Volume 11, Issue 6, Pages 1467-1478 (June 2003) Multiple Roles of Tap42 in Mediating Rapamycin-Induced Transcriptional Changes in Yeast  Katrin Düvel, Arti Santhanam, Stephen Garrett, Lisa Schneper, James R Broach  Molecular Cell  Volume 11, Issue 6, Pages 1467-1478 (June 2003) DOI: 10.1016/S1097-2765(03)00228-4

Figure 1 Characterization of New tap42 Mutants (A) Serial dilutions (1:10) of strains grown at 23°C were transferred onto SC plates and incubated at the indicated temperatures for 36 hr. The genotype of the strains are listed in Table 1, TAP42 (Y3033); tap42-106 (Y3034); tap42-109 (Y3035); TAP42/tap42-106 (Y3045); TAP42/tap42-109 (Y3046). (B) Rapamycin resistance of tap42-106 or tap42-109 strains in comparison to wild-type TAP42 and a previously isolated allele, tap42-11. Serial dilutions were transferred onto YEPD containing 100 ng/ml rapamycin. Growth at 23°C was recorded after 3 and 6 days, respectively. A strain carrying a rapamycin-resistant TOR2(S1975R) allele served as positive control. (C) Strains Y3033 (TAP42), Y3034 (tap42-106), and Y3035 (tap42-109) were grown in YEPD at 23°C and then shifted to 37°C. Cells were removed at the indicated times, lysed, fractionated by SDS PAGE, transferred to nitrocellulose, and probed with polyclonal antibodies to either Tap42 or Tpd3. (D) Protein extracts of strains Y3079 (PPH22(HA)3 TAP42), Y3080 (PPH22(HA)3 tap42-106), and Y3081 (PPH22(HA)3 tap42-109) were immunoprecipitated with anti-HA epitope antibodies. Samples of the immunoprecipitates (“HA-IP”) and the supernatant (“Sup”) were subjected to immunoblot analysis using anti-HA and anti-Tap42 antibodies. The first lane, depicted as “C,” shows the untagged control strain Y3033. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)

Figure 2 Microarray Analysis of Time Course Experiments Relative mRNA levels were determined from cDNA microarray analysis of wild-type TAP42 and tap42-109 strains following transfer of cultures from 23°C to 37°C. In a duplicate wt culture, rapamycin (100 ng/ml) was added immediately after transfer (TAP42 + rap). For each culture, RNA was isolated at 0, 15, 30, and 60 min after temperature shift. The RNA was converted to Cy5- or Cy3-labeled cDNA and cohybridized with the correspondingly labeled RNA from time 0 to cDNA microarrays. Induction ratios were converted to log (base 2) values and normalized as described. Scatter plots were derived from single representative experiments with each point the average of duplicate hybridizations. Each axis represents the log (base 2) ratio of mRNA levels at the indicated time point relative to those at time 0. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)

Figure 3 Msn2/Msn4-Responsive Genes Exhibit Sustained Expression in tap42 Mutants following Temperature Shift (A) Relative mRNA levels of 136 genes whose induction by stress is Msn2 dependent were determined from cDNA microarray analysis of wild-type TAP42 and tap42-109 strains following transfer of cultures from 23°C to 37°C and from TAP42 cultures to which rapamycin (100 ng/ml) was added (TAP42 + rap). Values plotted are the average mRNA levels of the collection of genes at the indicated time relative to the levels in the wild-type strain at time 0. (B) mRNA levels of stress response target genes were measured by Northern hybridization of RNA extracted from tap42 mutants following temperature shift and from wild-type TAP42 cells shifted to 37°C in the presence and absence of rapamycin. mRNA levels (in arbitrary units normalized to ACT1 mRNA and TAP42 time 0 value) are shown below each sample. (C) Msn2 localization in wild-type TAP42 and tap42 mutant cells. Msn2 was localized in the indicated strains by immunofluorescence microscopy using anti-Myc antibody against a (Myc)12-tagged Msn2 protein under control of its own promoter. The left panels show Msn2 immunofluorescence, DAPI staining, and phase contrast of cells grown at 23°C. Cells in the right panels were incubated at 37°C for 60 min. (D) Msn2 localization upon rapamycin treatment in cells grown at 23°C and 37°C, respectively. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)

