Regulation of Tec1 by rapamycin. (A) Tec1 protein levels in yeast strains RH2500 (tec1∆) and RH2754 (TEC1) 70 min after addition of rapamycin (+) or drug vehicle (−) to exponentially growing cultures. Tec1 and Cdc28 proteins were detected by immunoblot analysis using specific anti-Tec1 and anti-Cdc28 antibodies. Relative Tec1 levels (Tec1 norm) are indicated and were obtained by normalizing Tec1 to Cdc28 signals. (B) TEC1 transcript levels in yeast strain RH2754 70 min after addition of rapamycin (+) or drug vehicle (−). TEC1 and ACT1 transcripts were determined by Northern blot analysis and relative TEC1 transcript levels were obtained by using ACT1 as an internal standard. (C) TEC1-promoter activity. TEC1-lacZ expression was measured in yeast strain RH2754 carrying the pME2065 plasmid 0, 30, and 60 min after addition of rapamycin. Bars depict specific β-galactosidase activities and are means of three independent measurements. (D) Stability of Tec1 in the absence and presence of rapamycin. TEC1 expression was induced in yeast strain RH2500 carrying GAL1(P)-TEC1 (BHUM29) by growth in galactose containing medium for 6 hr. Glucose was added to repress TEC1 transcription. At time point zero rapamycin (+) or drug vehicle (−) were added and Tec1 protein levels were measured by immunoblot analysis at the indicated time points. Protein levels of Cdc28 served as an internal standard in all extracts. (E) Quantification of Tec1 protein assayed in D. Relative Tec1 levels in the absence (●) or presence (▪) of rapamycin are shown in arbitrary units and were obtained by normalizing Tec1 to Cdc28 signals. The Tec1/Cdc28 ratio at time point 0 was set to 100%. Half-life times (t_1/2) are indicated.

Rapamycin-induced Tec1 degradation depends on Sit4, Tip41, and Npr1. Tec1 protein levels were determined in the yeast strains of the indicated genotype as described in . (A) CY4029 (control), CY3938 (sit4∆), Y07045 (sap190∆), Y04596 (sap4∆), Y05459 (tip41∆), YP0607WH (pph21∆ pph22∆), and Y04010 (pph3∆). (B) YHUM482 (control), Y07006 (yak1∆), YHUM1089 (npr1∆), KT1960 (control), IP31 (rim15∆), Y2864 (control), and CDV316 (sch9∆). (C) YHUM119 (control), YHUM120 (ras2∆), YHUM407 (tpk2∆ tpk3∆), YHUM397 (tpk1∆ tpk3∆), and YHUM389 (tpk1∆ tpk2∆). Numbers indicate relative Tec1 levels (Tec1 norm) and were obtained by normalizing Tec1 to Cdc28 signals.

Rapamycin-induced down-regulation of Tec1 is independent of the Fus3/Kss1 MAPK cascade. (A) Tec1 protein levels were determined in yeast strains RH2754 (control), YM105 (kss1∆), YM106 (fus3∆), YM107 (kss1∆ fus3∆), and YHUM622 (ste7∆) as described in . Numbers indicate relative Tec1 levels (Tec1 norm) and were obtained by normalizing Tec1 to Cdc28 signals. (B) Influence of rapamycin on Fus3 and Kss1. Yeast strains RH2754 (FUS3) and YM106 (fus3∆) were grown in the absence (−) or presence (+) of either rapamycin or pheromone or both and protein extracts were prepared. Phosphorylated forms of Fus3 and Kss1 were detected by immunoblot analysis using phospho-specific antibodies. Cdc28 served as a loading control. (C) Concomitant effect of rapamycin and pheromone on Tec1. Tec1 protein levels were determined in yeast strain RH2754 grown for 70 min in the absence (−) or presence (+) of either rapamycin or pheromone or both. Relative Tec1 protein levels (Tec1 norm) are indicated.

Rapamycin-triggered Tec1 degradation does not appear to involve polyubiquitylation or proteasomal degradation. (A) Ubiquitylation of Tec1 in response to rapamycin. Cultures of strain YHUM1803 carrying plasmids pME2280 (3myc-TEC1) or BHUM1177 (TEC1) as well as YEplac181 (empty vector) or pHU821 (His-UBI), respectively, were grown in the presence of 0.1 mm CuSO₄ and treated with MG132 and/or rapamycin as indicated. Extracts were prepared under denaturing conditions, and total ubiquitin conjugates were isolated by Ni-NTA affinity chromatography. Ubiquitylated forms of Tec1 were detected in the isolated material by anti-myc Western blotting together with a whole cell extract (WCE) as a control (top). Total levels of Tec1 (anti-myc) and total ubiquitin conjugates (anti-Ubi) in the cell extracts were detected in parallel (bottom). Detection of Pgk1 (anti-Pgk1) served as a loading control. (B) Requirement of proteasome function. Tec1 protein levels were determined in temperature-sensitive yeast strains WCG4a (control), WCG4/11/22 (pre1-1 pre2-2), and YHI29/14 (pre1-1 pre4-1) carrying plasmid BHUM277 were grown for 70 min at 25° or 37° and in the absence (−) or presence (+) of rapamycin. Tec1 protein was then analyzed by immunoblot analysis.

