that showed the expected hybridization pattern were designated MH343 (tor2::LEU26/TOR2) and MH348 (tor2::ADE2-3/TOR2). These strains were sporulated, and 18 tetrads each from strains MH343 and MH348 were dissected. To test whether TOR2 complements a chromosomal 7OR2 disruption, strain MH348 was transformed, with the centromere- based plasmid pJK4 (TOR2) sporulated and dissected. Plasmid pJK4 was constructed by subcloning the 8.0 kb Smal restriction fragment derived from plasmid pJK3-3 into the low copy vector YCplacl II, amp’ CEN4 LEU2 (Gietz and Sugino, 1988); the 3.0 kb Smal-Pstl restriction fragment from plasmid pJK3-3 was then ligated into the resultant plas- mid to reconstruct the entire TORZ gene.

Expression of TOR2 under Control of the GAL1 Promoter To clone the TO/?2 gene under control of the GAL7 promoter, an ap- proximately 1.5 kb polymerase chain reaction product was generated using a forward primer that contained a Xhol site and was complemen- tary to the region of 7OR2 immediately upstream of the initiation codon (5’GACCTCGAGGAIlTCTGATCTlTACTlTC-3’) and a reverse primer that corresponded to downstream coding sequence (5GTCTCCTGC ATATGGTCG-3’). The polymerase chain reaction product was di- gested with Xhol and Sphl and cloned into the Sal1 and Sphl sites of pSEYC88galp. A complete 7OR2 gene was then regenerated by clon- ing a 8.5 kb Sac1 fragment containing the missing 3’ portion of TOR2 into the Sac1 site of this plasmid. The resulting plasmid, pJK5, con- tained the entire TOR2 coding sequence under control of the GAL7 promoter. pJK5 was transformed into yeast strain JK35Q and Ura+ transformants were sporulated and dissected. Segregants were germi- nated on YPGalGly and tested for leucine (torl::LEU2-4), adenine (tor2::ADE2-3), and uracil (pJK5) prototrophy. Haploid TORI TOR2/ pJK5, torl::LEU2-4 TOR2/pJK5, TO/?7 tor2::ADE2-3/pJK5, and tori:: LEU24 tor2::ADE2-3/pJK5 segregants were grown at 39% on media containing galactose and glycerol as the sole carbon sources (YP- GalGly or SGalGly). To test the phenotype of TOR depletion, the plas- mid-borne TOR2 gene was turned off by transferring the cells to YPD or SD solid medium and incubating at 30% cells were monitored at several time points up to 3 days after transfer to the glucose media. Growth of segregants containing a chromosomal 7OR2 disruption ar- rested after transfer to glucose media, whereas cells containing a wild-type 7OR2 gene continued to grow. Samples to be photographed were scraped from the solid media after overnight incubation at 39% and mounted in FluommountG (Southern Biitechnical Associates) on polyfysine-treated slides. Photographs were taken using a Zeiss Axiophot microscope with DIC optics.

DNA and RNA Manipulations Recombinant DNA manipulations were performed according to stan- dard methods (Sambrook et al., 1989). The DNA probes for Southern and Northern analysis were labeled using the random-primed labeling kit (Amersham International) following the recommendation of the manufacturer. DNA probes for the physical mapping of the TOR2 gene were made by random-primed “P-labeling using random hexanucleo- tides as primers, ja-YPjdATP, and the Klenow fragment of DNA poly merase I. Total yeast genomic DNA or RNA for Southern or Northern analyses was extracted as described (Guthrie and Fink, 1991) fraction- ated by gel electrophoresis on an agarose gel in Tris-borate-EDTA (DNA) or in MOPS-formaldehyde (RNA), and transferred to nylon membranes. Hybridization was according to the recommendation of the manufacturer (Amersham International). For sequencing, restric- tion fragments were cloned into the vector pGem7f(+) (Promega), and sets of nested deletions were obtained by the exonuclease Ill method using the Erase-aBasesystem (Promega). Custom-made oligonucleo- tides were used as primers for sequencing regions not accessible with the deletions. DNA sequencing was performed by the dideoxy chain termination method @anger et al., 19n) using the I7 sequencing system from Pharmacia. The DNA sequence of both strands was determined.

We thank Jose Garcia-Bustos and Bernard Dujon for the physical map- ping of TOR2, Scott Emr for valuable discussions and unpublished data, and the members of the laboratory for comments on the manu- script. The research was supported by a grant from the Swiss National Science Foundation awarded to M. N. H.

Received November 28, 1992; revised March 30, 1993.

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GsnBank Accession Number

The accession number for the sequence reported in this paper is X71416.