TOR2 Is Part of Two Related Signaling Pathways Coordinating Cell Growth in Saccharomyces cerevisiae

Corresponding author: Michael N. Hall, Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, hall{at}ubaclu.unibas.ch (E-mail).

Communicating editor: A. G. HINNEBUSCH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.

TOR1 and TOR2 are phosphatidylinositol (PI) kinase homologs originally identified genetically by dominant mutations that conferred rapamycin resistance in Saccharomyces cerevisiae (HEITMAN et al. 1991 ; CAFFERKEY et al. 1993 ; KUNZ et al. 1993 ; HELLIWELL et al. 1994 ). We have previously demonstrated that loss of both TOR1 and TOR2 causes a first-generation arrest in the early G1 phase of the cell cycle, as does rapamycin treatment (BARBET et al. 1996 ). The arrested cells exhibit characteristics of starved cells in stationary phase (G0), including glycogen accumulation and a decrease in protein synthesis. The block in protein synthesis occurs at the level of initiation and is the cause of the cell cycle arrest. Thus, it appears that TOR1 and TOR2 are part of a novel signal transduction pathway involved in regulating translation, cell growth, and progression through the G1 phase of the cell cycle in response to nutrients (BARBET et al. 1996 ; DI COMO and ARNDT 1996 ; THOMAS and HALL 1997 ). In mammalian cells, the TOR counterpart (FRAP/RAFT/mTOR) is part of a general mitogenic signaling pathway regulating translation and G1 progression (BROWN et al. 1994 , BROWN et al. 1995 ; CHIU et al. 1994 ; SABATINI et al. 1994 ; SABERS et al. 1995 ; BERETTA et al. 1996 ; THOMAS and HALL 1997 )

TOR2 has two functions. In contrast to cells lacking TOR1 and TOR2, cells lacking only TOR2 do not display a G0 arrest phenotype, but instead undergo a few cell divisions before arresting randomly in the cell cycle (KUNZ et al. 1993 ). Importantly, even overexpression of TOR1 cannot suppress the loss of TOR2. Cells lacking only TOR1 exhibit only mild growth defects (HELLIWELL et al. 1994 ). These phenotypes suggest that, in addition to having a redundant function with TOR1 in controlling G1 progression, TOR2 also has an essential, unique function that is not directly related to the cell cycle (KUNZ et al. 1993 ; HELLIWELL et al. 1994 ; ZHENG et al. 1995 ). Recent results have demonstrated that the TOR2-unique function is required for organization of the actin cytoskeleton. Mutants defective only in TOR2 do not exhibit the normal polarized distribution of the actin cytoskeleton and are rescued by overexpression of TCP20, an actin-specific chaperone (SCHMIDT et al. 1996 ).

Here we describe the isolation and characterization of different classes of TOR mutants that indicate that TOR2 indeed has two functions (or readouts). Suppressor analyses of representative mutants of these phenotypic classes suggest that the TOR2 functions are carried out by related signaling pathways.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, plasmids, and media:
The yeast strains used in this study are described in Table 1. Escherischia coli strain MH4 is (lac)X74 hsr^- hsm⁺ strA leuB600 galE galK. Plasmid pJK4 (YCplac111::TOR2) (KUNZ et al. 1993 ) was constructed as follows. The 8 kb SmaI fragment of pJK3-3 (pSEY18::TOR2) (KUNZ et al. 1993 ) was cloned into the SmaI site of YCplac111 (CEN LEU2), and the resultant plasmid was cut with BamHI and Pst I and ligated with the BamHI-Pst I fragment of pJK3-3. Plasmid YCplac111::tor2-21 was constructed as follows. Because the mutation in the tor2-21 allele (Ser-233 to Phe), originally isolated on YEplac181, eliminates the SacI site within the open reading frame (ORF), tor2-21 could be isolated as a single SacI-PstI fragment (T. BECK, personal communication). This SacI-PstI fragment was ligated into the multicloning site of YCplac111. Plasmid pSH22 is pSEY18 (2µ URA3) containing a 4.1 kb BamHI-XhoI fragment including MSS4. Plasmid pSH24 is pSEY18 with a 4.3 SphI fragment containing PKC1. pSH25 is pSEY18 containing a 3.4 kb BamHI fragment containing PLC1. pSH27 is a double-deletion construct of pSH121.30, an original suppressor, such that a 1.9 kb SmaI-SphI fragment remains containing SUR1. pEMY105 (gift of S. EMR) is pRS426 (2µ URA3) containing FAB1. YEp352-STT4 (YEp352-PSTB20) (gift of J. THORNER) is 2µ URA3 STT4. pGB1 is pSEY18 containing PIK1. pC-186 (2µ URA3 RHO2) and pAS30 (2µ URA3 ROM2) have been described previously (SCHMIDT et al. 1997 ). Media were prepared as described (SHERMAN 1991 ). Rich media contained 2% glucose (YPD), 2% galactose (YPG), 2% raffinose (YPR), or 3% ethanol and 3% glycerol (YPE/glycerol). Defined medium (SD) contained 2% glucose as the carbon source and, unless otherwise indicated, ammonium (0.01 M) as the nitrogen source. SD medium with proline (1 mg/ml) or glutamate (1 mg/ml) as the nitrogen source contained only tryptophan, histidine, and leucine as amino acid supplements (VANDENBOL et al. 1987 ); these three amino acids were required to satisfy the auxotrophies of our strains and cannot be used as a nitrogen source. SD-complete medium contains a complete complement of amino acids.

