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Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive in a sit4-deleted Strain *

Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided,... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 49, pp. 37980 –37992, December 8, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive □ S in a sit4-deleted Strain Received for publication, July 21, 2006, and in revised form, September 26, 2006 Published, JBC Papers in Press, October 2, 2006, DOI 10.1074/jbc.M606973200 ‡ § § ‡1 Jennifer J. Tate , Andre´ Feller , Evelyne Dubois , and Terrance G. Cooper ‡ § From the Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163 and the Institut de Recherches Microbiologiques J.-M. Wiame, Laboratoire de Microbiologie Universite´ Libre de Bruxelles, B1070 Brussels, Belgium Tor1,2 control of type 2A-related phosphatase activities in reporter in studies investigating Tor function (5, 6). A major Saccharomyces cerevisiae has been reported to be responsible function of Gln3 is to activate Nitrogen Catabolite Repression for the regulation of Gln3 phosphorylation and intracellular (NCR) -sensitive transcription needed to exploit poor nitrogen localization in response to the nature of the nitrogen source sources in the environment when nothing better is available available. According to the model, excess nitrogen stimulates (biology reviewed in Refs. 7–11). In the presence of good nitro- Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then com- gen sources (e.g. glutamine or ammonia in some strains) genes plexes with and inactivates Sit4 phosphatase, thereby prevent- encoding permeases and enzymes required for the transport ing it from dephosphorylating Gln3. Phosphorylated Gln3 com- and degradation of poor nitrogen sources (e.g. proline) are min- plexes with Ure2 and is sequestered in the cytoplasm. When imally expressed. In contrast, when good nitrogen sources are Tor1,2 kinase activities are inhibited by limiting nitrogen, or limiting or only poor sources are available, expression of these rapamycin- treatment, Tap42 can no longer complex with Sit4. genes increases (7–11). Active Sit4 dephosphorylates Gln3, which can then localize to Two pivotal observations opened a new era of investigation the nucleus and activate transcription. The paucity of experi- into the mechanisms regulating Gln3 function. (i) During times mental data directly correlating active Sit4 and Pph3 with Gln3 of high NCR, GATA sequences (binding sites for Gln3 and regulation prompted us to assay Gln3-Myc phosphorylation Gat1/Nil1) in the promoter of an NCR-sensitive gene (CAN1) and intracellular localization in isogenic wild type, sit4, pph3, are unoccupied by Gln3 and Gat1, and thus available to serve as and sit4pph3 deletion strains. We found that Sit4 actively surrogate TATA-binding protein (TBP) binding sites (12). brought about Gln3-Myc dephosphorylation in both good Gln3 and Gat1 occupancy of CAN1 GATA sequences corre- (glutamine or ammonia) and poor (proline) nitrogen sources. lates with Gln3 and Gat1 localization to the cytoplasm of cells in This Sit4 activity masked nitrogen source-dependent changes in which NCR-sensitive transcription is low, and to their nuclei Gln3-Myc phosphorylation which were clearly visible when when expression is high (12, 13). (ii) Treating YPD-grown wild- SIT4 was deleted. The extent of Sit4 requirement for Gln3 type cells with rapamycin induces Gln3 dephosphorylation, nuclear localization was both nitrogen source- and strain-de- Gln3 nuclear localization and high level NCR-sensitive tran- pendent. In some strains, Sit4 was not even required for Gln3 scription. In contrast, Gln3 is phosphorylated, localizes to the nuclear localization in untreated or rapamycin-treated, proline- cytoplasm, and NCR-sensitive transcription is low in similarly grown cells or Msx-treated, ammonia-grown cells. grown, untreated cells (14–17). The above correlations along with the protein-protein asso- ciation and phosphorylation relationships between Tor1,2, Applications of clinically important derivatives of the anti- Tap42, Tip41, Sit4, and Sit4-associated proteins (Saps), led to a inflammatory and anti-neoplastic drug rapamycin have steadily proposal describing Tor1,2 regulation of Gln3 phosphorylation grown in concert with our increased understanding of how cel- via control of the type 2A-related protein phosphatases, Sit4 lular processes are regulated by their target, mTor (mammalian and/or Pph3 (Fig. 1) (5, 14–22). In bare outline, the model pos- Target of Rapamycin) (1–4). The Saccharomyces cerevisiae its that signals of nitrogen excess (glutamine or a metabolite of GATA family transcription activator, Gln3 is widely used as a it) are sensed by Tor1,2, which become active and phospho- rylate Tap42 and/or Tip41 (14, 17–22). The outcome of these phosphorylations is the association of Tap42 with Sit4, thereby * This work was supported by National Institutes of Health Grant GM-35642 and NSF Collaborative Grant DMS-0443855 (to T. G. C.) and by a grant from inactivating the phosphatase (18–20). In this inactive form, Sit4 COCOF (Commission de la Communaute´ franc¸aise) (to E. D.). The costs of is unable to dephosphorylate Gln3, resulting in its sequestra- publication of this article were defrayed in part by the payment of page tion in the cytoplasm as a Gln3Ure2 complex (Fig. 1) (14, 17). charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. When cells are treated with rapamycin, or the glutamine syn- □ S The on-line version of this article (available at http://www.jbc.org) contains thetase inhibitor, methionine sulfoximine (Msx), which inhib- supplemental Fig. S1. To whom correspondence should be addressed: Dept. of Molecular Sci- ences, University of Tennessee, Memphis, TN 38163. Tel.: 901-448-6179; The abbreviations used are: NCR, nitrogen catabolite repression; Msx, L-me- Fax: 901-448-8462; E-mail: [email protected]. thionine sulfoximine; DAPI, 4,6-diamidino-2-phenylindole. 37980 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 This is an Open Access article under the CC BY license. Sit4 Phosphatase Active Irrespective of Nitrogen Source standing of Sit4 participation in the Tor1,2 control pathway regulating Gln3: (i) Is Sit4 required for Gln3 dephosphorylation and/or nuclear accumulation of Gln3 when cells are provided with a poor nitrogen source or treated with Msx?, (ii) Is Sit4 inactive under conditions where Gln3 is restricted to the cyto- plasm (i.e. nitrogen excess)? (iii) What are the relative require- ments of Sit4 and Pph3 for Gln3 dephosphorylation and nuclear localization in response to growth with various nitro- gen sources, rapamycin and Msx treatment? (iv) Does the Tap42-Sit4-requirement for Gln3 regulation found in some instances but not others reflect strain-specific differences when experiments are performed under identical conditions (14, 15)? To fill in some of the gaps in our understanding of type 2A-related phosphatases and their relationship to Gln3 regula- tion, we performed a systematic analysis of Gln3 phosphoryla- tion and intracellular localization, comparing these parameters in wild type, single and double sit4 and pph3 mutant cells. Our data demonstrate that: (i) Sit4 is active with respect to Gln3- Myc phosphorylation levels under conditions of excess nitro- gen in which Tor1,2 are active, (ii) Gln3-Myc phosphoryla- FIGURE 1. Abbreviated diagram of the model describing the regulation of tion is nitrogen source-dependent in a sit4, indicating that Gln3 phosphorylation and intracellular localization. The diagram was derived from Refs. 14, 20, and 21. Sit4 activity is unlikely to be the major determinant responsible for nitrogen-dependent changes in Gln3-Myc phosphoryla- its the synthesis of glutamine, Tor1,2 are inhibited and cease tion, (iii) rapamycin-induced alteration of Gln3-Myc phos- phosphorylating Tap42. Dephosphorylated Tap42 dissociates phorylation levels requires Sit4, whereas alterations elicited by from Sit4 thereby allowing Sit4 to be active and dephospho- Msx or the nitrogen source do not, (iv) the extent of the Sit4 rylate Gln3, resulting in or permitting Gln3 to dissociate from requirement for Msx- and rapamycin-induced nuclear localiza- Ure2 and enter the nucleus (14, 17). tion of Gln3-Myc is nitrogen source-dependent, (v) the Sit4 Experimentally, Sit4 was first implicated in Gln3 regulation requirement for nuclear Gln3-Myc localization is strain by the demonstration that neither rapamycin-induced Gln3 dependent, and (vi) demonstrable Pph3 influence on Gln3 reg- nuclear localization, nor dephosphorylation occurred in a YPD- ulation was minimal compared with that of Sit4. grown sit4 mutant (14). In a subsequent report, rapamycin was MATERIALS AND METHODS observed to induce modestly less GAP1 expression in a pph3 mutant than wild type, implicating the second type 2A-related Strains and Culture Conditions—S. cerevisiae strains used in phosphatase in Gln3 regulation (17). Most recently, Msx-in- this work are listed in Table 1. Strains were grown at 30 °C to duced Gln3 nuclear localization was reported not to occur in a mid-log phase (A  0.5) in YNB (without amino acids or 600 nm sit4 mutant growing in S.D. medium (22). ammonium sulfate) medium, containing 2% glucose, required Although impressive progress has been made in identifying auxotrophic supplements (120 g/ml leucine, 20 g/ml uracil, additional proteins involved in Tor1,2 global influence on cel- 20 g/ml histidine, 20 g/ml tryptophan, 20 g/ml arginine, lular processes, several observations do not fit comfortably with and the nitrogen source (0.1% final concentration) indicated. expectations generated by the model (23). Among them are: (i) YPD medium consisted of 10 g yeast extract, 20 g bactopeptone, In genetic studies, Tap42 behaves more like a positive than a and 20 g dextrose per 1000 ml. Rapamycin (Sigma-Aldrich) negative regulator of Sit4 (18). (ii) Sit4 association with Tap42 is (dissolved in 10% Tween 20  90% ethanol) was added to the required for Sit4 activity (18, 24, 25). (iii) Detectable Gln3- cultures, where indicated, to a final concentration of 0.2 g/ml Myc phosphorylation and intracellular localization do not for 20 or 30 min prior to cell harvest. Msx (Sigma-Aldrich) was correlate with one another in good versus poor nitrogen dissolved in water and added to the cultures, where indicated, sources, during nitrogen starvation, following Msx treatment, to a final concentration of 2 mM for 30 min prior to cell harvest and beyond 30 min of rapamycin treatment (26). (iv) Msx (by filtration). Where indicated, yeast were transferred (by fil- increases Gln3-Myc phosphorylation whereas rapamycin tration and resuspension) from one medium to another that decreases it even though both inhibitors elicit nuclear localiza- had been pre-warmed, pre-aerated, and contained the required tion of Gln3-Myc (27). auxotrophic supplements. As noted in Fig. 1, type 2A-related Sit4 (and Pph3) protein Strain Construction—Previous data, showing strain differ- phosphatase activities have been proposed to directly regulate ences can be critically important in evaluating the regulation of Gln3 phosphorylation and localization (14, 17, 22). The corre- Gln3 function (28, 29), recommended the use of isogenic lations upon which this conclusion rests are the failure of Gln3 strains. Recombinant methods were used to create specific dephosphorylation, nuclear localization, and NCR-sensitive deletions in two strain backgrounds: (i) TB123, used as the wild transcription to occur in YPD-grown, rapamycin-treated sit4 type in the original experiments demonstrating rapamycin-in- cells. There are, however, several important gaps in our under- duced alterations in Gln3 phosphorylation and intracellular DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37981 Sit4 Phosphatase Active Irrespective of Nitrogen Source TABLE 1 S. cerevisiaes trains used in this work Strains JK9-3da, TB123, TB50, and TB136 are described in Refs. 14 and 20 and BY4709 in Ref. 32. Strains JK9-3da and TB50 are isogenic except at the his3 and his4 loci. Strains TB123 and TB136 are isogenic, except for at the indicated loci, to JK9-3da. Strain Parent Genotype Primer coordinates TB123 JK9-3da MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL , HMLa, GLN3-Myc KanMX None TB50 MATa, leu2-3,112, ura3-52, trp1, his3, rme1, HMLa None JK9-3da MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa None TB136 MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL , HMLa, GLN3-Myc KanMX, None sit4::kanMX BY4709 MAT, ura3 None FV017 BY4709 Mat,ura3, GLN3-Myc KanMx FV1 BY4709 MAT, ura3, sit4::natMX, GLN3-MYC kanMX 5, 450 to 429 & 23 to 1 3 937 to 955 & 1380 to 1400 FV2 BY4709 MAT, ura3, pph3::natMX, GLN3-MYC kanMX 5, 400 to 379 & 22 to 1 3 927 to 950 & 1206 to 1228 FV3 TB123 MATa, his4, leu2-3,112, ura3, trp1, rme1, pph3::natMX, 5, 400 to 379 & 22 to 1 GLN3-myc kanMX 3 927 to 950 & 1206 to 1228 FV4 TB136 MATa, his4, leu2-3,112, ura3, trp1, rme1, sit4::kanMX, sit4:5, 450 to 429 & 23 to 1 pph3::natMX, GLN3-MYC kanMX 3 937 to 955 & 1380 to 1400 pph3:5, 400 to 379 & 22 to 1 3 927 to 950 & 1206 to 1228 localization (14), as well as in our own previous experiments (26, 27, 30, 31), and (ii) derivatives of BY4709, used in the sys- tematic deletion project (32). Deletion strains (FV series) were constructed using the long flanking homology strategy of Wach (33). The kanMX or natMX cassettes, flanked by about 500 bp corresponding to the promoter and terminator regions of the target genes, were syn- thesized by a two-step PCR procedure (primer coordinates are FIGURE 2. Intracellular distribution of Gln3-Myc in YNB-glutamine- in Table 1). DNA fragments (blunt-ended PCR products) con- grown sit4 (TB136) cells treated with rapamycin. Indirect immunofluo- taining the various constructs were used to transform the rescent staining of Gln3-Myc and DAPI-positive material appear in red and blue, respectively (see supplemental materials). appropriate strains. Transformants were selected on YPD medium containing 200 g per ml of geneticin or 100 g per ml of nourseothricin. Correct targeting of the deletions was veri- assess the relative amounts of various Gln3-Myc species fied by PCR analysis, using whole cells as the source of template within a particular lane and then compare the pattern of Gln3- DNA and two sets of primers: (i) a primer 5 of the deletion Myc distribution with that observed in another lane, rather cassette, a second at the 5-end of the Kan gene, and a third at than quantitatively comparing the amounts of particular Gln3- the beginning of the coding sequence of the gene that was Myc species observed in one lane with those in another. Fur- deleted, and (ii) a primer at the 3-end of the Kan gene, a second ther, although we occasionally compared the overall patterns of at the 3-end of the coding sequence of the deleted gene, and a data observed in one Western blot to that of another, detailed third 3 of the deletion cassette. comparisons were restricted to lane profiles contained within a Northern Blot Analysis—Total RNA was extracted as single Western blot membrane unless specifically indicated described earlier (34) and purified using the RNeasy kit (Qia- otherwise. gen). Northern blot analysis was performed as described by Indirect Immunofluorescence Microscopy—Cell preparation Foury and Talibi (35). DIG-DNA probes of about 500 bp were and assay of Gln3-Myc by indirect immunofluorescence was generated by PCR, using primers: 