Interaction between Tec1 and Rsp5. (A) The linear diagram of Tec1 indicates the TEA DNA-binding domain (TEAD), Cdc4-phosphodegron motif (CPD) and the C-terminal portion (shaded) conferring Ste12 binding, transactivation, and Tec1 destabilization (; ; ; ; ). The location of the conserved PxY motifs for Rsp5 interaction is indicated, and the three motifs are shown in boldface type in the protein sequence below. Underlined proline residues were exchanged for alanine in the AxY variant of Tec1. Numbers indicate amino acid residues of Tec1. (B) Two-hybrid interaction between Tec1 and Rsp5. Numbers indicate specific β-galactosidase activities obtained in strain EGY48-p1840 expressing lexA (pEG202) or lexA-TEC1^281-486 (BHUM752) in combination with B42 (pJG4-5) or B42-RSP5^372-809 (BHUM750). Values are the means of three independent measurements. (C) Copurification of different Tec1 variants with GST–Rsp5. Protein extracts were prepared from yeast strain YHUM793 expressing GST (BHUM1521) or GST–Rsp5 (BHUM1305) together with TEC1 (pME2068), TEC1^AxY (BHUM1306), or TEC1^∆C (pME2083). GST or GST–Rsp5 and associated proteins were purified using glutathione-sepharose. Presence of Tec1 protein in cell extracts (input) and in GST-purified complexes (beads) was analyzed by immunoblotting using anti-Tec1 antibodies. GST and GST–Rsp5 were detected by antibodies against GST. (D) Influence of rapamycin on Tec1–Rsp5 interaction. Copurification of Tec1 with GST–Rsp5 was determined in yeast strain RH2754 carrying plasmids BHUM1305 (GST-RSP5) and BHUM29 (TEC1) as described in C before (−) and 20 min after (+) addition of rapamycin.

Control of Tec1 by RSP5. (A) Tec1 protein levels were determined in yeast strains YHUM1104 (control), YHUM1341 (rsp5∆), YHUM1093 (bul1∆ bul2∆), Y05701 (rog3∆), and Y02394 (rup1∆) grown in the presence of rapamycin (+) or drug vehicle (−) as described in . (B) Rapamycin-induced destruction of different Tec1 variants. Tec1 protein levels were determined in RH2500 carrying TEC1 (pME2068), TEC1^∆355-486 (BHUM576), TEC1^∆258-486 (pME2083), or TEC1^AxY (BHUM1306) as described above. (C) Functionality of different TEC1 variants during adhesive growth. Yeast strain RH2500 carrying one of the plasmids YCplac33 (tec1∆), pME2068 (TEC1), BHUM570 (GFP-TEC1), or BHUM1306 (TEC1^AxY) was patched on SC −Ura medium. After 4 days the plate was photographed before (total growth) and after (adhesive growth), washing nonadhesive cells off the surface. (D) Rapamycin-induced destruction of GFP–Tec1 protein levels was determined in yeast strain RH2500 carrying GFP-TEC1 (BHUM570). (E) Localization of GFP–Tec1. Representative cells of yeast strains YHUM1104 (RSP5) and YHUM1341 (rsp5∆) carrying BHUM570 are shown. Living cells were visualized under a microscope by using fluorescence microscopy or Nomarski optics 0, 50, and 75 min after addition of rapamycin to exponentially growing cultures. Bar, 5 µm.

Degradation of Tec1 and Tec1 variants in response to rapamycin or pheromone. (A) Rapamycin-induced degradation of different Tec1 variants was analyzed in yeast strain RH2500 carrying TEC1 (pME2068), TEC1^T273M (pME2102), TEC1^P274S (pME2096), TEC1^K54A (BHUM1122), or TEC1^AxY (BHUM1306) on plasmids. (B) Pheromone-induced degradation of different Tec1 variants was analyzed in yeast strain RH2500 carrying TEC1, TEC1^AxY, or TEC1^T273M grown for 70 min in the absence (−) or presence (+) of yeast alpha factor. Numbers indicate relative Tec1 levels (Tec1 norm) and were obtained by normalizing Tec1 to Cdc28 signals. (C) Pheromone-induced degradation of Tec1 in yeast strains YHUM1104 (control) and YHUM1341 (rsp5∆).

Regulation of Tec1-target genes by rapamycin. (A) FLO11 and CLN1 transcript levels were determined in yeast strains RH2754 (TEC1) and RH2500 (tec1∆) 70 min after addition of rapamycin (+) or drug vehicle (−) to exponentially growing cultures by Northern blot analysis. FLO11 norm and CLN1 norm indicate relative transcript levels obtained by normalization to ACT1 levels. (B) Effect of rapamycin on FLO11 and CLN1 transcript levels was analyzed in yeast strain RH2500 carrying TEC1 (pME2068), TEC1^T273M (pME2102), or TEC1^AxY (BHUM1306) on plasmid.

Requirement of TEC1 for rapamycin-induced extension of yeast chronological lifespan. Survival (CFU ml⁻¹) was assessed in the absence (open bars) or presence (solid bars) of rapamycin after 8, 17, and 20 days in yeast strains expressing TEC1, no TEC1 (tec1∆), or TEC1^AxY. Bars depict relative values expressed in percentage of the TEC1-expressing strain.

Model for the control of different yeast developmental programs by the Fus3/Kss1 MAPK cascade, the TORC1 pathway, and by Tec1. For details see text.