Hydroxylamine mutagenesis, plasmid shuffling, and screening for tor2 ^ts mutants:
Mutagenesis was performed on YCplac111::TOR2 (CEN LEU2) (pJK4) plasmid DNA as described (SIKORSKI and BOEKE 1991 ). Samples were incubated at 75° for 12.5, 15, and 17.5 min in 0.1 M hydroxylamine, and then desalted with a Sephadex G-50 spin column. Aliquots from the three plasmid banks were transformed into the E. coli strain MH4, which is a leucine auxotroph. The percentage of Leu^- transformants indicated that between 2% and 5% of the plasmids in the three banks had lost the LEU2 marker by mutagenesis. These plasmid banks were then transformed into NB4-6a (tor1 tor2) or MH346-1a (TOR1 tor2). Transformants were plated at a density of approximately 500 per plate. After 2–3 days at 24°, colonies were replica plated onto medium containing 5-fluoroorotic acid (5-FOA) and incubated at 24° to select for cells that could lose the URA3-marked TOR2-containing plasmid pJK3-3. These were then replica plated (1) to SD-LEU and incubated at 24° and (2) to YPD and incubated at 37°. For colonies that appeared to have a growth defect on YPD at 37°, the corresponding colony on the master plate was restreaked for further analysis. Plasmid DNA was isolated from transformants that displayed temperature-sensitive growth, and these plasmids were retransformed into the original host strains and subjected to the plasmid shuffle procedure once again. Plasmids that conferred temperature-sensitive growth after this were deemed to contain tor2^ts alleles and were kept for further study. Allele tor2-156 ^ts was isolated from among (8000) screened transformants of MH346-1a (TOR1 tor2). The remaining 28 alleles were obtained from 30,000 transformants of NB4-6a (tor1 tor2).

Plasmids bearing each of the ts alleles were transformed into JK9-3da (TOR1 TOR2) and NB17-3d (tor1 TOR2) to determine whether these alleles were recessive to the genomic copy of TOR2. As expected, none of the plasmid-borne alleles exhibited any dominant effects in JK9-3da (TOR1 TOR2) as judged by a normal growth rate of transformants at 37° on solid minimal medium lacking leucine. However, the alleles tor2-21 ^ts and tor2-156 ^ts caused a reduction in growth rate at 37° when expressd in NB17-3d (tor1 TOR2), and additionally, the plasmid-borne tor2-156^ts slightly inhibited the growth of NB17-3d (tor1 TOR2) at 24° (data not shown). This suggests that in the absence of the genomic copy of TOR1, these CEN plasmid-based mutant alleles can have a dominant effect over the single genomic copy of TOR2.

Growth curves and FACS analysis:
Cultures were grown to log phase at 24° in SD medium, then diluted to give an OD₆₀₀ of approximately 0.05 in prewarmed media, and shaken at 37°. The OD₆₀₀ was then determined at the times indicated. To easily assess the number of generations each strain went through, we divided all the resultant OD readings by the OD₆₀₀ at the time of shift for any particular strain. Samples for fluorescence-activated cell sorting (FACS) were prepared and analyzed as described previously (HELLIWELL et al. 1994 )

³⁵S-methionine incorporation:
Strains were grown to log phase in SD-complete medium and diluted to an OD₆₀₀ of 0.05 in 4.5 ml of fresh prewarmed SD-complete medium; 1 ml was removed to determine the starting OD₆₀₀ and, to the remaining 3.5 ml, ³⁵S-methionine was added (in 100 µl SD-complete medium) to a final concentration of 10 µM (the concentration of cold methionine in SD-complete medium is 130 µM). The zero timepoint was taken immediately and the cultures were incubated at 37°. During the incubation, samples were taken at several timepoints. For each timepoint, 300 µl aliquots were taken and added directly to 700 µl of 10% trichloroacetic acid (TCA) on ice. A 20 µl aliquot was added directly to a glass fiber filter to act as a control for the total number of counts available in each labeling reaction. At the end of the experiment, the samples were heated at 95° for 4 min to deacylate tRNAs and were then filtered through TCA-soaked glass fiber filters, which were washed twice with ice-cold 10% TCA and once with acetone. The filters were then counted by liquid scintillation. Values for each strain were calculated by dividing the number of counts (1) by the starting OD₆₀₀ of the culture and (2) by the counts obtained from the control filters. Values were then plotted as normalized against a wild-type control strain.

Morphological observations:
Cells were stained with rhodamine phalloidin (stain for actin filaments) and 4',6'-diamidino-2-phenylindole (DAPI) (stain for DNA) as described (PRINGLE et al. 1989 ). Cells were fixed and treated 8 hr after shift to the nonpermissive temperature, as described (SCHMIDT et al. 1997 ). Microscopy was performed with a Zeiss Axiophot II fluorescence microscope.