5-CATAACCAGTTGGTG- initially performed as described earlier (31, 37). Although this AGCCC-3 and 5-ACCCCCGTTACTGTATGTGG-3 for method (used in Fig. 2 of this text and Fig. S1 of the supplemen- SIT4,5-TGGGCGATTTTGTGGATAGG-3 and 5-CTGTC- tal materials) performs well for wild-type strains, it was inade- ACTAATCCACCGTCG-3 for PPH3,5-AAACAGCAAGA- quate for analysis of Gln3-Myc intracellular distribution in AAGTCCACTGG-3 and 5-ACCTCTTAATCTTCTAGCC- phosphatase mutants. sit4, and to a lesser extent pph3, AAC-3 for HHT1, and labeled using a PCR DIG (digoxigenin) mutants possess characteristics that, if not circumvented, seri- probe synthesis kit (Roche Applied Science). Hybridizations ously compromise analysis of Gln3-Myc intracellular distri- were carried out according to standard procedures (36). Detec- bution. Gln3-Myc and DAPI stained material (DNA) were tion of DIG-labeled nucleic acids was performed by enzyme asymmetrically distributed to the daughters and mothers, immunoassay with luminescence following the suppliers pro- respectively, of sit4 cells with small to medium sized buds (Fig. cedure (Roche Applied Science). The Hybond-N nylon mem- 2). The frequency of this morphology was increased by rapamy- branes were exposed 120 min and analyzed with a chemilumi- cin treatment. This phenomenon and data suggesting that it nescence camera (Chemi-Smart from Vilbert-Lourmat). derives from differential sensitivity of mother and daughter Western Blot Analysis—Cells were harvested by the filtration cells walls to zymolyase digestion as well as differential affinity method of Tate et al. (27), and crude cell extracts prepared as for lysine-coated slides are described under supplemental described by Cox et al. (26). As noted earlier (27), we prefer to materials. Therefore, we used a modified form of another 37982 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source method (38) in all but Fig. 2. The modifications were: (i) fixation in the growth medium for 60 min, (ii) addition of -mercapto- ethanol (20 mM final concentration) to the zymolyase digestion mixture, and (iii) increased digestion of mutant cells (34–37 min) and decreased digestion (15–17 min) for the wild type. Digesting wild type cells for the same length of time as the sit4 resulted in significant degradation of cell integrity. Determination of Intracellular Gln3-Myc Distribution—To determine the intracellular distribution of Gln3-Myc gener- ated by the experimental conditions used, an average of 150– 200 cells, from randomly chosen fields of each experimental condition, were scored in a double blind manner to determine the distribution of Gln3-Myc . Experiments were independ- ently repeated from two to seven times. Cells were classified into three categories, those in which Gln3-Myc was cytoplas- FIGURE 3. A, Sit4 is active with respect to Gln3-Myc dephosphorylation in mic, nuclear-cytoplasmic, or nuclear. Recognizing that bound- cells provided with a good nitrogen source (YNB-glutamine). Western blot analysis of Gln3-Myc in wild-type (TB123) and sit4 (TB136) cells. Rapa- aries between the middle category and those flanking it would mycin was added for 30 min. prior to cell harvest where indicated (Rap ). be unavoidably subjective, we performed two control experi- B, steady state mRNA levels of SIT4 and PPH3 in wild type (TB123), sit4 ments to determine the precision with which we placed cells (TB136), and pph3 (FV3). Cells were cultured in YNB-glutamine (Gln) or -ammonia (Am. or NH ) medium in the presence ( Rap; 30 min) or absence into two adjacent categories. First, we categorized Gln3-Myc of rapamycin or Msx ( Msx; 20 min). Additionally, glutamine cultures were as being cytoplasmic or nuclear-cytoplasmic in cells from one transferred to proline (Gln to Pro) or nitrogen-free (Gln to N.S.) medium and incubated for 60 min before the cells were harvested. microscopic image and then repeated the count a second time using the same image. Next, we repeated this procedure using a second, randomly selected image derived from the same slide as ants of the Tor1,2 pathway model, i.e. (i) in the presence of a the first image. The cell sample used in this experiment con- good nitrogen source, Tor1,2 are active and (via Tap42 and/or tained approximately equal numbers of cells in which Gln3- Tip41) inhibit Sit4 activity, thus preventing it from dephospho- Myc was categorized as cytoplasmic and nuclear-cytoplas- rylating Gln3, and (ii) Sit4 is active when the Tor1,2 kinases are mic, respectively. The two counts yielded four sets of values, inhibited by growth in limiting nitrogen, thereby making it two from each image used above. The four sets of values varied available to dephosphorylate Gln3. Wild type and isogenic from one another by plus or minus 2–3%. Finally, we performed sit4 cultures were grown in YNB-glutamine medium in the an analogous experiment using two images of a cell sample presence and absence of rapamycin. As expected, and previ- which contained approximately equal numbers of cells in which ously shown by multiple laboratories, rapamycin treatment 13 13 Gln3-Myc was categorized as being nuclear-cytoplasmic and elicited Gln3-Myc dephosphorylation in wild-type cells (Fig. nuclear, respectively. The same variation (2–3%) was 3A, lanes A versus D). In sit4 cultures, two unexpected results observed in this second experiment as well. These results were observed: (i) Sit4 was clearly active in cells provided with a argued that we could categorize Gln3-Myc localization with good nitrogen source, because deleting SIT4 increased Gln3- 13 13 acceptable precision. The patterns of Gln3-Myc distribution Myc phosphorylation relative to wild type (Fig. 3A, lanes A between cell compartments in response to various experimen- versus B). (ii) Rapamycin induced partial Gln3-Myc dephos- tal conditions (strains, nitrogen source, inhibitor treatment) phorylation in a sit4 (Fig. 3A, lanes B versus C), indicating that were also reproducible as was the quantitative distribution of more than Sit4 alone was responsible for rapamycin-induced Gln3-Myc among the three intracellular categories in dephosphorylation. Together, these data indicated that type 2A repeated scoring of images from a single cell sample. Experi- phosphatase participation in the regulation of Gln3 phospho- ment-to-experiment variation was 2–10%, except when proline rylation was likely more complicated or different than previ- was used as nitrogen source. Here, experiment-to-experiment ously reported and motivated a more thorough investigation. values occasionally varied into the 20% range for isolated sam- Most importantly, these data showed that Sit4 is active in glu- ples. Therefore, our attempts to achieve as accurate and precise tamine-grown cells in which Tor1,2 were posited to actively data as possible notwithstanding, we attribute less significance phosphorylate Tap42 (and/or Tip41), thereby bringing about to the precise percentages of Gln3-Myc distribution among inhibition of Sit4 protein phosphatase. the three possible intracellular categories than to the patterns Influence of Sit4 and Pph3 Phosphatase on Steady State Levels of change observed in the distribution of Gln3-Myc when of SIT4 and PPH3 mRNA—Previous reports of both Pph3 and comparing one experimental condition to another, i.e. presence Sit4 functioning in rapamycin-induced Gln3 dephosphoryla- versus absence of inhibitor, nitrogen source identity, wild type tion and localization (14, 17, 22), prompted us to query whether versus mutant strains. the expression of their cognate genes was subject to Tor1,2 or NCR-sensitive regulation. Therefore, we assayed SIT4 and RESULTS PPH3 expression in wild type, sit4,and pph3 strains cultured Sit4 Phosphatase Brings about Gln3-Myc Dephosphoryla- under multiple conditions: the presence and absence of rapa- tion in Cells Provided with Good Nitrogen Sources—Our initial mycin (in glutamine medium) or Msx (in ammonia medium), experiment investigated central, but previously untested, ten- and following transfer from glutamine to proline or nitrogen- DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37983 Sit4 Phosphatase Active Irrespective of Nitrogen Source free medium. As shown in Fig. 3B, neither SIT4 nor PPH3 expression appeared to exhibit a response to the nitrogen source provided. As expected, there was no expression when the genes themselves were deleted (Fig. 3B). Type 2A-related Phosphatase Requirements for Nuclear Localization of Gln3-Myc —Next we evaluated the influence of defects in type 2A-related phosphatases, Sit4 and Pph3, on Gln3-Myc localization in cells cultured as described in Fig. 3B, lanes A–D and with proline as sole nitrogen source. Wild- type cells behaved as expected and previously reported, i.e. Gln3-Myc was cytoplasmic in glutamine-grown cells, and nuclear in proline-grown or rapamycin-treated cultures as well as glutamine-grown cells transferred to proline or nitrogen free medium (Fig. 4, column W.T.). Intracellular Gln3-Myc local- ization in pph3 mutants did not significantly differ from that in wild type (Fig. 4, column pph3). In contrast, Gln3-Myc was predominantly cytoplasmic in sit4 cells treated with rapa- mycin, transferred from glutamine to proline- or nitrogen-free medium, and grown with proline as sole nitrogen source (Fig. 4, column sit4). Results obtained with the sit4pph3 double mutant were similar to those observed with sit4 alone (data not shown). These data, at face value, supported the earlier contention that Sit4 was required for nuclear accumulation of Gln3-Myc when nitrogen was limiting or cells were treated with rapamycin. Contrasting Phosphatase Requirements for Nuclear Localiza- tion of Gln3-Myc in Response to Rapamycin and Msx Treatment—Careful inspection of many images such as those in Fig. 4 suggested the method of classification we had been using might be too crude to describe fully what occurred in response to various experimental perturbations. There were times when Gln3-Myc was neither completely nuclear nor cytoplasmic, i.e. one could see Gln3-Myc fluorescence in both cellular compartments. Therefore, we increased the resolution of our measurements by introducing a third scoring category, nuclear-cytoplasmic localization. Criteria used to place cells in each of the three categories were as follows: (i) cells in which Gln3-Myc could only be detected in the cytoplasm were scored cytoplasmic, (ii) those in which only nuclear localization was detected were scored nuclear, and (iii) those in which stain- ing could be clearly detected in both compartments were scored nuclear-cytoplasmic. The third category was clearly subjective. However, this potential for subjectivity did not prove to be problematic as shown by evaluation of the assay under “Materials and Methods.” Success and reliability of the assay most required consistency in scoring since it was the accurate and reproducible detection of changes in the patterns of Gln3-Myc distribution among the cellular compartments FIGURE 4. Intracellular distribution of Gln3-Myc . This was observed in wild-type (W.T.; TB123), sit4 (TB136), and pph3 (FV3) mutant cells grown that is most critical. in glutamine (Gln) or ammonia ( NH ) medium in the presence ( Rap) or Using these scoring criteria, we assayed Gln3-Myc intracel- absence of rapamycin or methionine sulfoximine (MSX), in proline (Pro) lular localization in wild type and three isogenic phosphatase medium, or in after transfer from glutamine to nitrogen-free (Gln to Nitro- gen Starvation; 60 min.) or proline (Gln to Pro; 60 min.) medium. Images are defective mutants. Gln3-Myc was cytoplasmic in all gluta- presented in pairs in which the Gln3-Myc (red) image appears above the mine-grown wild-type cells (Fig. 5A, red bar, W.T.). Following same one stained with DAPI (blue). rapamycin treatment, Gln3-Myc became nuclear-cytoplas- mic (yellow bar) in about 80% of the cells. In the remaining cells, ization of Gln3-Myc when glutamine-grown cultures were Gln3-Myc was about equally distributed between the nuclear transferred to minimal-proline or nitrogen-free medium (Fig. (green bar) and cytoplasmic (red bar) compartments (Fig. 5A, 5A, W.T.). However, Gln3-Myc was still cytoplasmic in about W.T.). The distribution shifted toward a greater nuclear local- 20% of the cells following nitrogen starvation, which may have 37984 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source Sit4 or Pph3 requirement follow- ing Msx treatment clearly distin- guishing it from the response to rapamycin. Sit4 Requirement of Gln3-Myc Localization Is Nitrogen Source- dependent—The Gln3-Myc dis- tribution data above were difficult to rectify with the idea that Sit4, per- formed the requisite dephosphoryl- ation of Gln3 in preparation for its transport into the nucleus. There- fore, we approached the putative Sit4 requirement from a different direction. If Sit4 functioned down- stream of the nitrogen supply signal in the Gln3 regulatory pathway, then the response to both rapamy- cin and Msx treatment in sit4 mutants should be independent of the nitrogen source provided. If, on the other hand, the nitrogen supply signal was situated downstream of Sit4, or in another regulatory path- way, then the nitrogen source pro- vided to the cells would be expected to influence Gln3 localization in wild-type and sit4 cells. To investigate these possibilities, FIGURE 5. A, intracellular Gln3-Myc distribution in wild type and type 2A-related phosphatase mutants cul- 13 we determined Gln3-Myc local- tured in glutamine (Gln) medium in the presence (Rap) or absence of rapamycin, or after transfer from ization, following rapamycin or Msx glutamine to nitrogen-free (Gln to N. S.; 60 min.) or proline (Gln to Pro; 60 min.) medium. Cells (TB123, TB136, FV3, and FV4) were scored as described under “Materials and Methods”: cytoplasmic (red), nuclear-cytoplasmic treatment in cultures provided with (yellow), and nuclear (green). B, strains identical to those used in A were cultured in ammonia medium in the nitrogen sources ranging from good presence (Msx) or absence of methionine sulfoximine. to poor: glutamine, YPD, ammonia, resulted from insufficient time being allowed for the nitrogen and proline (Fig. 6, A–D). Gln3-Myc was completely cytoplas- reserves of these cells to be completely exhausted before they mic in glutamine medium and this distribution was altered only were harvested. by rapamycin treatment which elicited Sit4-dependent, nucle- When sit4 and pph3 single and sit4pph3 double ar-cytoplasmic localization in most cells (Fig. 6A). Msx treat- mutants were subjected to the same perturbations as the wild ment failed to elicit a response in glutamine-grown cells con- type, quite unambiguous phenotypes were observed. The dis- curring with earlier reports (21, 27). Our experiments do not tribution of Gln3-Myc in a pph3 was nearly indistinguish- distinguish whether this result occurred because (i) providing able from wild type irrespective of the condition tested (Fig. glutamine as the nitrogen source eliminates the need for Msx- 5A). In contrast, Gln3-Myc was restricted to the cytoplasm of inhibited glutamine synthetase, and/or (ii) glutamine provided both sit4 and sit4pph3 cells cultured under comparable in the medium inhibits Msx uptake. conditions. This suggested, in agreement with other reports, In contrast, Gln3-Myc localization successively shifted in that Sit4 was required for nuclear localization of Gln3-Myc in Msx-treated cells from cytoplasmic to nuclear-cytoplasmic to cells exposed to