Figure 4 Induction of NDP and RTG Target Genes Requires Tap42 and Phosphatase Catalytic Subunits (A, upper panel) The average relative mRNA levels of strains Y3033 (TAP42, plus [diamonds] and minus [squares] rapamycin) and Y3035 (tap42-109 [triangles]) as a function of time following temperature shift of 27 Gln3-dependent genes (Shamji et al., 2000) were determined from cDNA microarray experiments described in Figure 2. (A, lower panel). Strains Y3033 (TAP42 [squares]), Y3035 (tap42-109 [diamonds]), Y1363 (sit4-102 [upward triangles]), Y3051 (pph21Δ pph3Δ PPH22 [downward triangles]), and Y3053 (pph21Δ pph3Δ pph22-172 [circles]) were grown into mid-log phase 23°C and transferred to 37°C. Rapamycin (100 ng/ml) was added after incubation at 37°C for 1 hr (indicated by arrow). Samples were taken at the time of the temperature shift (0), before addition of rapamycin (45 min), and 15, 30, and 60 min after rapamycin addition (75, 90, and 120 min, relative to the temperature shift). mRNA of post 0 time point samples were compared to mRNA from the 0 time point sample in cDNA microarray experiments. Values are the average change in expression of the 27 Gln3-dependent genes as in the upper panel. (B) The indicated strains were treated as in (A, lower panel) and GLN1 mRNA levels determined by Northern analysis. GLN1 mRNA levels (in arbitrary units normalized to ACT1 mRNA and TAP42 time 0 value) are shown below each sample. Arrows indicate time of addition of rapamycin. (C, upper panel) The average relative mRNA levels of 7 Rtg1/Rtg3-dpendent genes (subset of genes described in Dilova et al., 2002) were determined from cDNA microarray experiments as a function of time following temperature shift, plus or minus rapamycin (TAP42 —□—, TAP42 + rapamycin—♢—, tap42-109 —Δ—) (C, lower panel) Values for the 7 Rtg1/Rtg3-dependent genes were extracted from the dataset described in (A). The strains correspond to the ones in the lower panel of (B). (D) CIT2 mRNA levels are shown for the indicated strains treated as described in (B). Arrows indicate time of rapamycin addition. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)

Figure 5 Ribosomal Gene Expression Is Independent of Tap42 (A) The average relative mRNA levels of cytoplasmic ribosomal protein genes were determined from cDNA microarray experiments as a function of time following temperature shift, plus or minus rapamycin. (B) Expression levels of RPS6A were measured in Northern hybridization experiments of RNA extracted from tap42 mutants following a shift from 23°C to 37°C and from wild-type TAP42 cells subject to the same temperature shift with and without addition of rapamycin. (C) Rapamycin (100 ng/ml) was added to the indicated tap42 mutants and wild-type TAP42 cultures grown at 23°C. Expression levels of RPS6A at the indicated times following rapamycin addition were determined by Northern analysis. (D) RPS6A mRNA levels are shown for strains Y3033 (TAP42), Y3034 (tap42-106), Y3035 (tap42-109), and Y3032 (tap42-11) treated as described in Figure 4B. The triangles indicate time of addition of rapamycin. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)

Figure 6 Model for the Role of Tap42 in Gene Expression Tap42 exists in either a phosphorylated (black circle) or nonphosphorylated form due to the competing activities of the Tor kinase and the PP2A phosphatases, Pph31/22 (Pph), and Sit4. Our results suggest that the phosphorylated form of Tap42 inhibits PP2A phosphatase activity while the nonphosphorylated serves as an essential coactivator of the phosphatases. Stress induces nuclear entry of the transcription factors Msn2/4 to activate stress response (SR) gene transcription and active phosphatase acts to retain the transcription factors in the nucleus. Phosphorylated Tap42 inhibits this phosphatase activity, so that loss of Tap42 or inhibition of Tor by rapamycin causes retention of Msn2/4 in the nucleus. In contrast, PP2A phosphatase in conjunction with nonphosphorylated Tap42 promotes dephosphorylation of the transcription of Gln3 and Rtg1/3, resulting in nuclear import and subsequent activation of nitrogen discrimination pathway (NDP) and retrograde target (RT) genes. Thus, Tap42 inactivation does not activate NDP and RT transcription, but Tap42 is required for rapamycin induction of these gene families. Finally, we propose that ribosomal protein (RP) gene expression occurs independently of Tap42 function. Solid arrows represent interconversion reactions, and dotted lines indicated stimulation or inhibition of the corresponding reaction. Molecular Cell 2003 11, 1467-1478DOI: (10.1016/S1097-2765(03)00228-4)