Multicopy suppressor isolation:
Strains SH121 (class A) and SH229 (class B) were used to isolate high-copy suppressors of unique and shared function defects, respectively. Class D mutant SH129 (tor2-29 ^ts) grows slower at nonpermissive temperature than class D mutant SH130 (tor2-30 ^ts), indicating that the unique function encoded by tor2-29 ^ts is not completely intact, but still sufficient for growth. We did not use class B mutants SH230 or SH2119 to isolate suppressors because they had high reversion rates. The isolation of the multicopy suppressors was performed using a 2µ based library derived from strain R1 (HELLIWELL et al. 1994 ). Cells were allowed to recover from the transformation in liquid YPD at the permissive temperature for 5 hr before being plated on selective media at the nonpermissive temperature. Transformants growing at 37° were passaged through 5-FOA plates to demonstrate that the suppression was plasmid linked. Plasmids conferring suppression were rescued and retransformed into the parental strain to recheck suppression. Deletions and subclones were made using standard techniques to ascertain which ORF on each clone was responsible for suppression.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of temperature sensitive tor2 mutants:
A strain disrupted in TOR2 arrests after three to four generations with no previously observed cell cycle phenotype. In contrast, strains disrupted in TOR1 and TOR2 arrest rapidly, within one cell cycle, and in early G1 (KUNZ et al. 1993 ; HELLIWELL et al. 1994 ; BARBET et al. 1996 ). Furthermore, overexpression of TOR1 cannot suppress a tor2-null or tor2^ts mutation (HELLIWELL et al. 1994 ; T. SCHMELZLE and M. N. HALL, unpublished results). These differing arrest phenotypes suggest that TOR2 has at least two distinct essential functions. The first is involved in G1 progression and can be provided by TOR1; the second is not involved in cell cycle control and cannot be provided by TOR1. We refer to these two functions throughout this study as the TOR-shared and the TOR2-unique functions, respectively. The TOR-shared function is required for the activation of translation initiation. The TOR2-unique function is required for the organization of the actin cytoskeleton.

To define further the two functions of TOR2, we attempted to isolate temperature-sensitive tor2 mutants that were defective for only one of the two TOR2 functions. We isolated 29 independent plasmid-borne tor2^ts alleles using a screen based on in vitro mutagenesis and plasmid shuffling (see MATERIALS AND METHODS; SIKORSKI and BOEKE 1991 ). All plasmids containing a temperature-senstive allele and the starting plasmid used for the mutagenesis (YCplac111::TOR2) were transformed into both MH346-1a (TOR1 tor2/pSEY18::TOR2) and NB4-6a (tor1 tor2/pSEY18::TOR2), and the pSEY18-borne wild-type TOR2 gene was eliminated using 5-FOA (see MATERIALS AND METHODS). Our nomenclature convention for the resulting TOR1 tor2^ts and tor1 tor2^ts strains is as follows. MH346-1a carrying substitute plasmid YCplac111::TOR2 (relevant genotype TOR1 tor2/pTOR2) is named SH100. The same host strain carrying a tor2^ts plasmid instead of the TOR2 wild-type plasmid is named SH1x, where x is the number of the ts allele; e.g., the TOR1 tor2 strain carrying tor2-21^ts is SH121. NB4-6a (tor1 tor2) kept viable with YCplac111::TOR2 is called SH200; when its plasmid-borne TOR2 allele is replaced with a tor2^ts allele, it is named SH2x, where x is again the number of the ts allele (Table 1).

The growth of the TOR1 tor2^ts strains (SH1x series) and tor1 tor2^ts strains (SH2x series) at 24° and 37° on solid rich medium containing glucose as the carbon source (YPD) was assessed. Table 2 summarizes these results, from which we have classified the mutant strains into four classes, A through D, based on whether a mutant was defective for the unique, the shared, or both functions of TOR2 (Table 3). Class A mutants, represented by strains SH121 (tor2-21^ts) and SH1156 (tor2-156^ts), did not form colonies after 3 days at 37° and were thus temperature sensitive for the TOR2-unique function (Figure 1). The shared function is provided in these mutants by the chromosomal wild-type TOR1 gene. Interestingly, we never obtained a class A mutant in which the shared function is provided by TOR2, i.e., a TOR2 allele that is defective only for the unique function. Class B mutants, represented by strains SH230 (tor1 tor2-30 ^ts) and SH2119 (tor1 tor2-119 ^ts), also arrested growth at 37° and were temperature sensitive only for the shared function (see class D below). Class C mutants, represented by strains SH221 (tor1 tor2-21^ts) and SH2156 (tor1 tor2-156^ts), were temperature sensitive for both TOR2 functions. Class D consists of pseudomutants and is represented by strains SH130 (tor2-30 ^ts) and SH1119 (tor2-119 ^ts). These pseudomutants formed normal-sized colonies at all temperatures on solid medium, indicating that the class B mutants, which contain the same TOR2 alleles but in a tor1 background, were defective only in the shared function. Some class D mutants had a partial growth defect indicating that, in these strains, the TOR2-unique function was partly defective.

View larger version (83K): [in this window] [in a new window]   Figure 1. —Growth of class A through D mutants. Mutant and control strains were streaked on rich medium (YPD) and incubated at 24° for 3 days or 37° for 2.5 days. Strains are as follows: control (TOR1 TOR2) is SH100; control (tor1 TOR2) is SH200; class A (TOR1 tor2-21ts) is SH121; class B (tor1 tor2-30 t s) is SH230; class C (tor1 tor2-21ts) is SH221; class D (TOR1 tor2-30 ts) is SH130.