these three conditions. However, the Sit4 nuclear first in the wild type and then in the sit4 as the nitro- requirement was nearly absent when analogous experiments gen source changed from glutamine (Fig. 6A) to YPD (Fig. 6B) were performed with the metabolic inhibitor, Msx. As shown in to ammonia (Fig. 6C) to proline (Fig. 6D). Note that a similar Fig. 5B, Gln3-Myc was largely cytoplasmic in ammonia- but less pronounced shift occurred after rapamycin treatment grown cultures, but was nuclear and/or nuclear-cytoplasmic in in ammonia- versus proline-grown cells (Fig. 6, C and D). The all but a few sit4 or sit4pph3 mutant cells following Msx same results were observed in the sit4pph3 double mutant treatment. Again, as seen in Fig. 5A, the Gln3-Myc intra- (Fig. 6E). These data suggested that Msx and rapamycin-in- cellular distribution profile in a pph3 was indistinguishable duced Gln3-Myc nuclear localization exhibited a nitrogen from that of wild type (Fig. 5B). These data demonstrated source-dependent Sit4 requirement, which paralleled that of that, although treating cells with rapamycin or Msx had been NCR, i.e. the more severe the NCR, the greater the Sit4-require- reported to have similar inhibitory effects on Tor1,2 activity ment. However, even in proline-grown cells, a limited Sit4-re- 13 13 (22, 27), nuclear localization of Gln3-Myc did not possess a quirement remained (Fig. 6D, Gln3-Myc was cytoplasmic in DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37985 Sit4 Phosphatase Active Irrespective of Nitrogen Source appeared (14, 15). The data presented above suggested that intracellular localization profiles of Gln3-Myc might differ from one strain to another as a result of strain-dependent dif- ferences in nitrogen catabolism and/or sensitivity to NCR anal- ogous to those reported in earlier studies (40, 42). We decided to test this hypothesis by comparing the intracellular distribu- tion of Gln3-Myc in two genetic backgrounds, TB123 (the background in which the Sit4 requirement was first reported (14)) and BY4709 (from the yeast genome deletion project (32)). Gln3-Myc localization following rapamycin treatment of glu- tamine-grown wild type and sit4 strains were independent of the genetic background (Fig. 7A). In YNB-ammonia medium, Gln3-Myc localization shifted from the cytoplasm to the nuclei of Msx-treated cells in both genetic backgrounds (Fig. 7B). Additionally, Gln3-Myc was more nuclear-cytoplasmic in untreated ammonia-grown BY4709-derived than TB123-de- rived cells. An even more striking difference occurred when cells were grown in YNB-proline medium (Fig. 7C). In a TB123 genetic background, Gln3-Myc localization in proline medium required Sit4, whereas in a BY4709 background no such requirement was observed. A clear difference between the TB123 and BY4709 genetic backgrounds is an rme1 mutation present in the former strain but not in the latter. To determine whether this mutation accounted for the two strains strikingly different Sit4 require- ments with proline as nitrogen source, we transformed TB123 and sit4pph3 (FV4) strains with CEN-plasmid yCplac22- RME1. Untransformed strains and corresponding transfor- mants were grown in glutamine, glutamine  rapamycin, and proline media and Gln3-Myc localization measured. Gln3- Myc localization in the transformants was not detectably dif- ferent from that observed in the untransformed recipients (data not shown). These observations argued that the rme1 mutation did not account for differing Gln3-Myc distributions observed in the two genetic backgrounds. Together, data presented above demonstrate the extent to which Sit4 is required for inhibitor-induced nuclear localiza- tion of Gln3-Myc qualitatively correlates with the degree of NCR elicited by the nitrogen source provided to the cells. The Sit4 requirement was greater with repressive nitrogen sources than with those that were non-repressive. This also occurs for rapamycin-induced Gln3-Myc nuclear localiza- tion. At face value, the data are more consistent with the suggestion that the cellular signal generated in response to its nitrogen supply is situated at or below the level of Sit4 function because a poor nitrogen source, such as proline, decreased and completely bypassed the requirement for Sit4 FIGURE 6. Sit4 requirement for nuclear localization of Gln3-Myc follow- ing rapamycin (Rap) or Msx (MSX) treatment decreases with nitrogen in the TB123 and BY4709 genetic backgrounds, respectively. sources of decreasing from good to poor quality. The nitrogen source(s) Alternatively, Sit4 and the nitrogen signal that influenced are indicated in the panels along with the inhibitor treatments and pertinent genotype of the cells used; strains were the same as described in the legend Gln3 localization derived from separate branches of the reg- to Fig. 5. ulatory pathway. Gln3-Myc Phosphorylation/Dephosphorylation in Wild 37% of the rapamycin-treated cells), which decreased further Type and Type 2A-related Phosphatase-defective Strains—Data if pph3 was also deleted (Fig. 6E, Gln3-Myc was cytoplasmic in Fig. 3 demonstrated that Sit4 is clearly active and influences in 26% of the rapamycin-treated cells). Gln3-Myc dephosphorylation levels even in cultures growing Strain-dependent Effects of sit4 Mutations—Reports reach- with a good nitrogen source, glutamine. We reasoned that ing differing conclusions with respect to Tap42 and Sit4 partic- deleting the cognate gene of an enzyme whose activity was ipation in the regulation of NCR-sensitive transcription have reported to be inactivated in the model describing Tor1,2 reg- 37986 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source FIGURE 8. Nitrogen source-dependent Gln3-Myc phosphorylation occurs in a sit4 mutant. A, percent of wild-type (W.T.; TB123) or sit4 (TB136) cells in which Gln3-Myc is cytoplasmic. The cells were grown in YNB medium with the indicated nitrogen sources (glutamine (Gln), proline (Pro), ammonia ( NH )) or in YPD medium. B, Western blot analysis of Gln3-Myc phosphorylation in wild-type and sit4 cells cultured as described in A. phorylation varied greatly in the sit4 (Fig. 8B). It was lowest in ammonia- (lane C) or proline- (lane B) grown sit4 cells and reached its highest levels with glutamine (lane D)or YPD (lane E) as nitrogen source (Fig. 8B). The relationship was just opposite that observed when a parallel experiment was performed several years ago using wild-type cells (26). In that case, Gln3-Myc intracellular localization ranged from completely cytoplasmic with glutamine to nuclear with pro- line, while Gln3-Myc was almost uniformly phosphoryla- ted (Fig. 3 and Ref. 26). Although Gln3-Myc phosphoryla- FIGURE 7. Strain background and nitrogen source dependence of intra- cellular Gln3-Myc distribution observed in wild-type (TB123 and tion changed with the nitrogen source in a sit4, these FV017) and sit4 (TB136 and FV1) cells. Culture conditions were as changes did not correlate with whether the nitrogen source described in the legend to Figs. 5 and 6. was a good or poor one. If it had, then the Gln3-Myc phos- phorylation profile observed with ammonia as nitrogen ulation of Gln3 would not be expected, in the most straightfor- source should have been more similar to that observed with ward case, to detectably affect Gln3-Myc phosphorylation glutamine than with proline (Fig. 8B). 13 13 levels. This reasoning and the Gln3-Myc nitrogen source-de- The above data convincingly demonstrated Gln3-Myc was pendent distribution data described above prompted us to phosphorylated to different extents in sit4 cells provided with determine the relationship between Gln3-Myc localization, various nitrogen sources, but did not distinguish whether it was 13 13 Gln3-Myc phosphorylation, and the nitrogen source pro- sit4 or the nitrogen source that altered Gln3-Myc phospho- vided to wild-type and mutant cells. rylation. Therefore, we compared Gln3-Myc phosphoryla- Investigating these relationships, we found that Gln3-Myc tion in wild-type and sit4 cells cultured under various condi- was almost uniformly cytoplasmic in a sit4 (TB123 genetic tions. Deletion of SIT4 increased Gln3-Myc phosphorylation background) irrespective of the nitrogen source provided (Fig. irrespective of the nitrogen source provided (Fig. 9, A–D, lanes 8A). Only in proline medium was Gln3-Myc minimally A and B, note the black dots between lanes A and B). These ( 10%) nuclear-cytoplasmic. In contrast, Gln3-Myc phos- effects appeared in one or both of two ways: (i) disappearance of DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37987 Sit4 Phosphatase Active Irrespective of Nitrogen Source phorylation in a sit4. If both Msx and rapamycin act at or above the level of Tor1,2 in the regulatory pathway (22), and hence above the level of Sit4 dephosphorylation of Gln3- Myc (14) then the loss of Sit4 should eliminate responses to the inhibitor-generated signals in parallel and irrespective of the nitrogen source provided. On the other hand, if this is not the case, loss of Sit4 would not be expected to cause such uniform changes in Gln3-Myc phosphorylation levels. Treating a sit4 with Msx increased Gln3-Myc phosphoryla- tion in both ammonia- and proline-grown cells (Fig. 9, A and B, lanes B and C). Increased Gln3-Myc phosphorylation was not observed with YPD- or glutamine-grown sit4 cells (Fig. 9, C and D, lanes B and C). When evaluating these results, however, it is important to keep two things in mind: (i) Msx is reported (and we have confirmed) to be ineffective in wild type, glu- tamine-grown cells (22, 27). On the other hand, the inhibitor shifted Gln3-Myc localization from cytoplasmic to nuclear- cytoplasmic and nuclear in wild type YPD-grown cells indicat- ing that it did function in this medium. (ii) Gln3-Myc was already highly phosphorylated in glutamine and YPD grown cells. Such high levels of Gln3-Myc phosphorylation at the outset may have masked whatever effects that might have occurred when the ability to dephosphorylate Gln3-Myc was lost in the sit4. In sum, the level of Gln3-Myc phosphoryla- tion either remained the same or increased following Msx treat- ment of a sit4. This compares with increased Gln3-Myc phosphorylation observed upon Msx treatment of wild-type cells regardless of the nitrogen source. The responses of Gln3-Myc phosphorylation in rapamy- cin-treated, sit4 cells did not parallel the effects described above. First, in the sit4, rapamycin did not elicit as extensive dephosphorylation of Gln3-Myc as in wild-type cells, where treating cells with rapamycin or cell extracts with calf intestinal alkaline phosphatase yield similarly dephosphorylated Gln3- Myc species (Fig. 4A of Ref. 26). This is easily observed com- paring the wild type and sit4 Gln3-Myc profiles (Fig. 9, B–D, lanes D and E). On the other hand, rapamycin clearly caused some Gln3-Myc dephosphorylation in YPD- and glutamine- grown sit4 cells (Fig. 9, C and D, lanes B and D). Rapamycin- induced Gln3-Myc dephosphorylation was not apparent in an ammonia-grown sit4 (Fig. 9B, lanes B and D), and Gln3-Myc phosphorylation actually increased in proline-grown sit4 as previously observed in wild-type cells (Fig. 9A, lanes B and D and in Fig. 4D of Ref. 27). In sum, Gln3-Myc phosphoryla- tion following rapamycin treatment decreased, remained FIGURE 9. Sit4 is active with respect to Gln3-Myc phosphorylation irre- unchanged, or increased depending upon the nitrogen spective of the nitrogen source. Western blot analyses of Gln3-Myc phos- phorylation in wild type (W.T.; TB123) and sit4 (TB136) grown in the pres- source used. ence () or absence () of rapamycin (Rap) or Msx (Msx) as described It was not difficult to envision that Gln3-Myc dephospho- under “Materials and Methods.” The nitrogen source in which each experi- ment was conducted is indicated above the blots. rylation noted above in a sit4 might derive from the closely related Pph3 phosphatase. To evaluate this possibility we assayed Gln3-Myc phosphorylation in pph3 single and a faster migrating species and/or appearance of a slower sit4pph3 double mutants. As shown in Fig. 10A, the Gln3- migrating Gln3-Myc species (Fig. 9, A, B, and D, lanes A and Myc phosphorylation profiles in untreated as well as rapamy- B), or (ii) a shift in the relative amounts of Gln3-Myc species, with a slower mobility species increasing and the faster migrat- cin- and Msx-treated pph3 cells were indistinguishable from ing species decreasing (Fig. 9, A, B, and D). wild type. This parallels the results observed with Gln3-Myc We next determined whether the nitrogen source affected intracellular localization (Fig. 5). In addition, the sit4pph3 rapamycin- or Msx-mediated changes in Gln3-Myc phos- double mutant possessed a phenotype that was indistinguish- 37988 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source variations in Gln3-Myc phosphorylation. The overall Gln3- Myc phosphorylation profiles were similar, though not iden- tical in wild-type cells from a TB123 versus BY4709 genetic background (Fig. 10C). There was overall greater Gln3-Myc phosphorylation in the BY4709 background. This is observed as: (i) a slight increase in the upper-most and decrease in the lower-most Gln3-Myc species in ammonia-grown FV017 (BY4709) cells (Fig. 10C, lanes E versus F), or (ii) the appearance of a slower migrating species (Fig. 10C, lanes A versus B), and a decrease in the fastest migrating species in glutamine-grown BY4709 cells (lanes A and B) relative to results in the TB123 background. Similarly, rapamycin induced Gln3-Myc dephosphorylation in FV017 (BY4709), but not to the level observed earlier for TB123. Compare the relative positions of Gln3-Myc species in Fig. 9D, lanes E and F with those in Fig. 10C, lanes A and C, i.e. the positions in TB123  rapamycin (Fig. 9) and FV017  rapamycin (Fig. 10) relative to the posi- tions in untreated TB123. The increased percentage of untreated, ammonia-grown FV017 (BY4709) cells containing nuclear-cytoplasmic Gln3-Myc relative to TB123 (Fig. 7B) was not paralleled by decreased Gln3-Myc phosphorylation as would be expected from the current model describing Tor1,2 regulation of Gln3. If anything, Gln3-Myc phosphorylation remained the same or increased modestly in the BY4709 back- ground (Fig 10C, lanes A and B and E and F). Changes in Gln3-Myc phosphorylation resulting from the deletion of SIT4 in the BY4709 genetic background were the same as those in the TB123 background. However, this experi- ment again emphasized another difference in the behavior of Gln3-Myc in the two genetic backgrounds, i.e. a significant decrease in the amount of detectable Gln3-Myc observed in ammonia-grown BY4709 wild-type and sit4 cells. This neces- sitated the use of longer exposures during development of the Western blot depicted in Fig. 10D (lanes D–F) even though all six lanes were derived from a single membrane. A similarly diminished Gln3-Myc signal also occurred in Fig. 10C, lanes D and E. Our last objective was to compare the phosphorylation pro- files of proline-grown sit4 cells in the TB123 and BY4709 genetic backgrounds, because nuclear localization of Gln3- Myc was Sit4-dependent in the former and Sit4-independent in the latter (Fig. 7C). To this end, we repeated the experiment described in Fig. 9A using wild-type (FV017) and sit4 (FV1) FIGURE 10. A and B, effects of defects in phosphatase Pph3 on Gln3 phospho- rylation. Wild type (W.T. TB123), pph3 (FV3), and sit4pph3 (FV4) were strains. Unfortunately, the Gln3-Myc signals were so dimin- grown in YNB-glutamine (Gln) or -ammonia ( NH ) medium in the presence ished and of such poor quality that our observations can be or absence of rapamycin or Msx. C and D, effects of the genetic background on Gln3-Myc phosphorylation. Strains used in these experiments were considered only tentative at best. That said, we were unable to derived from JK9 –3da (TB123), i.e. pph3 (FV3), pph3sit4 (FV4), or BY4709 detect a difference between the phosphorylation of Gln3-Myc (FV017), and sit4 (FV1). The experimental format and conditions were as described in the legend to Fig. 9. in the proline-grown wild type (FV017) and sit4 (FV1) strains. We could, however, detect modestly increased Gln3-Myc phosphorylation upon addition of rapamycin to the sit4 cul- able from that of a sit4 both with respect to the effects elicited ture similar to that observed in the TB123 genetic background by nitrogen source and the two inhibitors (Fig. 10B). in Fig. 9A, lanes B and D (data not shown). In sum, although Influence of Genetic Background on Gln3-Myc Phos- there were modest differences in the phosphorylation profiles phorylation—Differences in intracellular Gln3-Myc distribu- observed in the TB123 and BY4709 genetic backgrounds, none tion in different strains, especially in untreated, ammonia- of them appeared as drastic as the differences observed in intra- grown wild type and proline-grown sit4 cells prompted us to ascertain whether these differences correlated with parallel cellular Gln3-Myc distribution in the two strains. DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37989 Sit4 Phosphatase Active Irrespective of Nitrogen Source DISCUSSION Correlating with this loss of requirement, BY4709-derived FV017 is less NCR-sensitive than TB123 with at least ammonia This work was undertaken to gain a better understanding of as nitrogen source. Whether this correlation derives from a how the type 2A-related protein phosphatases, Sit4 and Pph3, cause-effect relationship between the strains and nitrogen participate in the regulation of Gln3 phosphorylation and intra- source provided or alternatively from an unrelated event, can- cellular localization. Conceptually, we expected these enzymes, not be ascertained at present. Further, existing data do not per- being multifunctional, would be active toward one or another mit us to evaluate whether the lack of difference in Gln3-Myc substrate under most conditions. The pivotal question was phosphorylation profiles observed for the two strains derives whether they were specifically active with respect to Gln3 phos- from phosphorylation/dephosphorylation events that might phorylation in a nitrogen source-dependent manner. In other exist, but are not detected in a total phosphorylation profile. words, was the extent of Gln3 phosphorylation dictated by the The decreasing Sit4 requirement in glutamine versus ammo- degree to which a nitrogen source resulted in active versus inac- nia versus proline medium adds to a growing list of situations in tive Sit4 and Pph3? For Pph3, we were unable to demonstrate an which the effects of the nitrogen source and NCR control active role in Gln3 control, i.e. detectable Gln3-Myc phos- appear to feed into the regulation of Gln3 downstream of phorylation and intracellular localization were minimally if at Tor1,2 and its inhibition by rapamycin. This relation of the all affected by deleting PPH3. Sit4, on the other hand, clearly nitrogen signal to Tor1,2 is also consistent with the observa- influenced Gln3-Myc phosphorylation levels, but did not do tions that Sit4 is not required for Msx-induced nuclear local- so in a nitrogen source-dependent manner as expected. Delet- ization of Gln3-Myc . They join the earlier observation that ing SIT4 increased Gln3-Myc phosphorylation irrespective of Tor1,2 control of retrograde gene expression is also nitrogen the nitrogen source provided. Sit4 was active with all nitrogen source-dependent in that the ability of rapamycin to induce sources, and most importantly, with nitrogen sources that are retrograde gene expression (CIT2 is the reporter) occurs with the most repressive for NCR-sensitive transcription. In fact, glutamine as nitrogen source, but not proline (28, 40). The sep- differences in Gln3-Myc phosphorylation observed when arability of retrograde regulation from Tor1,2 activity has been comparing wild type and a sit4 were greatest with good nitro- recently confirmed by Giannattasio et al. (39). It is important to gen sources such as glutamine or in YPD medium, conditions note, however, that while these observations support the con- where Sit4 would have been expected to be least active and tention of a nitrogen source-derived signal feeding into the therefore a sit4 to have the least effect. These results are just Gln3 regulatory pathway downstream of the rapamycin-inhib- opposite of predictions emanating from the idea that Tor1,2, ited step, they do not exclude a more complicated possibility Tap42, and/or Tip41 control Gln3 phosphorylation and local- hypothesizing the existence of two nitrogen signals, one ization by regulating Sit4 activity (14, 20). impinging on regulation above the rapamycin-inhibited step A second experimental finding, which pointed to a less direct and the other below it. role for Sit4 in Gln3 regulation, was its declining requirement Our investigations identify an interesting set of relation- for rapamycin- and Msx-induced nuclear Gln3 localization in ships that offer insights into the nitrogen-responsive control response to the quality of the nitrogen source, i.e. good versus pathway. Earlier studies reported the only time that demon- poor (Fig. 6). Under the current view of Tor1,2 control, Sit4 is strable dephosphorylation of Gln3-Myc correlated with its posited to be most active following loss of the positive nitrogen nuclear localization was shortly after treating cells with signal activating Tor1,2. The positive signal (glutamine or one rapamycin (26). In ammonia- or glutamine-grown wild-type of its metabolites) is lost when cells are provided with a poor cells, Gln3-Myc was cytoplasmic and in proline-grown nitrogen source, treating them with Msx, or inhibiting Tor1,2 cells it was nuclear. Yet Gln3-Myc was almost uniformly itself with rapamycin. Therefore, Sit4 activity, and hence its phosphorylated; Gln3-Myc was slightly more phosphoryl- requirement for nuclear Gln3-Myc localization, should ated with proline than with ammonia or glutamine as nitro- increase as the quality of available nitrogen decreases. In con- gen source (26, 27). In present studies, just the opposite trast to these predictions, the Sit4 requirement for Msx-in- occurred, i.e. Gln3-Myc was cytoplasmic in the sit4 mutant duced nuclear localization of Gln3 disappears as one moves regardless of whether the nitrogen source was good or poor from YPD to ammonia or proline media. Analogously, the Sit4- (Fig. 8). However, Gln3-Myc phosphorylation levels requirement is significantly reduced for rapamycin-induced responded markedly to the nitrogen source provided. In nuclear Gln3 localization in proline-grown cells, i.e. Gln3 is other words, deleting SIT4 unmasked the response of Gln3- nuclear-cytoplasmic or nuclear in the majority of rapamycin- Myc phosphorylation to the nitrogen source. This is the treated sit4 ( 60%) or sit4pph3 ( 75%) cells (Fig. 6, D and first time Gln3 phosphorylation has been shown to be asso- E). In other words, the poorer the nitrogen source the less Sit4 is ciated with a nitrogen source provided to the cells. If Sit4 was required for nuclear Gln3-Myc localization. the molecule responsible for dephosphorylating Gln3-Myc Our data, describing a relationship between nitrogen source, in response to nitrogen source, then the nitrogen source-de- strain background, and Sit4 requirement for Gln3-Myc local- pendent signal would a priori have been expected to be lost ization, are consistent with some studies finding a Tap42-Sit4 when SIT4 was deleted. This suggests that the nitrogen requirement for Gln3 control (14), whereas others do not (15). source specificity of NCR control is probably more directly We observed, for example, that Sit4 is necessary for Gln3 to connected to the response of one or more protein kinases accumulate in the nuclei of proline-grown cells in the TB123 responsible for Gln3-Myc phosphorylation than to the genetic background, but not for BY4709-derived cells (Fig. 7C). type 2A-related protein phosphatases (Sit4 and Pph3). 37990 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source 3. Morgensztern, D., and McLeod, H. L. (2005) Anticancer Drugs 16, By this reasoning, Gln3 phosphorylation would occur in the 797–803 presence of nitrogen source-independent Sit4 phosphatase 4. Lorber, M. I., Mulgaonkar, S., Butt, K. M., Elkhammas, E., Mendez, R., activity, the latter buffering or masking the effects of nitrogen Rajagopalan, P. R., Kahan, B., Sollinger, H., Li, Y., Cretin, N., Tedesco, H., source-responsive phosphorylation from being observed. Here, and B251 Study Group (2005) Transplantation 80, 244–252 type 2A-related phosphatases rather than generating the signal 5. Thomas, G., Sabatini, D., and Hall, M. N. (2004) TOR: Target of Rapamy- that results in Gln3-Myc accumulating in the nucleus, would cin: Current Topics in Microbiology & Immunology, Vol. 279, Springer- Verlag, Berlin serve a stabilizing role to reset the system in preparation to 6. Inoki, K., Ouyang, H., Li, Y., and Guan, K. L. Microbiol. Mol. Biol. Rev. receive such signals. In this model, the observed Gln3-Myc (2005) 69, 79–100 phosphorylation would derive from the ratio of kinase to phos- 7. Hofman-Bang, J. (1999) Mol. Biotechnol. 12, 35–73 phatase activities acting on Gln3, the former being the one pre- 8. ter Schure, E. G., van Riel, N. A., and Verrips, C. T. (2000) FEMS Microbiol. dominantly modulated by the nitrogen source provided. Rev. 24, 67–83 Although the above ideas would explain Gln3 phosphoryl- 9. Cooper. T. G. (2002) FEMS Microbiol. Rev. 26, 223–238 10. Cooper, T. G. (2004) Topics in Current Genetics (Winderickx, J. and Tay- ation and localization in the sit4, present data are insuffi- lor, P. M., eds) Vol. 7, pp. 225–257, Springer-Verlag, Berlin cient to provide details of the precise link(s) between the 11. Magasanik, B., and Kaiser, C. A. (2002) Gene (Amst.) 290, 1–18 nitrogen source, observable Gln3 phosphorylation levels and 12. Cox, K. H., Rai, R., Distler, M., Daugherty, J. R., Coffman, J. A., and Cooper, NCR-sensitive transcription. For example, they do not T. G. (2000) J. Biol. Chem. 275, 17611–17618 exclude the possibility that a nitrogen source may regulate 13. Cunningham, T. S., Andhare, R., and Cooper, T. G. (2000) J. Biol. Chem. 275, 14408–14414 NCR-sensitive transcription via influencing Gln3 binding to 14. Beck, T., and Hall, M. N. (1999) Nature 402, 689–692 the DNA in addition to controlling its intracellular localiza- 15. Cardenas, M. E., Cutler, N. S., Lorenz, M. C., Di Como, C. J., and Heitman, tion. Further, they do not identify the physiological advan- J. (1999) Genes Dev. 13, 3271–3279 tage gained when functional Sit4 brings Gln3-Myc phos- 16. Hardwick, J. S., Kuruvilla, F. G., Tong, J. K., Shamji, A. F., and Schreiber, phorylation to the same level irrespective of the nitrogen S. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14866–14870 source provided and the intracellular localization of Gln3- 17. Bertram, P. G., Choi, J. H., Carvalho, J., Ai, W., Zeng, C., Chan, T. F., and Zheng, X. F. (2000) J. Biol. Chem. 275, 35727–35733 Myc . And finally, they are also insufficient to distinguish 18. Di Como, C. J., and Arndt, K. T. (1996) Genes Dev. 10, 1904–1916 the possibility that Gln3-Myc phosphorylation increases in 19. Jiang, Y., and Broach, J. R. (1999) EMBO J. 18, 2782–2792 a sit4 because Sit4 phosphatase is no longer dephospho- 20. Jacinto, E., Guo, B., Arndt, K. T., Schmelzle, T., and Hall, M. N. (2001) Mol. rylating Gln3-Myc from the alternative that loss of Sit4 Cell. 8, 1017–1026 activity increases protein kinase activity which in turn 21. Rohde, J. R., Campbell, S., Zurita-Martinez, S. A., Cutler, N. S., Ashe, M., increases Gln3-Myc phosphorylation. and Cardenas, M. E. (2004) Mol. Cell. Biol. 24, 8332–8341 22. Crespo, J. L., Powers, T., Fowler, B., and Hall, M. N. (2002) Proc. Natl. Data presented in Fig. 9 also indicate that loss of Sit4 and Acad. Sci. U. S. A. 99, 6784–6789 Pph3 activities are not sufficient to abrogate the influence of 23. Magasanik, B. (2005) Proc. Natl. Acad. Sci. U. S. A. 2005 102, Tor1,2 on Gln3-Myc phosphorylation level. Addition of rapa- 16537–16538 mycin to YPD- glutamine- or ammonia-grown sit4 cells 24. Duvel, K., Santhanam, A., Garrett, S., Schneper, L., and Broach, J. R. (2003) clearly decreases Gln3-Myc phosphorylation. The most likely Mol. Cell. 11, 1467–1478 candidates to mediate this dephosphorylation are Pph21 and 25. Wang, H., Wang, X., and Jiang, Y. (2003) Mol. Biol. Cell 14, 4342–4351 26. Cox, K. H., Kulkarni, A., Tate, J. J., and Cooper, T. G. (2004) J. Biol. Chem. Pph22 (19, 26, 39). Although we have not yet investigated the 279, 10270–10278 roles of Pph21 and Pph22 in the regulation of Gln3 because of 27. Tate, J. J., Rai, R., and Cooper, T. G. (2005) J. Biol. Chem. 280, technical difficulties derived from the severe growth defect and 27195–27204 other difficult phenotypes of the pph21,22 mutations, the rela- 28. Tate, J. J., Cox, K. H., Rai, R., and Cooper, T. G. (2002) J. Biol. Chem. 277, tionships are unlikely to be straightforward. One expression of 20477–20482 29. Dilova, I., and Powers, T. (2006) FEMS Yeast Res. 6, 112–119 this expectation is the fact that although rapamycin induced 30. Cox, K. H., Tate, J. J., and Cooper, T. G. (2002) J. Biol. Chem. 277, dephosphorylation of Gln3-Myc in the sit4, i.e. a strain in 37559–37566 which Pph3, Pph21 and Pph22 are all functional, it did not 31. Cox, K. H., Tate, J. J., and Cooper, T. G. (2004) J. Biol. Chem. 279, induce its nuclear localization with any of the good nitrogen 19294–19301 sources. Further, treating cells with Msx increases Gln3-Myc 32. Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, phosphorylation and yet results in nuclear localization of Gln3- S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., Astromoff, A., El-Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Cur- Myc (27). tiss, M., Davis, K., Deutschbauer, A., Entian, K. D., Flaherty, P., Foury, F., Garfinkel, D. J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J. H., Acknowledgments—We thank Dr. Michael Hall for strains and Drs. Hempel, S., Herman, Z., Jaramillo, D. F., Kelly, D. E., Kelly, S. L., Kotter, P., Florian Bauer and Dewald van Dyk for the RME1 plasmid, Fabienne LaBonte, D., Lamb, D. C., Lan, N., Liang, H., Liao, H., Liu, L., Luo, C., Vierendeels for excellent technical assistance, Tim Higgins for prepar- Lussier, M., Mao, R., Menard, P., Ooi, S. L., Revuelta, J. L., Roberts, C. J., ing the artwork, and UT Yeast Group for suggestions to improve the Rose, M., Ross-Macdonald, P., Scherens, B., Schimmack, G., Shafer, B., manuscript. Shoemaker, D. D., Sookhai-Mahadeo, S., Storms, R. K., Strathern, J. N., Valle, G., Voet, M., Volckaert, G., Wang, C. Y., Ward, T. R., Wilhelmy, J., Winzeler, E. A., Yang, Y., Yen, G., Youngman, E., Yu, K., Bussey, H., Boeke, REFERENCES J. D., Snyder, M., Philippsen, P., Davis, R. W., and Johnston, M. (2002) Nature 418, 387–391 1. Schluter, M., and Schofer, J. (2005) Am. Heart Hosp. J. 3, 182–186 33. Wach, A. (1996) Yeast 12, 259–265 2. Boulay, A., Rudloff, J., Ye, J., Zumstein-Mecker, S., O’Reilly, T., Evans, 34. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids D. B., Chen, S., and Lane, H. A. (2005) Clin. Cancer Res. 11, 5319–5328 DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37991 Sit4 Phosphatase Active Irrespective of Nitrogen Source Res. 18, 3091–3092 Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., 35. Foury, F., and Talibi, D. (2001) J. Biol. Chem. 276, 7762–7768 Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., 36. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, York M., M’Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Re- 37. Pringle, J. R., Adams, A. E. M., Drubin, D. G., and Haarer, B. K. (1991) vuelta, J. L., Riles, L., Roberts, C. 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Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive in a sit4-deleted Strain *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 49, pp. 37980 –37992, December 8, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive □ S in a sit4-deleted Strain Received for publication, July 21, 2006, and in revised form, September 26, 2006 Published, JBC Papers in Press, October 2, 2006, DOI 10.1074/jbc.M606973200 ‡ § § ‡1 Jennifer J. Tate , Andre´ Feller , Evelyne Dubois , and Terrance G. Cooper ‡ § From the Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163 and the Institut de Recherches Microbiologiques J.-M. Wiame, Laboratoire de Microbiologie Universite´ Libre de Bruxelles, B1070 Brussels, Belgium Tor1,2 control of type 2A-related phosphatase activities in reporter in studies investigating Tor function (5, 6). A major Saccharomyces cerevisiae has been reported to be responsible function of Gln3 is to activate Nitrogen Catabolite Repression for the regulation of Gln3 phosphorylation and intracellular (NCR) -sensitive transcription needed to exploit poor nitrogen localization in response to the nature of the nitrogen source sources in the environment when nothing better is available available. According to the model, excess nitrogen stimulates (biology reviewed in Refs. 7–11). In the presence of good nitro- Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then com- gen sources (e.g. glutamine or ammonia in some strains) genes plexes with and inactivates Sit4 phosphatase, thereby prevent- encoding permeases and enzymes required for the transport ing it from dephosphorylating Gln3. Phosphorylated Gln3 com- and degradation of poor nitrogen sources (e.g. proline) are min- plexes with Ure2 and is sequestered in the cytoplasm. When imally expressed. In contrast, when good nitrogen sources are Tor1,2 kinase activities are inhibited by limiting nitrogen, or limiting or only poor sources are available, expression of these rapamycin- treatment, Tap42 can no longer complex with Sit4. genes increases (7–11). Active Sit4 dephosphorylates Gln3, which can then localize to Two pivotal observations opened a new era of investigation the nucleus and activate transcription. The paucity of experi- into the mechanisms regulating Gln3 function. (i) During times mental data directly correlating active Sit4 and Pph3 with Gln3 of high NCR, GATA sequences (binding sites for Gln3 and regulation prompted us to assay Gln3-Myc phosphorylation Gat1/Nil1) in the promoter of an NCR-sensitive gene (CAN1) and intracellular localization in isogenic wild type, sit4, pph3, are unoccupied by Gln3 and Gat1, and thus available to serve as and sit4pph3 deletion strains. We found that Sit4 actively surrogate TATA-binding protein (TBP) binding sites (12). brought about Gln3-Myc dephosphorylation in both good Gln3 and Gat1 occupancy of CAN1 GATA sequences corre- (glutamine or ammonia) and poor (proline) nitrogen sources. lates with Gln3 and Gat1 localization to the cytoplasm of cells in This Sit4 activity masked nitrogen source-dependent changes in which NCR-sensitive transcription is low, and to their nuclei Gln3-Myc phosphorylation which were clearly visible when when expression is high (12, 13). (ii) Treating YPD-grown wild- SIT4 was deleted. The extent of Sit4 requirement for Gln3 type cells with rapamycin induces Gln3 dephosphorylation, nuclear localization was both nitrogen source- and strain-de- Gln3 nuclear localization and high level NCR-sensitive tran- pendent. In some strains, Sit4 was not even required for Gln3 scription. In contrast, Gln3 is phosphorylated, localizes to the nuclear localization in untreated or rapamycin-treated, proline- cytoplasm, and NCR-sensitive transcription is low in similarly grown cells or Msx-treated, ammonia-grown cells. grown, untreated cells (14–17). The above correlations along with the protein-protein asso- ciation and phosphorylation relationships between Tor1,2, Applications of clinically important derivatives of the anti- Tap42, Tip41, Sit4, and Sit4-associated proteins (Saps), led to a inflammatory and anti-neoplastic drug rapamycin have steadily proposal describing Tor1,2 regulation of Gln3 phosphorylation grown in concert with our increased understanding of how cel- via control of the type 2A-related protein phosphatases, Sit4 lular processes are regulated by their target, mTor (mammalian and/or Pph3 (Fig. 1) (5, 14–22). In bare outline, the model pos- Target of Rapamycin) (1–4). The Saccharomyces cerevisiae its that signals of nitrogen excess (glutamine or a metabolite of GATA family transcription activator, Gln3 is widely used as a it) are sensed by Tor1,2, which become active and phospho- rylate Tap42 and/or Tip41 (14, 17–22). The outcome of these phosphorylations is the association of Tap42 with Sit4, thereby * This work was supported by National Institutes of Health Grant GM-35642 and NSF Collaborative Grant DMS-0443855 (to T. G. C.) and by a grant from inactivating the phosphatase (18–20). In this inactive form, Sit4 COCOF (Commission de la Communaute´ franc¸aise) (to E. D.). The costs of is unable to dephosphorylate Gln3, resulting in its sequestra- publication of this article were defrayed in part by the payment of page tion in the cytoplasm as a Gln3Ure2 complex (Fig. 1) (14, 17). charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. When cells are treated with rapamycin, or the glutamine syn- □ S The on-line version of this article (available at http://www.jbc.org) contains thetase inhibitor, methionine sulfoximine (Msx), which inhib- supplemental Fig. S1. To whom correspondence should be addressed: Dept. of Molecular Sci- ences, University of Tennessee, Memphis, TN 38163. Tel.: 901-448-6179; The abbreviations used are: NCR, nitrogen catabolite repression; Msx, L-me- Fax: 901-448-8462; E-mail: [email protected]. thionine sulfoximine; DAPI, 4,6-diamidino-2-phenylindole. 37980 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 This is an Open Access article under the CC BY license. Sit4 Phosphatase Active Irrespective of Nitrogen Source standing of Sit4 participation in the Tor1,2 control pathway regulating Gln3: (i) Is Sit4 required for Gln3 dephosphorylation and/or nuclear accumulation of Gln3 when cells are provided with a poor nitrogen source or treated with Msx?, (ii) Is Sit4 inactive under conditions where Gln3 is restricted to the cyto- plasm (i.e. nitrogen excess)? (iii) What are the relative require- ments of Sit4 and Pph3 for Gln3 dephosphorylation and nuclear localization in response to growth with various nitro- gen sources, rapamycin and Msx treatment? (iv) Does the Tap42-Sit4-requirement for Gln3 regulation found in some instances but not others reflect strain-specific differences when experiments are performed under identical conditions (14, 15)? To fill in some of the gaps in our understanding of type 2A-related phosphatases and their relationship to Gln3 regula- tion, we performed a systematic analysis of Gln3 phosphoryla- tion and intracellular localization, comparing these parameters in wild type, single and double sit4 and pph3 mutant cells. Our data demonstrate that: (i) Sit4 is active with respect to Gln3- Myc phosphorylation levels under conditions of excess nitro- gen in which Tor1,2 are active, (ii) Gln3-Myc phosphoryla- FIGURE 1. Abbreviated diagram of the model describing the regulation of tion is nitrogen source-dependent in a sit4, indicating that Gln3 phosphorylation and intracellular localization. The diagram was derived from Refs. 14, 20, and 21. Sit4 activity is unlikely to be the major determinant responsible for nitrogen-dependent changes in Gln3-Myc phosphoryla- its the synthesis of glutamine, Tor1,2 are inhibited and cease tion, (iii) rapamycin-induced alteration of Gln3-Myc phos- phosphorylating Tap42. Dephosphorylated Tap42 dissociates phorylation levels requires Sit4, whereas alterations elicited by from Sit4 thereby allowing Sit4 to be active and dephospho- Msx or the nitrogen source do not, (iv) the extent of the Sit4 rylate Gln3, resulting in or permitting Gln3 to dissociate from requirement for Msx- and rapamycin-induced nuclear localiza- Ure2 and enter the nucleus (14, 17). tion of Gln3-Myc is nitrogen source-dependent, (v) the Sit4 Experimentally, Sit4 was first implicated in Gln3 regulation requirement for nuclear Gln3-Myc localization is strain by the demonstration that neither rapamycin-induced Gln3 dependent, and (vi) demonstrable Pph3 influence on Gln3 reg- nuclear localization, nor dephosphorylation occurred in a YPD- ulation was minimal compared with that of Sit4. grown sit4 mutant (14). In a subsequent report, rapamycin was MATERIALS AND METHODS observed to induce modestly less GAP1 expression in a pph3 mutant than wild type, implicating the second type 2A-related Strains and Culture Conditions—S. cerevisiae strains used in phosphatase in Gln3 regulation (17). Most recently, Msx-in- this work are listed in Table 1. Strains were grown at 30 °C to duced Gln3 nuclear localization was reported not to occur in a mid-log phase (A  0.5) in YNB (without amino acids or 600 nm sit4 mutant growing in S.D. medium (22). ammonium sulfate) medium, containing 2% glucose, required Although impressive progress has been made in identifying auxotrophic supplements (120 g/ml leucine, 20 g/ml uracil, additional proteins involved in Tor1,2 global influence on cel- 20 g/ml histidine, 20 g/ml tryptophan, 20 g/ml arginine, lular processes, several observations do not fit comfortably with and the nitrogen source (0.1% final concentration) indicated. expectations generated by the model (23). Among them are: (i) YPD medium consisted of 10 g yeast extract, 20 g bactopeptone, In genetic studies, Tap42 behaves more like a positive than a and 20 g dextrose per 1000 ml. Rapamycin (Sigma-Aldrich) negative regulator of Sit4 (18). (ii) Sit4 association with Tap42 is (dissolved in 10% Tween 20  90% ethanol) was added to the required for Sit4 activity (18, 24, 25). (iii) Detectable Gln3- cultures, where indicated, to a final concentration of 0.2 g/ml Myc phosphorylation and intracellular localization do not for 20 or 30 min prior to cell harvest. Msx (Sigma-Aldrich) was correlate with one another in good versus poor nitrogen dissolved in water and added to the cultures, where indicated, sources, during nitrogen starvation, following Msx treatment, to a final concentration of 2 mM for 30 min prior to cell harvest and beyond 30 min of rapamycin treatment (26). (iv) Msx (by filtration). Where indicated, yeast were transferred (by fil- increases Gln3-Myc phosphorylation whereas rapamycin tration and resuspension) from one medium to another that decreases it even though both inhibitors elicit nuclear localiza- had been pre-warmed, pre-aerated, and contained the required tion of Gln3-Myc (27). auxotrophic supplements. As noted in Fig. 1, type 2A-related Sit4 (and Pph3) protein Strain Construction—Previous data, showing strain differ- phosphatase activities have been proposed to directly regulate ences can be critically important in evaluating the regulation of Gln3 phosphorylation and localization (14, 17, 22). The corre- Gln3 function (28, 29), recommended the use of isogenic lations upon which this conclusion rests are the failure of Gln3 strains. Recombinant methods were used to create specific dephosphorylation, nuclear localization, and NCR-sensitive deletions in two strain backgrounds: (i) TB123, used as the wild transcription to occur in YPD-grown, rapamycin-treated sit4 type in the original experiments demonstrating rapamycin-in- cells. There are, however, several important gaps in our under- duced alterations in Gln3 phosphorylation and intracellular DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37981 Sit4 Phosphatase Active Irrespective of Nitrogen Source TABLE 1 S. cerevisiaes trains used in this work Strains JK9-3da, TB123, TB50, and TB136 are described in Refs. 14 and 20 and BY4709 in Ref. 32. Strains JK9-3da and TB50 are isogenic except at the his3 and his4 loci. Strains TB123 and TB136 are isogenic, except for at the indicated loci, to JK9-3da. Strain Parent Genotype Primer coordinates TB123 JK9-3da MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL , HMLa, GLN3-Myc KanMX None TB50 MATa, leu2-3,112, ura3-52, trp1, his3, rme1, HMLa None JK9-3da MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa None TB136 MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL , HMLa, GLN3-Myc KanMX, None sit4::kanMX BY4709 MAT, ura3 None FV017 BY4709 Mat,ura3, GLN3-Myc KanMx FV1 BY4709 MAT, ura3, sit4::natMX, GLN3-MYC kanMX 5, 450 to 429 & 23 to 1 3 937 to 955 & 1380 to 1400 FV2 BY4709 MAT, ura3, pph3::natMX, GLN3-MYC kanMX 5, 400 to 379 & 22 to 1 3 927 to 950 & 1206 to 1228 FV3 TB123 MATa, his4, leu2-3,112, ura3, trp1, rme1, pph3::natMX, 5, 400 to 379 & 22 to 1 GLN3-myc kanMX 3 927 to 950 & 1206 to 1228 FV4 TB136 MATa, his4, leu2-3,112, ura3, trp1, rme1, sit4::kanMX, sit4:5, 450 to 429 & 23 to 1 pph3::natMX, GLN3-MYC kanMX 3 937 to 955 & 1380 to 1400 pph3:5, 400 to 379 & 22 to 1 3 927 to 950 & 1206 to 1228 localization (14), as well as in our own previous experiments (26, 27, 30, 31), and (ii) derivatives of BY4709, used in the sys- tematic deletion project (32). Deletion strains (FV series) were constructed using the long flanking homology strategy of Wach (33). The kanMX or natMX cassettes, flanked by about 500 bp corresponding to the promoter and terminator regions of the target genes, were syn- thesized by a two-step PCR procedure (primer coordinates are FIGURE 2. Intracellular distribution of Gln3-Myc in YNB-glutamine- in Table 1). DNA fragments (blunt-ended PCR products) con- grown sit4 (TB136) cells treated with rapamycin. Indirect immunofluo- taining the various constructs were used to transform the rescent staining of Gln3-Myc and DAPI-positive material appear in red and blue, respectively (see supplemental materials). appropriate strains. Transformants were selected on YPD medium containing 200 g per ml of geneticin or 100 g per ml of nourseothricin. Correct targeting of the deletions was veri- assess the relative amounts of various Gln3-Myc species fied by PCR analysis, using whole cells as the source of template within a particular lane and then compare the pattern of Gln3- DNA and two sets of primers: (i) a primer 5 of the deletion Myc distribution with that observed in another lane, rather cassette, a second at the 5-end of the Kan gene, and a third at than quantitatively comparing the amounts of particular Gln3- the beginning of the coding sequence of the gene that was Myc species observed in one lane with those in another. Fur- deleted, and (ii) a primer at the 3-end of the Kan gene, a second ther, although we occasionally compared the overall