Suppression of growth defects by nutrient source or medium supplements:
Because the TORs have previously been implicated in nutrient sensing (BARBET et al. 1996 ; DI COMO and ARNDT 1996 ), we examined the effect of altering the carbon or nitrogen source on growth of various mutants. Mutants from all four classes were assessed for growth at 37° on rich medium containing galactose (YPG), raffinose (YPR), or ethanol/glycerol (YPE/glycerol) as the sole carbon source; growth was assessed previously (Table 2) on YPD medium, containing glucose as the carbon source. The growth defect of class A mutants, strains SH121 (tor2-21^ts) and SH1156 (tor2-156^ts), was fully suppressed on the raffinose medium (data not shown), was partly suppressed on the galactose medium (Table 4), and was not suppressed on the ethanol/glycerol medium (data not shown). The growth defect of class B and C mutants was not suppressed by any carbon source, except for strains SH214 (tor1 tor2-14^ts), SH298 (tor1 tor2-98^ts), SH2140 (tor1 tor2-140 ^ts), SH2143 (tor1 tor2-143^ts), SH2150 (tor1 tor2-150 ^ts), and SH2152 (tor1 tor2-152^ts). These mutants eventually formed very small colonies after 3 days, but only on the galactose medium (Table 4). Class D mutants that exhibited a partial growth defect all grew like wild type on galactose and raffinose medium, but ethanol/glycerol medium was not able to suppress the slower growth of these mutants. Mutants from all four classes were also assessed for growth at 37° on defined medium containing glucose as the carbon source and either proline, glutamate, or ammonium as the nitrogen source. Proline, glutamate, and ammonium are poor, intermediate, and good nitrogen sources, respectively. Unlike the carbon source, the nitrogen source had no effect on the growth of any of the mutants; none of the nitrogen sources suppressed the growth defect of any mutant.

Growth at 37° was also assessed on YPD medium supplemented with 1 M sorbitol, 0.5 M NaCl or 0.1 M CaCl₂. The growth defect of Class A mutants was fully suppressed by 1 M sorbitol or 0.5 M NaCl, and was partly suppressed by 0.1 M CaCl₂. Allele-specific effects were seen for class B mutants grown on medium containing one of the three supplements, ranging from complete suppression to no suppression (Table 4). The growth defect of class C mutants was not suppressed by 1 M sorbitol, 0.5 M NaCl, or 0.1 M CaCl₂. Class D slow-growing mutants all grew like wild type on medium supplemented with 1 M sorbitol, 0.5 M NaCl, or 0.1 M CaCl₂, with the exception of SH1152 (TOR1 tor2-152^ts), for which CaCl₂ had no effect. Thus, of all the mutants, class A mutants were most susceptible to suppression by either the carbon source or media supplements.

Class A mutants arrest in G2/M, and class B and C mutants arrest in G1:
We characterized further the growth and cell cycle phenotypes of the class A, B, and C mutants. The growth and cell cycle arrests of the mutants were analyzed by determining growth curves (OD₆₀₀) and by FACS analysis. Class A mutants SH121 (tor2-21^ts) and SH1156 (tor2-156^ts) ceased to grow after 7–8 hr at 37°, after having completed two to three divisions (Figure 3 and data not shown). Most of the arrested cells (>80%) contained a 2n DNA content, indicating a block in the G2/M phase of the replicative cycle (Figure 2 and data not shown). Scoring the cells morphologically for cell cycle distribution showed that these strains arrested with an increased number of cells in different stages of budding, as described in greater detail below (Table 5 and data not shown). This postreplicative budded-cell arrest upon shifting a tor2^ts mutant to nonpermissive temperature differs from an apparently random arrest that was observed when TOR2 was depleted by down-regulating its transcription using the regulatable GAL1 promoter (KUNZ et al. 1993 ; HELLIWELL et al. 1994 ). At present, we cannot provide an explanation for this apparent discrepancy. However, it may be related to the fact that depleting TOR2 by down-regulating its transcription is a much more gradual depletion than that obtained by shifting the temperature-sensitive mutants to high temperature; cells containing TOR2 under control of the glucose-repressed galactose promoter arrest growth, at 30°, only after approximately 14 hr in glucose medium.

View larger version (57K): [in this window] [in a new window]   Figure 2. —FACS analysis of wild-type and tor ts strains at nonpermissive temperature. Strains are as follows: control (TOR1 TOR2) is SH100; class A (TOR1 tor2-21ts) is SH121; class B (tor1 tor2-30 ts) is SH230; class C (tor1 tor2-21ts) is SH221. The DNA content in cell populations of each strain was analyzed at hourly intervals after a shift to the nonpermissive temperature; 50,000 events were analyzed. Class B and C strains arrest with 1n DNA content; class A strains arrest with 2n DNA content.

View larger version (18K): [in this window] [in a new window]   Figure 3. —Growth rates of TOR2 (wild type, SH100), tor2-21ts (class A, SH121), tor1 tor2-30 ts (class B, SH230), tor1 tor2-21ts (class C, SH221), and tor2-30 ts (class D, SH130) strains. At time zero, exponentially growing cells were diluted to an OD600 of approximately 0.05 in prewarmed media and shaken at 37°. All values for a particular strain were normalized to its zero timepoint.