patterns of at the 3-end of the coding sequence of the deleted gene, and a data observed in one Western blot to that of another, detailed third 3 of the deletion cassette. comparisons were restricted to lane profiles contained within a Northern Blot Analysis—Total RNA was extracted as single Western blot membrane unless specifically indicated described earlier (34) and purified using the RNeasy kit (Qia- otherwise. gen). Northern blot analysis was performed as described by Indirect Immunofluorescence Microscopy—Cell preparation Foury and Talibi (35). DIG-DNA probes of about 500 bp were and assay of Gln3-Myc by indirect immunofluorescence was generated by PCR, using primers: 5-CATAACCAGTTGGTG- initially performed as described earlier (31, 37). Although this AGCCC-3 and 5-ACCCCCGTTACTGTATGTGG-3 for method (used in Fig. 2 of this text and Fig. S1 of the supplemen- SIT4,5-TGGGCGATTTTGTGGATAGG-3 and 5-CTGTC- tal materials) performs well for wild-type strains, it was inade- ACTAATCCACCGTCG-3 for PPH3,5-AAACAGCAAGA- quate for analysis of Gln3-Myc intracellular distribution in AAGTCCACTGG-3 and 5-ACCTCTTAATCTTCTAGCC- phosphatase mutants. sit4, and to a lesser extent pph3, AAC-3 for HHT1, and labeled using a PCR DIG (digoxigenin) mutants possess characteristics that, if not circumvented, seri- probe synthesis kit (Roche Applied Science). Hybridizations ously compromise analysis of Gln3-Myc intracellular distri- were carried out according to standard procedures (36). Detec- bution. Gln3-Myc and DAPI stained material (DNA) were tion of DIG-labeled nucleic acids was performed by enzyme asymmetrically distributed to the daughters and mothers, immunoassay with luminescence following the suppliers pro- respectively, of sit4 cells with small to medium sized buds (Fig. cedure (Roche Applied Science). The Hybond-N nylon mem- 2). The frequency of this morphology was increased by rapamy- branes were exposed 120 min and analyzed with a chemilumi- cin treatment. This phenomenon and data suggesting that it nescence camera (Chemi-Smart from Vilbert-Lourmat). derives from differential sensitivity of mother and daughter Western Blot Analysis—Cells were harvested by the filtration cells walls to zymolyase digestion as well as differential affinity method of Tate et al. (27), and crude cell extracts prepared as for lysine-coated slides are described under supplemental described by Cox et al. (26). As noted earlier (27), we prefer to materials. Therefore, we used a modified form of another 37982 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source method (38) in all but Fig. 2. The modifications were: (i) fixation in the growth medium for 60 min, (ii) addition of -mercapto- ethanol (20 mM final concentration) to the zymolyase digestion mixture, and (iii) increased digestion of mutant cells (34–37 min) and decreased digestion (15–17 min) for the wild type. Digesting wild type cells for the same length of time as the sit4 resulted in significant degradation of cell integrity. Determination of Intracellular Gln3-Myc Distribution—To determine the intracellular distribution of Gln3-Myc gener- ated by the experimental conditions used, an average of 150– 200 cells, from randomly chosen fields of each experimental condition, were scored in a double blind manner to determine the distribution of Gln3-Myc . Experiments were independ- ently repeated from two to seven times. Cells were classified into three categories, those in which Gln3-Myc was cytoplas- FIGURE 3. A, Sit4 is active with respect to Gln3-Myc dephosphorylation in mic, nuclear-cytoplasmic, or nuclear. Recognizing that bound- cells provided with a good nitrogen source (YNB-glutamine). Western blot analysis of Gln3-Myc in wild-type (TB123) and sit4 (TB136) cells. Rapa- aries between the middle category and those flanking it would mycin was added for 30 min. prior to cell harvest where indicated (Rap ). be unavoidably subjective, we performed two control experi- B, steady state mRNA levels of SIT4 and PPH3 in wild type (TB123), sit4 ments to determine the precision with which we placed cells (TB136), and pph3 (FV3). Cells were cultured in YNB-glutamine (Gln) or -ammonia (Am. or NH ) medium in the presence ( Rap; 30 min) or absence into two adjacent categories. First, we categorized Gln3-Myc of rapamycin or Msx ( Msx; 20 min). Additionally, glutamine cultures were as being cytoplasmic or nuclear-cytoplasmic in cells from one transferred to proline (Gln to Pro) or nitrogen-free (Gln to N.S.) medium and incubated for 60 min before the cells were harvested. microscopic image and then repeated the count a second time using the same image. Next, we repeated this procedure using a second, randomly selected image derived from the same slide as ants of the Tor1,2 pathway model, i.e. (i) in the presence of a the first image. The cell sample used in this experiment con- good nitrogen source, Tor1,2 are active and (via Tap42 and/or tained approximately equal numbers of cells in which Gln3- Tip41) inhibit Sit4 activity, thus preventing it from dephospho- Myc was categorized as cytoplasmic and nuclear-cytoplas- rylating Gln3, and (ii) Sit4 is active when the Tor1,2 kinases are mic, respectively. The two counts yielded four sets of values, inhibited by growth in limiting nitrogen, thereby making it two from each image used above. The four sets of values varied available to dephosphorylate Gln3. Wild type and isogenic from one another by plus or minus 2–3%. Finally, we performed sit4 cultures were grown in YNB-glutamine medium in the an analogous experiment using two images of a cell sample presence and absence of rapamycin. As expected, and previ- which contained approximately equal numbers of cells in which ously shown by multiple laboratories, rapamycin treatment 13 13 Gln3-Myc was categorized as being nuclear-cytoplasmic and elicited Gln3-Myc dephosphorylation in wild-type cells (Fig. nuclear, respectively. The same variation (2–3%) was 3A, lanes A versus D). In sit4 cultures, two unexpected results observed in this second experiment as well. These results were observed: (i) Sit4 was clearly active in cells provided with a argued that we could categorize Gln3-Myc localization with good nitrogen source, because deleting SIT4 increased Gln3- 13 13 acceptable precision. The patterns of Gln3-Myc distribution Myc phosphorylation relative to wild type (Fig. 3A, lanes A between cell compartments in response to various experimen- versus B). (ii) Rapamycin induced partial Gln3-Myc dephos- tal conditions (strains, nitrogen source, inhibitor treatment) phorylation in a sit4 (Fig. 3A, lanes B versus C), indicating that were also reproducible as was the quantitative distribution of more than Sit4 alone was responsible for rapamycin-induced Gln3-Myc among the three intracellular categories in dephosphorylation. Together, these data indicated that type 2A repeated scoring of images from a single cell sample. Experi- phosphatase participation in the regulation of Gln3 phospho- ment-to-experiment variation was 2–10%, except when proline rylation was likely more complicated or different than previ- was used as nitrogen source. Here, experiment-to-experiment ously reported and motivated a more thorough investigation. values occasionally varied into the 20% range for isolated sam- Most importantly, these data showed that Sit4 is active in glu- ples. Therefore, our attempts to achieve as accurate and precise tamine-grown cells in which Tor1,2 were posited to actively data as possible notwithstanding, we attribute less significance phosphorylate Tap42 (and/or Tip41), thereby bringing about to the precise percentages of Gln3-Myc distribution among inhibition of Sit4 protein phosphatase. the three possible intracellular categories than to the patterns Influence of Sit4 and Pph3 Phosphatase on Steady State Levels of change observed in the distribution of Gln3-Myc when of SIT4 and PPH3 mRNA—Previous reports of both Pph3 and comparing one experimental condition to another, i.e. presence Sit4 functioning in rapamycin-induced Gln3 dephosphoryla- versus absence of inhibitor, nitrogen source identity, wild type tion and localization (14, 17, 22), prompted us to query whether versus mutant strains. the expression of their cognate genes was subject to Tor1,2 or NCR-sensitive regulation. Therefore, we assayed SIT4 and RESULTS PPH3 expression in wild type, sit4,and pph3 strains cultured Sit4 Phosphatase Brings about Gln3-Myc Dephosphoryla- under multiple conditions: the presence and absence of rapa- tion in Cells Provided with Good Nitrogen Sources—Our initial mycin (in glutamine medium) or Msx (in ammonia medium), experiment investigated central, but previously untested, ten- and following transfer from glutamine to proline or nitrogen- DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37983 Sit4 Phosphatase Active Irrespective of Nitrogen Source free medium. As shown in Fig. 3B, neither SIT4 nor PPH3 expression appeared to exhibit a response to the nitrogen source provided. As expected, there was no expression when the genes themselves were deleted (Fig. 3B). Type 2A-related Phosphatase Requirements for Nuclear Localization of Gln3-Myc —Next we evaluated the influence of defects in type 2A-related phosphatases, Sit4 and Pph3, on Gln3-Myc localization in cells cultured as described in Fig. 3B, lanes A–D and with proline as sole nitrogen source. Wild- type cells behaved as expected and previously reported, i.e. Gln3-Myc was cytoplasmic in glutamine-grown cells, and nuclear in proline-grown or rapamycin-treated cultures as well as glutamine-grown cells transferred to proline or nitrogen free medium (Fig. 4, column W.T.). Intracellular Gln3-Myc local- ization in pph3 mutants did not significantly differ from that in wild type (Fig. 4, column pph3). In contrast, Gln3-Myc was predominantly cytoplasmic in sit4 cells treated with rapa- mycin, transferred from glutamine to proline- or nitrogen-free medium, and grown with proline as sole nitrogen source (Fig. 4, column sit4). Results obtained with the sit4pph3 double mutant were similar to those observed with sit4 alone (data not shown). These data, at face value, supported the earlier contention that Sit4 was required for nuclear accumulation of Gln3-Myc when nitrogen was limiting or cells were treated with rapamycin. Contrasting Phosphatase Requirements for Nuclear Localiza- tion of Gln3-Myc in Response to Rapamycin and Msx Treatment—Careful inspection of many images such as those in Fig. 4 suggested the method of classification we had been using might be too crude to describe fully what occurred in response to various experimental perturbations. There were times when Gln3-Myc was neither completely nuclear nor cytoplasmic, i.e. one could see Gln3-Myc fluorescence in both cellular compartments. Therefore, we increased the resolution of our measurements by introducing a third scoring category, nuclear-cytoplasmic localization. Criteria used to place cells in each of the three categories were as follows: (i) cells in which Gln3-Myc could only be detected in the cytoplasm were scored cytoplasmic, (ii) those in which only nuclear localization was detected were scored nuclear, and (iii) those in which stain- ing could be clearly detected in both compartments were scored nuclear-cytoplasmic. The third category was clearly subjective. However, this potential for subjectivity did not prove to be problematic as shown by evaluation of the assay under “Materials and Methods.” Success and reliability of the assay most required consistency in scoring since it was the accurate and reproducible detection of changes in the patterns of Gln3-Myc distribution among the cellular compartments FIGURE 4. Intracellular distribution of Gln3-Myc . This was observed in wild-type (W.T.; TB123), sit4 (TB136), and pph3 (FV3) mutant cells grown that is most critical. in glutamine (Gln) or ammonia ( NH ) medium in the presence ( Rap) or Using these scoring criteria, we assayed Gln3-Myc intracel- absence of rapamycin or methionine sulfoximine (MSX), in proline (Pro) lular localization in wild type and three isogenic phosphatase medium, or in after transfer from glutamine to nitrogen-free (Gln to Nitro- gen Starvation; 60 min.) or proline (Gln to Pro; 60 min.) medium. Images are defective mutants. Gln3-Myc was cytoplasmic in all gluta- presented in pairs in which the Gln3-Myc (red) image appears above the mine-grown wild-type cells (Fig. 5A, red bar, W.T.). Following same one stained with DAPI (blue). rapamycin treatment, Gln3-Myc became nuclear-cytoplas- mic (yellow bar) in about 80% of the cells. In the remaining cells, ization of Gln3-Myc when glutamine-grown cultures were Gln3-Myc was about equally distributed between the nuclear transferred to minimal-proline or nitrogen-free medium (Fig. (green bar) and cytoplasmic (red bar) compartments (Fig. 5A, 5A, W.T.). However, Gln3-Myc was still cytoplasmic in about W.T.). The distribution shifted toward a greater nuclear local- 20% of the cells following nitrogen starvation, which may have 37984 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source Sit4 or Pph3 requirement follow- ing Msx treatment clearly distin- guishing it from the response to rapamycin. Sit4 Requirement of Gln3-Myc Localization Is Nitrogen Source- dependent—The Gln3-Myc dis- tribution data above were difficult to rectify with the idea that Sit4, per- formed the requisite dephosphoryl- ation of Gln3 in preparation for its transport into the nucleus. There- fore, we approached the putative Sit4 requirement from a different direction. If Sit4 functioned down- stream of the nitrogen supply signal in the Gln3 regulatory pathway, then the response to both rapamy- cin and Msx treatment in sit4 mutants should be independent of the nitrogen source provided. If, on the other hand, the nitrogen supply signal was situated downstream of Sit4, or in another regulatory path- way, then the nitrogen source pro- vided to the cells would be expected to influence Gln3 localization in wild-type and sit4 cells. To investigate these possibilities, FIGURE 5. A, intracellular Gln3-Myc distribution in wild type and type 2A-related phosphatase mutants cul- 13 we determined Gln3-Myc local- tured in glutamine (Gln) medium in the presence (Rap) or absence of rapamycin, or after transfer from ization, following rapamycin or Msx glutamine to nitrogen-free (Gln to N. S.; 60 min.) or proline (Gln to Pro; 60 min.) medium. Cells (TB123, TB136, FV3, and FV4) were scored as described under “Materials and Methods”: cytoplasmic (red), nuclear-cytoplasmic treatment in cultures provided with (yellow), and nuclear (green). B, strains identical to those used in A were cultured in ammonia medium in the nitrogen sources ranging from good presence (Msx) or absence of methionine sulfoximine. to poor: glutamine, YPD, ammonia, resulted from insufficient time being allowed for the nitrogen and proline (Fig. 6, A–D). Gln3-Myc was completely cytoplas- reserves of these cells to be completely exhausted before they mic in glutamine medium and this distribution was altered only were harvested. by rapamycin treatment which elicited Sit4-dependent, nucle- When sit4 and pph3 single and sit4pph3 double ar-cytoplasmic localization in most cells (Fig. 6A). Msx treat- mutants were subjected to the same perturbations as the wild ment failed to elicit a response in glutamine-grown cells con- type, quite unambiguous phenotypes were observed. The dis- curring with earlier reports (21, 27). Our experiments do not tribution of Gln3-Myc in a pph3 was nearly indistinguish- distinguish whether this result occurred because (i) providing able from wild type irrespective of the condition tested (Fig. glutamine as the nitrogen source eliminates the need for Msx- 5A). In contrast, Gln3-Myc was restricted to the cytoplasm of inhibited glutamine synthetase, and/or (ii) glutamine provided both sit4 and sit4pph3 cells cultured under comparable in the medium inhibits Msx uptake. conditions. This suggested, in agreement with other reports, In contrast, Gln3-Myc localization successively shifted in that Sit4 was required for nuclear localization of Gln3-Myc in Msx-treated cells from cytoplasmic to nuclear-cytoplasmic to cells exposed to these three conditions. However, the Sit4 nuclear first in the wild type and then in the sit4 as the nitro- requirement was nearly absent when analogous experiments gen source changed from glutamine (Fig. 6A) to YPD (Fig. 6B) were performed with the metabolic inhibitor, Msx. As shown in to ammonia (Fig. 6C) to proline (Fig. 6D). Note that a similar Fig. 5B, Gln3-Myc was largely cytoplasmic in ammonia- but less pronounced shift occurred after rapamycin treatment grown cultures, but was nuclear and/or nuclear-cytoplasmic in in ammonia- versus proline-grown cells (Fig. 6, C and D). The all but a few sit4 or sit4pph3 mutant cells following Msx same results were observed in the sit4pph3 double mutant treatment. Again, as seen in Fig. 5A, the Gln3-Myc intra- (Fig. 6E). These data suggested that Msx and rapamycin-in- cellular distribution profile in a pph3 was indistinguishable duced Gln3-Myc nuclear localization exhibited a nitrogen from that of wild type (Fig. 5B). These data demonstrated source-dependent Sit4 requirement, which paralleled that of that, although treating cells with rapamycin or Msx had been NCR, i.e. the more severe the NCR, the greater the Sit4-require- reported to have similar inhibitory effects on Tor1,2 activity ment. However, even in proline-grown cells, a limited Sit4-re- 13 13 (22, 27), nuclear localization of Gln3-Myc did not possess a quirement remained (Fig. 6D, Gln3-Myc was cytoplasmic in DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37985 Sit4 Phosphatase Active Irrespective of Nitrogen Source appeared (14, 15). The data presented above suggested that intracellular localization profiles of Gln3-Myc might differ from one strain to another as a result of strain-dependent dif- ferences in nitrogen catabolism and/or sensitivity to NCR anal- ogous to those reported in earlier studies (40, 42). We decided to test this hypothesis by comparing the intracellular distribu- tion of Gln3-Myc in two genetic backgrounds, TB123 (the background in which the Sit4 requirement was first reported (14)) and BY4709 (from the yeast genome deletion project (32)). Gln3-Myc localization following rapamycin treatment of glu- tamine-grown wild type and sit4 strains were independent of the genetic background (Fig. 7A). In YNB-ammonia medium, Gln3-Myc localization shifted from the cytoplasm to the nuclei of Msx-treated cells in both genetic backgrounds (Fig. 7B). Additionally, Gln3-Myc was more nuclear-cytoplasmic in untreated ammonia-grown BY4709-derived than TB123-de- rived cells. An even more striking difference occurred when cells were grown in YNB-proline medium (Fig. 7C). In a TB123 genetic background, Gln3-Myc localization in proline medium required Sit4, whereas in a BY4709 background no such requirement was observed. A clear difference between the TB123 and BY4709 genetic backgrounds is an rme1 mutation present in the former strain but not in the latter. To determine whether this mutation accounted for the two strains strikingly different Sit4 require- ments with proline as nitrogen source, we transformed TB123 and sit4pph3 (FV4) strains with CEN-plasmid yCplac22- RME1. Untransformed strains and corresponding transfor- mants were grown in glutamine, glutamine  rapamycin, and proline media and Gln3-Myc localization measured. Gln3- Myc localization in the transformants was not detectably dif- ferent from that observed in the untransformed recipients (data not shown). These observations argued that the rme1 mutation did not account for differing Gln3-Myc distributions observed in the two genetic backgrounds. Together, data presented above demonstrate the extent to which Sit4 is required for inhibitor-induced nuclear localiza- tion of Gln3-Myc qualitatively correlates with the degree of NCR elicited by the nitrogen source provided to the cells. The Sit4 requirement was greater with repressive nitrogen sources than with those that were non-repressive. This also occurs for rapamycin-induced Gln3-Myc nuclear localiza- tion. At face value, the data are more consistent with the suggestion that the cellular signal generated in response to its nitrogen supply is situated at or below the level of Sit4 function because a poor nitrogen source, such as proline, decreased and completely bypassed the requirement for Sit4 FIGURE 6. Sit4 requirement for nuclear localization of Gln3-Myc follow- ing rapamycin (Rap) or Msx (MSX) treatment decreases with nitrogen in the TB123 and BY4709 genetic backgrounds, respectively. sources of decreasing from good to poor quality. The nitrogen source(s) Alternatively, Sit4 and the nitrogen signal that influenced are indicated in the panels along with the inhibitor treatments and pertinent genotype of the cells used; strains were the same as described in the legend Gln3 localization derived from separate branches of the reg- to Fig. 5. ulatory pathway. Gln3-Myc Phosphorylation/Dephosphorylation in Wild 37% of the rapamycin-treated cells), which decreased further Type and Type 2A-related Phosphatase-defective Strains—Data if pph3 was also deleted (Fig. 6E, Gln3-Myc was cytoplasmic in Fig. 3 demonstrated that Sit4 is clearly active and influences in 26% of the rapamycin-treated cells). Gln3-Myc dephosphorylation levels even in cultures growing Strain-dependent Effects of sit4 Mutations—Reports reach- with a good nitrogen source, glutamine. We reasoned that ing differing conclusions with respect to Tap42 and Sit4 partic- deleting the cognate gene of an enzyme whose activity was ipation in the regulation of NCR-sensitive transcription have reported to be inactivated in the model describing Tor1,2 reg- 37986 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source FIGURE 8. Nitrogen source-dependent Gln3-Myc phosphorylation occurs in a sit4 mutant. A, percent of wild-type (W.T.; TB123) or sit4 (TB136) cells in which Gln3-Myc is cytoplasmic. The cells were grown in YNB medium with the indicated nitrogen sources (glutamine (Gln), proline (Pro), ammonia ( NH )) or in YPD medium. B, Western blot analysis of Gln3-Myc phosphorylation in wild-type and sit4 cells cultured as described in A. phorylation varied greatly in the sit4 (Fig. 8B). It was lowest in ammonia- (lane C) or proline- (lane B) grown sit4 cells and reached its highest levels with glutamine (lane D)or YPD (lane E) as nitrogen source (Fig. 8B). The relationship was just opposite that observed when a parallel experiment was performed several years ago using wild-type cells (26). In that case, Gln3-Myc intracellular localization ranged from completely cytoplasmic with glutamine to nuclear with pro- line, while Gln3-Myc was almost uniformly phosphoryla- ted (Fig. 3 and Ref. 26). Although Gln3-Myc phosphoryla- FIGURE 7. Strain background and nitrogen source dependence of intra- cellular Gln3-Myc distribution observed in wild-type (TB123 and tion changed with the nitrogen source in a sit4, these FV017) and sit4 (TB136 and FV1) cells. Culture conditions were as changes did not correlate with whether the nitrogen source described in the legend to Figs. 5 and 6. was a good or poor one. If it had, then the Gln3-Myc phos- phorylation profile observed with ammonia as nitrogen ulation of Gln3 would not be expected, in the most straightfor- source should have been more similar to that observed with ward case, to detectably affect Gln3-Myc phosphorylation glutamine than with proline (Fig. 8B). 13 13 levels. This reasoning and the Gln3-Myc nitrogen source-de- The above data convincingly demonstrated Gln3-Myc was pendent distribution data described above prompted us to phosphorylated to different extents in sit4 cells provided with determine the relationship between Gln3-Myc localization, various nitrogen sources, but did not distinguish whether it was 13 13 Gln3-Myc phosphorylation, and the nitrogen source pro- sit4 or the nitrogen source that altered Gln3-Myc phospho- vided to wild-type and mutant cells. rylation. Therefore, we compared Gln3-Myc phosphoryla- Investigating these relationships, we found that Gln3-Myc tion in wild-type and sit4 cells cultured under various condi- was almost uniformly cytoplasmic in a sit4 (TB123 genetic tions. Deletion of SIT4 increased Gln3-Myc phosphorylation background) irrespective of the nitrogen source provided (Fig. irrespective of the nitrogen source provided (Fig. 9, A–D, lanes 8A). Only in proline medium was Gln3-Myc minimally A and B, note the black dots between lanes A and B). These ( 10%) nuclear-cytoplasmic. In contrast, Gln3-Myc phos- effects appeared in one or both of two ways: (i) disappearance of DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37987 Sit4 Phosphatase Active Irrespective of Nitrogen Source phorylation in a sit4. If both Msx and rapamycin act at or above the level of Tor1,2 in the regulatory pathway (22), and hence above the level of Sit4 dephosphorylation of Gln3- Myc (14) then the loss of Sit4 should eliminate responses to the inhibitor-generated signals in parallel and irrespective of the nitrogen source provided. On the other hand, if this is not the case, loss of Sit4 would not be expected to cause such uniform changes in Gln3-Myc phosphorylation levels. Treating a sit4 with Msx increased Gln3-Myc phosphoryla- tion in both ammonia- and proline-grown cells (Fig. 9, A and B, lanes B and C). Increased Gln3-Myc phosphorylation was not observed with YPD- or glutamine-grown sit4 cells (Fig. 9, C and D, lanes B and C). When evaluating these results, however, it is important to keep two things in mind: (i) Msx is reported (and we have confirmed) to be ineffective in wild type, glu- tamine-grown cells (22, 27). On the other hand, the inhibitor shifted Gln3-Myc localization from cytoplasmic to nuclear- cytoplasmic and nuclear in wild type YPD-grown cells indicat- ing that it did function in this medium. (ii) Gln3-Myc was already highly phosphorylated in glutamine and YPD grown cells. Such high levels of Gln3-Myc phosphorylation at the outset may have masked whatever effects that might have occurred when the ability to dephosphorylate Gln3-Myc was lost in the sit4. In sum, the level of Gln3-Myc phosphoryla- tion either remained the same or increased following Msx treat- ment of a sit4. This compares with increased Gln3-Myc phosphorylation observed upon Msx treatment of wild-type cells regardless of the nitrogen source. The responses of Gln3-Myc phosphorylation in rapamy- cin-treated, sit4 cells did not parallel the effects described above. First, in the sit4, rapamycin did not elicit as extensive dephosphorylation of Gln3-Myc as in wild-type cells, where treating cells with rapamycin or cell extracts with calf intestinal alkaline phosphatase yield similarly dephosphorylated Gln3- Myc species (Fig. 4A of Ref. 26). This is easily observed com- paring the wild type and sit4 Gln3-Myc profiles (Fig. 9, B–D, lanes D and E). On the other hand, rapamycin clearly caused some Gln3-Myc dephosphorylation in YPD- and glutamine- grown sit4 cells (Fig. 9, C and D, lanes B and D). Rapamycin- induced Gln3-Myc dephosphorylation was not apparent in an ammonia-grown sit4 (Fig. 9B, lanes B and D), and Gln3-Myc phosphorylation actually increased in proline-grown sit4 as previously observed in wild-type cells (Fig. 9A, lanes B and D and in Fig. 4D of Ref. 27). In sum, Gln3-Myc phosphoryla- tion following rapamycin treatment decreased, remained FIGURE 9. Sit4 is active with respect to Gln3-Myc phosphorylation irre- unchanged, or increased depending upon the nitrogen spective of the nitrogen source. Western blot analyses of Gln3-Myc phos- phorylation in wild type (W.T.; TB123) and sit4 (TB136) grown in the pres- source used. ence () or absence () of rapamycin (Rap) or Msx (Msx) as described It was not difficult to envision that Gln3-Myc dephospho- under “Materials and Methods.” The nitrogen source in which each experi- ment was conducted is indicated above the blots. rylation noted above in a sit4 might derive from the closely related Pph3 phosphatase. To evaluate this possibility we assayed Gln3-Myc phosphorylation in pph3 single and a faster migrating species and/or appearance of a slower sit4pph3 double mutants. As shown in Fig. 10A, the Gln3- migrating Gln3-Myc species (Fig. 9, A, B, and D, lanes A and Myc phosphorylation profiles in untreated as well as rapamy- B), or (ii) a shift in the relative amounts of Gln3-Myc species, with a slower mobility species increasing and the faster migrat- cin- and Msx-treated pph3 cells were indistinguishable from ing species decreasing (Fig. 9, A, B, and D). wild type. This parallels the results observed with Gln3-Myc We next determined whether the nitrogen source affected intracellular localization (Fig. 5). In addition, the sit4pph3 rapamycin- or Msx-mediated changes in Gln3-Myc phos- double mutant possessed a phenotype that was indistinguish- 37988 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source variations in Gln3-Myc phosphorylation. The overall Gln3- Myc phosphorylation profiles were similar, though not iden- tical in wild-type cells from a TB123 versus BY4709 genetic background (Fig. 10C). There was overall greater Gln3-Myc phosphorylation in the BY4709 background. This is observed as: (i) a slight increase in the upper-most and decrease in the lower-most Gln3-Myc species in ammonia-grown FV017 (BY4709) cells (Fig. 10C, lanes E versus F), or (ii) the appearance of a slower migrating species (Fig. 10C, lanes A versus B), and a decrease in the fastest migrating species in glutamine-grown BY4709 cells (lanes A and B) relative to results in the TB123 background. Similarly, rapamycin induced Gln3-Myc dephosphorylation in FV017 (BY4709), but not to the level observed earlier for TB123. Compare the relative positions of Gln3-Myc species in Fig. 9D, lanes E and F with those in Fig. 10C, lanes A and C, i.e. the positions in TB123  rapamycin (Fig. 9) and FV017  rapamycin (Fig. 10) relative to the posi- tions in untreated TB123. The increased percentage of untreated, ammonia-grown FV017 (BY4709) cells containing nuclear-cytoplasmic Gln3-Myc relative to TB123 (Fig. 7B) was not paralleled by decreased Gln3-Myc phosphorylation as would be expected from the current model describing Tor1,2 regulation of Gln3. If anything, Gln3-Myc phosphorylation remained the same or increased modestly in the BY4709 back- ground (Fig 10C, lanes A and B and E and F). Changes in Gln3-Myc phosphorylation resulting from the deletion of SIT4 in the BY4709 genetic background were the same as those in the TB123 background. However, this experi- ment again emphasized another difference in the behavior of Gln3-Myc in the two genetic backgrounds, i.e. a significant decrease in the amount of detectable Gln3-Myc observed in ammonia-grown BY4709 wild-type and sit4 cells. This neces- sitated the use of longer exposures during development of the Western blot depicted in Fig. 10D (lanes D–F) even though all