In contrast to class A mutants, all the class B and C mutants arrested within one generation (within 3–4 hr) with >80% of the cells in G1 (1n DNA content). Results are shown for the class B mutant SH230 (tor1 tor2-30 ^ts) and the class C mutant SH221 (tor1 tor2-21^ts) (Figure 2; Figure 3). In addition, the average cell size of the strains increased after shift to 37°, for unknown reasons. The G1 arrest is in agreement with previous observations (KUNZ et al. 1993 ; HELLIWELL et al. 1994 ; BARBET et al. 1996 ) and furthermore demonstrates that all the isolated tor2^ts alleles are temperature sensitive for the shared TOR function, regardless of the state of the other function of TOR2. Thus, no tor2^ts allele was isolated that was defective for only the unique function.

The class D mutants SH130 (tor2-30 ^ts) and SH1119 (tor2-119 ^ts) formed normal-sized colonies on solid medium. In liquid medium, strain SH130 exhibited a slight reduction in growth rate, as compared to the control strain, but this pseudomutant eventually reached a stationary phase cell density similar to that of control strain SH100.

The unique function of TOR2 is involved in the organization of the actin cytoskeleton:
We have shown recently that the unique function of TOR2 is required for the organization of the actin cytoskeleton. Class A strain SH121 is defective in the cell-cycle-dependent, polarized distribution of the actin cytoskeleton, and overexpression of the actin- and tubulin-specific chaperone TCP20 suppresses both the growth and actin defects of SH121 (tor2-21^ts) (SCHMIDT et al. 1996 ). To confirm and extend the finding that the unique function of TOR2 is involved in actin organization, we examined the budding index and actin distribution of the class A mutant SH121 (tor2-21^ts), the class D mutant SH130 (tor2-30^ts), and the control strain SH100 (Table 5; Figure 4). Class B and C mutants were not examined because these mutants arrest as unbudded G1 cells in which the actin cytoskeleton is normally not polarized. In agreement with the FACS analysis, which indicated that SH121 cells accumulated in G2/M, SH121 had more cells in the budding stages of the cell cycle. In particular, SH121 preferentially accumulated small-budded cells, indicating that this mutant had a defect in the early stages of bud growth. Interestingly, class D mutant SH130 (tor2-30^ts) also showed an altered distribution, which, although not as severe as for the class A mutant, also favored budded cells.

View larger version (119K): [in this window] [in a new window]   Figure 4. —Loss of only the unique function of TOR2 causes a severe actin defect. Analysis of the actin distribution (Actin), nuclear distribution (DAPI), and cell morphology (Nomarski) of wild-type TOR2 (SH100), class A tor2-21ts (SH121), and class D tor2-30 ts (SH130). Cells were fixed for actin and DNA staining 8 hr after shift to the nonpermissive temperature in rich medium.

The budded cells in this analysis were assessed for actin distribution (Table 5; Figure 4). Actin was considered to be abnormally distributed (apolar) if more than five actin patches could still be seen in the mother cell and if these patches were not adjacent to the bud neck. Class A mutant SH121 (tor2-21^ts) exhibited poor polarization of actin patches, with 80% of the cells analyzed having improperly distributed actin (Figure 4). Additionally, very few or no actin cables could be seen in the mutant cells, implying defects in cable assembly. The other class A mutant SH1156 (tor2-156^ts) exhibited a similar defect in actin distribution (data not shown). Interestingly, 47% of the class D mutant (SH130) cells had an altered actin distribution as compared with 16% of the control cells (SH100). This intermediate effect was apparently not sufficient to severely affect growth, since SH130 (tor2-30 ^ts) formed normal colonies. Yeast cells can tolerate a certain amount of cytoskeletal perturbation and still grow relatively normally, as observed previously with mutants defective in actin itself or in actin-binding proteins (NOVICK and BOTSTEIN 1985 ). Thus, it appears that in a class A mutant there is a severe defect in actin polarization that slows down or prevents bud growth without affecting DNA replication. This defect leads to a cell population dominated by small-budded cells with a replicated genome. The unique function of TOR2 is thus involved in the correct localization of actin and in bud growth. The defect in bud growth is presumably a secondary consequence of the actin defect.

The shared function of TOR2 is involved in protein synthesis:
We have recently demonstrated that depletion of TOR1 and TOR2 or rapamycin treatment of wild-type cells causes an immediate and severe block in protein synthesis. Furthermore, we have shown that this block occurs at the level of translation initiation and is the cause of the early G1 arrest seen with the loss of TOR or rapamycin treatment (BARBET et al. 1996 ). We wished to confirm these observations for class B and C mutants (tor1 tor2^ts). Figure 5 shows the results of continuous labeling experiments that measure the amount of labeled methionine incorporated into protein in these mutants at times after a shift to nonpermissive temperature, thereby giving an indication of the rate of protein synthesis. As expected, class B (SH230 and SH2119) and C (SH221 and SH2156) mutants exhibited a rapid and severe defect in the amount of label incorporated into protein (Figure 5 and data not shown), in agreement with previous data and congruent with the fact that these mutants arrested within one cell cycle and in G1 (Figure 2, Figure 3, and data not shown). As both types of mutants exhibited a similarly severe translation defect, the unique function of TOR2 did not appear to have a role in translation. To assess further whether the unique function of TOR2 could be involved in the regulation of translation, we examined the rates of label incorporation in the two class A mutants. The class A mutants, SH121 (tor2-21^ts) and SH1156 (tor2-156^ts), showed a relatively mild reduction in incorporation rates, suggesting that the unique function of TOR2 is not directly involved in the regulation of translation. This mild loss of translation seen with SH121 and SH1156 is presumably an indirect consequence of the reduction in growth of these strains after shift to nonpermissive temperature.

View larger version (22K): [in this window] [in a new window]   Figure 5. —Protein synthesis of tor ts strains after shift to nonpermissive temperature. 35S-methionine incorporation rates of tor2-21ts (class A, SH121), tor2-156ts (class A, SH1156), tor1 tor2-30 ts (class B, SH230), tor1 tor2-21ts (class C, SH221), tor1 tor2-156 ts (class C, SH2156), and tor2-30 ts (class D, SH130) mutants normalized to a TOR2 (wild type, SH100) strain. The experiment was performed as described in MATERIALS AND METHODS. The data from a representative experiment are shown. The label was added, and the cells were shifted to 37° at time zero.

Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 rescues a class A mutant:
The above analysis of class A, class B, and class C mutants supports the existence of at least two functions for TOR2 (Table 3). Class A mutants arrest within a few generations as small-budded cells in G2/M and with an actin defect, whereas class B and C mutants arrest within one generation in G1 because of a translation initiation defect. To identify the two pathways by which TOR2 may control actin organization and translation initiation, we isolated multicopy suppressors of class A mutant SH121 and of class B mutant SH229 (see MATERIALS AND METHODS).

To identify the pathway mediating the TOR2-unique function, we isolated high-copy suppressors that rescue class A mutant SH121 (tor2-21^ts). Plasmids isolated as multicopy suppressors were sequenced and subcloned to identify the ORF with suppressor activity. TOR2 itself was isolated as a suppressor four times. Other multicopy suppressors were MSS4, PKC1, PLC1, RHO2, and SUR1 (Figure 6; Table 6).

View larger version (90K): [in this window] [in a new window]   Figure 6. —Suppressor analysis of tor ts strains lacking either the unique (class A), the shared (class B), or both (class C) functions of TOR2. Class A (TOR1 tor2-21ts) strain is SH121; class B (tor1 tor2-29 ts) strain is SH229; class C (tor1 tor2-21ts) strain is SH221. The strains were transformed with 2µ URA3 plasmids bearing the genes denoted in the figure (RHO2, pC-186; ROM2, pAS30; MSS4, pSH22; PKC1, pSH24; SUR1, pSH27; PLC1, pSH25; TOR2, pJK3-3), streaked on rich medium, and incubated for 2.5 (plates A and C) or 3.5 (plate B) days at 37°. Table 6 summarizes these data.

MSS4, a strong suppressor obtained once (clone pSH121.10), encodes an essential PI-4-P 5-kinase homolog (YOSHIDA et al. 1994B ). FAB1 encodes the only other PI-4-P 5-kinase homolog in yeast (YAMAMOTO et al. 1995 ), and we thus examined whether overexpression of FAB1 could also rescue SH121. FAB1 on a high-copy-number vector (pEMY105) was unable to suppress the growth defect of SH121 at nonpermissive temperature (data not shown). We also examined whether overexpression of either STT4 (YEp352-STT4) or PIK1 (pGB1) could rescue SH121; these two genes encode the two PI 4-kinases in yeast, and MSS4 was first isolated as a multicopy suppressor of an STT4 mutation (YOSHIDA et al. 1994A , YOSHIDA et al. 1994B ). Neither STT4 or PIK1 on high copy number vectors suppressed the growth arrest of SH121. Finally, we examined if overexpression of MSS4 could rescue a strain disrupted for TOR2. Strain MH346 (tor2::ADE2/TOR2) containing the MSS4 multicopy suppressor was sporulated and dissected. For each tetrad, only the two TOR2 segregants of the four progeny were viable, and of these, most had retained the high-copy-number MSS4 plasmid. Thus, overexpression of MSS4 could not suppress a tor2-null allele, unlike the tor2^ts allele, suggesting that suppression by MSS4 requires residual TOR2 activity.

PKC1, a strong suppressor obtained twice (clones pSH121.8 and pSH121.25), encodes the yeast homolog of protein kinase C (LEVIN et al. 1990 ). Multiple copies of PKC1 could not suppress a tor2-null allele (tor2:: ADE2), as assayed as described above for MSS4.

PLC1, a moderate suppressor obtained once (clone pSH121.56), encodes the yeast homolog of phospholipase C (FLICK and THORNER 1993 ; PAYNE and FITZGERALD-HAYES 1993 ; YOKO-O et al. 1993 ). A subclone of pSH121.56 (pSH25) containing only PLC1 was also only a moderate suppressor of tor2-21^ts. The PLC1 plasmid pSH25 did not suppress a tor2-null allele (tor2:: ADE2).

RHO2, a strong suppressor obtained once (clone pSH121.33), encodes a small Rho-like GTPase. We also found RHO2 as a multicopy suppressor of a tor2 mutation by an independent and concurrent approach (SCHMIDT et al. 1997 ). In this other study, we also found that overexpression of ROM2, encoding the GDP/GTP exchange factor (GEF) for RHO2, rescues strain SH121, although we never obtained ROM2 in the multicopy suppressor isolation performed here.

SUR1, a weak suppressor obtained three times (clones pSH121.28, pSH121.30, and pSH121.59), encodes a protein with no homology to known proteins. The function of SUR1 is unknown, but it is possibly involved in the maintenance of phospholipid levels (see DISCUSSION).