six lanes were derived from a single membrane. A similarly diminished Gln3-Myc signal also occurred in Fig. 10C, lanes D and E. Our last objective was to compare the phosphorylation pro- files of proline-grown sit4 cells in the TB123 and BY4709 genetic backgrounds, because nuclear localization of Gln3- Myc was Sit4-dependent in the former and Sit4-independent in the latter (Fig. 7C). To this end, we repeated the experiment described in Fig. 9A using wild-type (FV017) and sit4 (FV1) FIGURE 10. A and B, effects of defects in phosphatase Pph3 on Gln3 phospho- rylation. Wild type (W.T. TB123), pph3 (FV3), and sit4pph3 (FV4) were strains. Unfortunately, the Gln3-Myc signals were so dimin- grown in YNB-glutamine (Gln) or -ammonia ( NH ) medium in the presence ished and of such poor quality that our observations can be or absence of rapamycin or Msx. C and D, effects of the genetic background on Gln3-Myc phosphorylation. Strains used in these experiments were considered only tentative at best. That said, we were unable to derived from JK9 –3da (TB123), i.e. pph3 (FV3), pph3sit4 (FV4), or BY4709 detect a difference between the phosphorylation of Gln3-Myc (FV017), and sit4 (FV1). The experimental format and conditions were as described in the legend to Fig. 9. in the proline-grown wild type (FV017) and sit4 (FV1) strains. We could, however, detect modestly increased Gln3-Myc phosphorylation upon addition of rapamycin to the sit4 cul- able from that of a sit4 both with respect to the effects elicited ture similar to that observed in the TB123 genetic background by nitrogen source and the two inhibitors (Fig. 10B). in Fig. 9A, lanes B and D (data not shown). In sum, although Influence of Genetic Background on Gln3-Myc Phos- there were modest differences in the phosphorylation profiles phorylation—Differences in intracellular Gln3-Myc distribu- observed in the TB123 and BY4709 genetic backgrounds, none tion in different strains, especially in untreated, ammonia- of them appeared as drastic as the differences observed in intra- grown wild type and proline-grown sit4 cells prompted us to ascertain whether these differences correlated with parallel cellular Gln3-Myc distribution in the two strains. DECEMBER 8, 2006• VOLUME 281 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 37989 Sit4 Phosphatase Active Irrespective of Nitrogen Source DISCUSSION Correlating with this loss of requirement, BY4709-derived FV017 is less NCR-sensitive than TB123 with at least ammonia This work was undertaken to gain a better understanding of as nitrogen source. Whether this correlation derives from a how the type 2A-related protein phosphatases, Sit4 and Pph3, cause-effect relationship between the strains and nitrogen participate in the regulation of Gln3 phosphorylation and intra- source provided or alternatively from an unrelated event, can- cellular localization. Conceptually, we expected these enzymes, not be ascertained at present. Further, existing data do not per- being multifunctional, would be active toward one or another mit us to evaluate whether the lack of difference in Gln3-Myc substrate under most conditions. The pivotal question was phosphorylation profiles observed for the two strains derives whether they were specifically active with respect to Gln3 phos- from phosphorylation/dephosphorylation events that might phorylation in a nitrogen source-dependent manner. In other exist, but are not detected in a total phosphorylation profile. words, was the extent of Gln3 phosphorylation dictated by the The decreasing Sit4 requirement in glutamine versus ammo- degree to which a nitrogen source resulted in active versus inac- nia versus proline medium adds to a growing list of situations in tive Sit4 and Pph3? For Pph3, we were unable to demonstrate an which the effects of the nitrogen source and NCR control active role in Gln3 control, i.e. detectable Gln3-Myc phos- appear to feed into the regulation of Gln3 downstream of phorylation and intracellular localization were minimally if at Tor1,2 and its inhibition by rapamycin. This relation of the all affected by deleting PPH3. Sit4, on the other hand, clearly nitrogen signal to Tor1,2 is also consistent with the observa- influenced Gln3-Myc phosphorylation levels, but did not do tions that Sit4 is not required for Msx-induced nuclear local- so in a nitrogen source-dependent manner as expected. Delet- ization of Gln3-Myc . They join the earlier observation that ing SIT4 increased Gln3-Myc phosphorylation irrespective of Tor1,2 control of retrograde gene expression is also nitrogen the nitrogen source provided. Sit4 was active with all nitrogen source-dependent in that the ability of rapamycin to induce sources, and most importantly, with nitrogen sources that are retrograde gene expression (CIT2 is the reporter) occurs with the most repressive for NCR-sensitive transcription. In fact, glutamine as nitrogen source, but not proline (28, 40). The sep- differences in Gln3-Myc phosphorylation observed when arability of retrograde regulation from Tor1,2 activity has been comparing wild type and a sit4 were greatest with good nitro- recently confirmed by Giannattasio et al. (39). It is important to gen sources such as glutamine or in YPD medium, conditions note, however, that while these observations support the con- where Sit4 would have been expected to be least active and tention of a nitrogen source-derived signal feeding into the therefore a sit4 to have the least effect. These results are just Gln3 regulatory pathway downstream of the rapamycin-inhib- opposite of predictions emanating from the idea that Tor1,2, ited step, they do not exclude a more complicated possibility Tap42, and/or Tip41 control Gln3 phosphorylation and local- hypothesizing the existence of two nitrogen signals, one ization by regulating Sit4 activity (14, 20). impinging on regulation above the rapamycin-inhibited step A second experimental finding, which pointed to a less direct and the other below it. role for Sit4 in Gln3 regulation, was its declining requirement Our investigations identify an interesting set of relation- for rapamycin- and Msx-induced nuclear Gln3 localization in ships that offer insights into the nitrogen-responsive control response to the quality of the nitrogen source, i.e. good versus pathway. Earlier studies reported the only time that demon- poor (Fig. 6). Under the current view of Tor1,2 control, Sit4 is strable dephosphorylation of Gln3-Myc correlated with its posited to be most active following loss of the positive nitrogen nuclear localization was shortly after treating cells with signal activating Tor1,2. The positive signal (glutamine or one rapamycin (26). In ammonia- or glutamine-grown wild-type of its metabolites) is lost when cells are provided with a poor cells, Gln3-Myc was cytoplasmic and in proline-grown nitrogen source, treating them with Msx, or inhibiting Tor1,2 cells it was nuclear. Yet Gln3-Myc was almost uniformly itself with rapamycin. Therefore, Sit4 activity, and hence its phosphorylated; Gln3-Myc was slightly more phosphoryl- requirement for nuclear Gln3-Myc localization, should ated with proline than with ammonia or glutamine as nitro- increase as the quality of available nitrogen decreases. In con- gen source (26, 27). In present studies, just the opposite trast to these predictions, the Sit4 requirement for Msx-in- occurred, i.e. Gln3-Myc was cytoplasmic in the sit4 mutant duced nuclear localization of Gln3 disappears as one moves regardless of whether the nitrogen source was good or poor from YPD to ammonia or proline media. Analogously, the Sit4- (Fig. 8). However, Gln3-Myc phosphorylation levels requirement is significantly reduced for rapamycin-induced responded markedly to the nitrogen source provided. In nuclear Gln3 localization in proline-grown cells, i.e. Gln3 is other words, deleting SIT4 unmasked the response of Gln3- nuclear-cytoplasmic or nuclear in the majority of rapamycin- Myc phosphorylation to the nitrogen source. This is the treated sit4 ( 60%) or sit4pph3 ( 75%) cells (Fig. 6, D and first time Gln3 phosphorylation has been shown to be asso- E). In other words, the poorer the nitrogen source the less Sit4 is ciated with a nitrogen source provided to the cells. If Sit4 was required for nuclear Gln3-Myc localization. the molecule responsible for dephosphorylating Gln3-Myc Our data, describing a relationship between nitrogen source, in response to nitrogen source, then the nitrogen source-de- strain background, and Sit4 requirement for Gln3-Myc local- pendent signal would a priori have been expected to be lost ization, are consistent with some studies finding a Tap42-Sit4 when SIT4 was deleted. This suggests that the nitrogen requirement for Gln3 control (14), whereas others do not (15). source specificity of NCR control is probably more directly We observed, for example, that Sit4 is necessary for Gln3 to connected to the response of one or more protein kinases accumulate in the nuclei of proline-grown cells in the TB123 responsible for Gln3-Myc phosphorylation than to the genetic background, but not for BY4709-derived cells (Fig. 7C). type 2A-related protein phosphatases (Sit4 and Pph3). 37990 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 •DECEMBER 8, 2006 Sit4 Phosphatase Active Irrespective of Nitrogen Source 3. Morgensztern, D., and McLeod, H. L. (2005) Anticancer Drugs 16, By this reasoning, Gln3 phosphorylation would occur in the 797–803 presence of nitrogen source-independent Sit4 phosphatase 4. Lorber, M. I., Mulgaonkar, S., Butt, K. M., Elkhammas, E., Mendez, R., activity, the latter buffering or masking the effects of nitrogen Rajagopalan, P. R., Kahan, B., Sollinger, H., Li, Y., Cretin, N., Tedesco, H., source-responsive phosphorylation from being observed. Here, and B251 Study Group (2005) Transplantation 80, 244–252 type 2A-related phosphatases rather than generating the signal 5. Thomas, G., Sabatini, D., and Hall, M. N. (2004) TOR: Target of Rapamy- that results in Gln3-Myc accumulating in the nucleus, would cin: Current Topics in Microbiology & Immunology, Vol. 279, Springer- Verlag, Berlin serve a stabilizing role to reset the system in preparation to 6. Inoki, K., Ouyang, H., Li, Y., and Guan, K. L. Microbiol. Mol. Biol. Rev. receive such signals. In this model, the observed Gln3-Myc (2005) 69, 79–100 phosphorylation would derive from the ratio of kinase to phos- 7. Hofman-Bang, J. (1999) Mol. Biotechnol. 12, 35–73 phatase activities acting on Gln3, the former being the one pre- 8. ter Schure, E. G., van Riel, N. A., and Verrips, C. T. (2000) FEMS Microbiol. dominantly modulated by the nitrogen source provided. Rev. 24, 67–83 Although the above ideas would explain Gln3 phosphoryl- 9. Cooper. T. G. (2002) FEMS Microbiol. Rev. 26, 223–238 10. Cooper, T. G. (2004) Topics in Current Genetics (Winderickx, J. and Tay- ation and localization in the sit4, present data are insuffi- lor, P. M., eds) Vol. 7, pp. 225–257, Springer-Verlag, Berlin cient to provide details of the precise link(s) between the 11. Magasanik, B., and Kaiser, C. A. (2002) Gene (Amst.) 290, 1–18 nitrogen source, observable Gln3 phosphorylation levels and 12. Cox, K. H., Rai, R., Distler, M., Daugherty, J. R., Coffman, J. A., and Cooper, NCR-sensitive transcription. For example, they do not T. G. (2000) J. Biol. Chem. 275, 17611–17618 exclude the possibility that a nitrogen source may regulate 13. Cunningham, T. S., Andhare, R., and Cooper, T. G. (2000) J. Biol. Chem. 275, 14408–14414 NCR-sensitive transcription via influencing Gln3 binding to 14. Beck, T., and Hall, M. N. (1999) Nature 402, 689–692 the DNA in addition to controlling its intracellular localiza- 15. Cardenas, M. E., Cutler, N. S., Lorenz, M. C., Di Como, C. J., and Heitman, tion. Further, they do not identify the physiological advan- J. (1999) Genes Dev. 13, 3271–3279 tage gained when functional Sit4 brings Gln3-Myc phos- 16. Hardwick, J. S., Kuruvilla, F. G., Tong, J. K., Shamji, A. F., and Schreiber, phorylation to the same level irrespective of the nitrogen S. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14866–14870 source provided and the intracellular localization of Gln3- 17. Bertram, P. G., Choi, J. H., Carvalho, J., Ai, W., Zeng, C., Chan, T. F., and Zheng, X. F. (2000) J. Biol. Chem. 275, 35727–35733 Myc . And finally, they are also insufficient to distinguish 18. Di Como, C. J., and Arndt, K. T. (1996) Genes Dev. 10, 1904–1916 the possibility that Gln3-Myc phosphorylation increases in 19. Jiang, Y., and Broach, J. R. (1999) EMBO J. 18, 2782–2792 a sit4 because Sit4 phosphatase is no longer dephospho- 20. Jacinto, E., Guo, B., Arndt, K. T., Schmelzle, T., and Hall, M. N. (2001) Mol. rylating Gln3-Myc from the alternative that loss of Sit4 Cell. 8, 1017–1026 activity increases protein kinase activity which in turn 21. Rohde, J. R., Campbell, S., Zurita-Martinez, S. A., Cutler, N. S., Ashe, M., increases Gln3-Myc phosphorylation. and Cardenas, M. E. (2004) Mol. Cell. Biol. 24, 8332–8341 22. Crespo, J. L., Powers, T., Fowler, B., and Hall, M. N. (2002) Proc. Natl. Data presented in Fig. 9 also indicate that loss of Sit4 and Acad. Sci. U. S. A. 99, 6784–6789 Pph3 activities are not sufficient to abrogate the influence of 23. Magasanik, B. (2005) Proc. Natl. Acad. Sci. U. S. A. 2005 102, Tor1,2 on Gln3-Myc phosphorylation level. Addition of rapa- 16537–16538 mycin to YPD- glutamine- or ammonia-grown sit4 cells 24. Duvel, K., Santhanam, A., Garrett, S., Schneper, L., and Broach, J. R. (2003) clearly decreases Gln3-Myc phosphorylation. The most likely Mol. Cell. 11, 1467–1478 candidates to mediate this dephosphorylation are Pph21 and 25. Wang, H., Wang, X., and Jiang, Y. (2003) Mol. Biol. Cell 14, 4342–4351 26. Cox, K. H., Kulkarni, A., Tate, J. J., and Cooper, T. G. (2004) J. Biol. Chem. Pph22 (19, 26, 39). Although we have not yet investigated the 279, 10270–10278 roles of Pph21 and Pph22 in the regulation of Gln3 because of 27. Tate, J. J., Rai, R., and Cooper, T. G. (2005) J. Biol. Chem. 280, technical difficulties derived from the severe growth defect and 27195–27204 other difficult phenotypes of the pph21,22 mutations, the rela- 28. Tate, J. J., Cox, K. H., Rai, R., and Cooper, T. G. (2002) J. Biol. Chem. 277, tionships are unlikely to be straightforward. One expression of 20477–20482 29. Dilova, I., and Powers, T. (2006) FEMS Yeast Res. 6, 112–119 this expectation is the fact that although rapamycin induced 30. Cox, K. H., Tate, J. J., and Cooper, T. G. (2002) J. Biol. Chem. 277, dephosphorylation of Gln3-Myc in the sit4, i.e. a strain in 37559–37566 which Pph3, Pph21 and Pph22 are all functional, it did not 31. Cox, K. H., Tate, J. J., and Cooper, T. G. (2004) J. Biol. Chem. 279, induce its nuclear localization with any of the good nitrogen 19294–19301 sources. Further, treating cells with Msx increases Gln3-Myc 32. Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, phosphorylation and yet results in nuclear localization of Gln3- S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., Astromoff, A., El-Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Cur- Myc (27). tiss, M., Davis, K., Deutschbauer, A., Entian, K. D., Flaherty, P., Foury, F., Garfinkel, D. J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J. H., Acknowledgments—We thank Dr. Michael Hall for strains and Drs. Hempel, S., Herman, Z., Jaramillo, D. F., Kelly, D. E., Kelly, S. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Dec 8, 2006

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