Overexpression of PLC1 or MSS4 rescues a class B mutant:
To identify the pathway mediating the TOR-shared function, we isolated multicopy suppressors that rescue class B mutant SH229 (tor1 tor2-29 ^ts). TOR2 itself was isolated as a multicopy suppressor 12 times. Five other suppressor plasmids were obtained and found to have overlapping fragments containing the PLC1 gene in common. We confirmed that overexpression of PLC1 alone was sufficient for suppression by transforming the class B mutants SH229 (tor1 tor2-29 ^ts) and SH2103 (tor1 tor2-103^ts) with pSH25 (pSEY18::PLC1) and assessing growth on rich medium at the nonpermissive temperature (Figure 6 and data not shown). The suppression by PLC1 was moderate.

We then examined whether any of the multicopy suppressors, other than PLC1, that rescued a class A mutant could also rescue a class B mutant (Figure 6B; Table 6). Neither PKC1, RHO2, ROM2, nor SUR1 could rescue class B mutant SH229, as judged by growth on solid rich medium at 37°, even after prolonged incubation. MSS4 was able to rescue strain SH229 at high temperature; however, the suppression by MSS4 was weaker than that by PLC1 and evident only after prolonged incubation.

As PLC1 was isolated as a multicopy supppressor of a TOR-shared function defect, we also examined whether high-copy PLC1 could suppress a tor1 mutation. tor1 strains have a slow-growth phenotype that is exacerbated at low and high temperatures, and at least at 39° tor1 cells accumulate in the G1 phase of the cell cycle (S. B. HELLIWELL and M. N. HALL, unpublished results). We transformed plasmids bearing TOR1, PLC1, and an empty vector into MH349-3d (tor1::LEU2-4) and examined the growth of the resulting transformants at 24° and 39°. At both temperatures, multicopy PLC1 was able to partially suppress the growth defect of the tor1 strain (data not shown), confirming that overexpression of PLC1 could suppress a defect in the TOR-shared function. As PLC1 was able to suppress defects in either the shared or the unique function of TOR2, we examined whether it could rescue a class C mutant that lacks all TOR function. pSH25 (pSEY18::PLC1) was transformed into class C mutant SH221 (tor1 tor2-21^ts ) and assessed for growth at the nonpermissive temperature. After 2.5 days incubation at 37°, transformants formed small colonies, indicating that overexpression of PLC1 could weakly suppress a complete TOR deficiency (Figure 6C). Overexpression of MSS4, PKC1, RHO2, ROM2, or SUR1 did not rescue strain SH221.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

TOR1 and TOR2 encode 67% identical PI kinase homologs, and mutations in either gene confer rapamycin resistance (HEITMAN et al. 1991 ; KUNZ et al. 1993 ; HELLIWELL et al. 1994 ). TOR2 has two essential functions, only one of which can be performed by TOR1. We have shown previously that the TOR-shared function is involved in the control of translation initiation; loss of this function causes cells to arrest in early G1 within one cell cycle (BARBET et al. 1996 ). Recent evidence indicates that the unique function of TOR2 is involved in the control of the actin cytoskeleton (SCHMIDT et al. 1996 ; SCHMIDT et al. 1997 ). In this study, we sought to genetically dissect these two functions at the level of the TOR2 gene by generating a large number of tor2^ts mutants. We expected that some of these mutants would be conditional for one function while being unaffected in the other. This would support the model that TOR2 is part of two separate signaling pathways and would facilitate the analysis of each pathway individually.

Twenty-nine tor2^ts alleles were obtained and characterized. All of the tor2^ts alleles were defective in the TOR-shared function such that all tor1 tor2^ts strains arrested growth like strains depleted for TOR. However, the same alleles varied in their ability to provide the TOR2-unique function such that they caused growth defects of varying severity when in a strain background containing a wild-type TOR1 gene. The alleles varied from being unaffected (tor2-30 ^ts and tor2-119 ^ts) to being fully defective for the unique function (tor2-21^ts and tor2-156^ts). No TOR2 allele was isolated that was temperature sensitive for only the unique function, for unknown reasons.

The alleles obtained allowed the construction of three classes of tor mutants that arrest growth for different reasons. Class A mutants, strains SH121 (tor2-21^ts) and SH1156 (tor2-156^ts), are tor2-unique ^ts and arrest within a few generations, primarily as small-budded cells with a 2n DNA content. These mutants are defective in the organization of the actin cytoskeleton. The actin defect is presumably the cause of growth arrest (SCHMIDT et al. 1996 ), and may account for the terminal phenotype of small-budded cells. Class B mutants, strains SH230 (tor1 tor2-30 ^ts) and SH229 (tor1 tor2-29 ^ts), and class C mutants, strains SH221 (tor1 tor2-21^ts) and SH2156 (tor1 tor2-156^ts), are tor-shared^ts and tor-null^ts, respectively. These mutants arrest rapidly, in G1, and are defective in translation. We have previously shown that the loss of translation is responsible for the G1 arrest phenotype. Importantly, class D strains, SH130 (tor2-30 ^ts) and SH129 (tor2-29 ^ts), are viable pseudomutants, indicating that these tor2^ts alleles encode a TOR2 protein that can carry out the unique function, but not the shared function. Thus, the mutants support the model that TOR2 performs two different growth-controlling functions: one function is involved in G1 progression via control of translation initiation, and the other function is involved in bud growth via control of the actin cytoskeleton. These two functions, or readouts, of TOR2 are presumably mediated by two separate TOR2 signaling pathways.

Suppressor analyses with the mutants deficient in either the shared function or the unique function provided insight into the nature of the possible signaling events downstream of TOR2. Many genes were isolated that when overexpressed allow growth of a tor2^ts strain lacking the TOR2-unique function. These genes encode proteins (MSS4, PKC1, PLC1, RHO2, ROM2, and SUR1) directly or indirectly implicated in signaling and, in particular, in protein kinase C signaling. MSS4 is a PI-4-P 5-kinase homolog (YOSHIDA et al. 1994B ) and may be upstream of the yeast protein kinase C PKC1, which was also isolated as a multicopy suppressor. PLC1 is the yeast homolog of mammalian PI-phospholipase C, which has been shown to exhibit a calcium dependence with respect to its hydrolytic activity toward PI and PI-4,5-P₂ (PIP₂) (FLICK and THORNER 1993 ). RHO2 and ROM2 are components of a Rho-type GTPase switch that also controls PKC1 (NONAKA et al. 1995 ; OZAKI et al. 1996 ). SUR1 is a protein of unkown function involved in maintenance of phospholipid levels. SUR1 was originally identified as a mutation that suppresses the growth defect of strains lacking RVS161 or RVS167 (DESFARGES et al. 1993 ; TAKITA et al. 1995 ). Importantly, rvs161 and rvs167 mutants exhibit an altered actin cytoskeleton and an inability to maintain viability upon carbon or nitrogen starvation (CROUZET et al. 1991 ; BAUER et al. 1993 ). Analysis of a sur1-1 mutant demonstrated reduced levels and altered ratios of membrane phospholipids (DESFARGES et al. 1993 ). Overexpression of MSS4 or PLC1 were also found to rescue a mutant lacking the TOR-shared function. Additional studies will be required to determine whether MSS4, PKC1, PLC1, and SUR1 are in a signaling pathway with TOR2 or whether they are in a parallel pathway and are suppressing via a bypass mechanism. However, the suppressors strongly suggest that there are signaling events downstream of TOR2 with regard to both the shared and the unique functions of TOR2.

Furthermore, we anticipate that at least some of the suppressors, including PKC1, are in a TOR2 pathway. First, the growth arrest phenotype of the class A mutant SH121 (tor2-21^ts) is similar to the arrest phenotype of strains expressing a conditional allele of PKC1; both tor2-21^ts and pkc1^ts mutants arrest with increased numbers of small-budded cells containing replicated DNA, and both mutants are rescued by the addition of 0.1 M CaCl₂, 0.5 M NaCl, or the osmotic stabilizer sorbitol to the growth medium (LEVIN and BARTLETT-HEUBUSCH 1992 ). Second, we have recently shown that TOR2 signals to the actin cytoskeleton by activating a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7 (SCHMIDT et al. 1997 ), and others have shown recently that PKC1 is regulated by RHO1 (NONAKA et al. 1995 ; KAMADA et al. 1996 ). However, pkc1 mutants have not been shown to have actin defects, although mutants altered in MPK1, a mitogen-activated protein kinase (MAPK) regulated by PKC1, exhibit delocalized actin (MAZZONI et al. 1993 ). PKC1 is responsible for the regulation of genes involved in the remodeling and synthesis of the cell wall at the growing bud tip (IGUAL et al. 1996 ), suggesting that TOR2 is a global controller of both actin reorganization and cell wall biosynthesis. More careful analysis of the roles played by TOR2, RHO1, PKC1, and the cell integrity pathway will shed light on the importance of TOR2 in both cell wall biosynthesis and actin reorganization in polarized cell growth.

A surprising observation was that two of the multicopy suppressors, MSS4 and PLC1, rescue both class A and class B mutants. Regardless of whether MSS4 and PLC1 are in a TOR2 pathway or in a parallel pathway, this finding suggests that the two signaling functions of TOR2 are more related than previously thought. It remains to be determined how the two different readouts of TOR2—translation initiation and organization of the actin cytoskeleton—are affected by the same signaling proteins. There may be crosstalk between two TOR2 signaling pathways such that overexpressing a protein in one pathway affects the other. Alternatively, two pathways may have common components or the common suppressors may have two functions. A sharing of components between two TOR2 signaling pathways could ensure that temporal (protein synthesis) and spatial (actin cytoskeleton organization) control of cell growth are coordinated (SCHMIDT et al. 1997 ).

	FOOTNOTES

¹ Present address: Institute for Biochemistry and Molecular Biology, University of Bern, CH-3012 Bern, Switzerland.

	ACKNOWLEDGMENTS

We thank THOMAS BECK, GERHARD PARAVICINI, JEREMY THORNER and SCOTT EMR for plasmids, ANJA SCHMIDT and TOBIAS SCHMELZLE for comments on the manuscript, and members of the lab for helpful discussions. N. B. was the recipient of a long-term European Molecular Biology Organization fellowship. This work was supported by grants from the Swiss National Science Foundation and the Canton of Basel to M.N.H.

Manuscript received June 13, 1997; Accepted for publication October 6, 1997.

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