The yeast elongator histone acetylase requires Sit4-dependent dephosphorylation for toxin-target capacity

Abstract

Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase. Here, we demonstrate that Elongator is a phospho-complex. Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action. Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190. Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation. Excess Sit4.Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4. Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin. Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Delta-nuclei retain Tot4. Together with the findings that sit4Delta and totDelta cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics.

Figure 1.

Sit4 can be found in the nucleus and sit4Δ and Tot^- mutants resist ceramide. (A) The Sit4 C terminus confers zymocin sensitivity. Killer eclipse assays used K. lactis killer (AWJ137) and nonkiller (NK40) strains (Table 1) and S. cerevisiae strains expressing wild-type Sit4 (FY1679-08A: SIT4) or Sit4 tagged with the HA epitope at either the C terminus (DJYS4H: SIT4-(HA)₃), or at the N terminus (DJYHS4: (HA)₃-SIT4) under GAL promoter control. Eclipse formation around the killer strain indicates zymocin sensitivity (zym^S), whereas lack of inhibition indicates a resistant (zym^R) response. The Elongator mutant LFY5 (tot3Δ) served as zym^R control. Growth was on glucose (YPD) or galactose (YPG) medium. (B) Sit4 is nucleocytoplasmic. Total extracts (TE) from lysed CY3938 (sit4Δ) spheroplasts carrying pCB243 (UAS_SIT4::SIT4-HA) (Sutton et al., 1991) and DJYHS4 (UAS_GAL1::(HA)₃-SIT4) spheroplasts grown in YPD (left) or YPG (right) were immunoprobed together with separated cytoplasmic (Cy) and nuclear (Nu) fractions by using Sit4- and HA-specific antibodies to detect the PP2A-type Sit4. Marker distribution used anti-Nop1 (nucleus), anti-RFA (cytoplasm and nucleus), and anti-Cdc19 (predominantly cytoplasm) antibodies. To test effects of promoter-dependent SIT4 expression on zymocin sensitivity, both promoter scenarios (UAS_SIT4 vs. UAS_GAL1) were assayed by killer eclipse assays (top; also see A). (C) Ceramide assay. Tenfold dilutions of S. cerevisiae strains (W303-1A: wild-type, CY3938: sit4Δ, LFY3: tot1Δ, LFY4: tot2Δ, LFY5: tot3Δ and LFY6: tot4Δ) were spotted on YPD plates without (control) and with ceramide. Sensitivity and resistance to ceramide are indicated (cer^S and cer^R, respectively).

Figure 2.

Elongator expression and complex assembly in correlation to SIT4 and SAP155. (A) Elongator gene transcription (TOT1-4) is unaffected by sit4Δ. RT-PCR was used to compare expression of TOT1-4 in CY4029 (SIT4) and CY3938 (sit4Δ) strains. Histone H3 (HHT1) served as control. Numbers refer to kilobases (Gene Ruler; MBI Fermentas). (B) Elongator expression is unaffected by multicopy SAP155. Identical amounts of protein extracts from c-Myc-tagged Elongator strains DJY1t-a (TOT1-(c-myc)₃), FFY3t (TOT3-(cmyc)₃) and FFY4t-a (TOT4-(c-myc)₃) carrying multi-(mc) or single copy (sc) SAP155 were immunoprobed by 9E10 (anti-c-Myc). Loading was followed using anti-Pfk1 antibody (recognizing phosphofructokinase α and β subunits as indicated). Positions of c-Myc-tagged Tot proteins are marked by arrows. Tot1 separates (at least) in two forms, with the faster (^*) being N-terminally truncated (Fichtner et al., 2003). (C). Loss of SIT4 does not affect Elongator assembly. Equal amounts of protein extracts obtained from wild-type S1T4 DJY108, DJY110, and DJY112 and sit4Δ DJY107, DJY109, and DJY111 strains expressing the indicated tagged Tot proteins were subjected to 9E10 (anti-c-Myc) immunoprecipitations. Detection of Tot3 and Tot5 used 9E10 (anti-c-Myc), monitoring HA-tagged Tot5 or Tot4 used 3F10 (anti-HA). Numbering (B and C) refers to kilodaltons of molecular markers (Invitrogen, Carlsbad, CA).

Figure 3.

Elongator is a phospho-complex. (A) 9E10 (anti-c-Myc) immunoprecipitates of strains FFY2t-a (TOT2-(c-myc)₃), FFY2/1dt (TOT2-(c-myc)₃ TOT1-(HA)₆), and DJY2t1d-a (TOT2-(c-myc)₃ tot1Δ) were subjected to 6% SDS-PAGE analysis and probed with Q5 and Q7 antibodies, respectively. Phosphoforms () of Tot1 and Tot1-HA are shown by arrows. (B) Phosphorylation of Tot1 requires full-length protein. 9E10 (anti-c-Myc) immunoprecipitates from strains FFY3/1dt (TOT3-(c-myc)₃ TOT1-(HA)₆) and FFY3/2dt (TOT3-(c-myc)₃ TOT2-(HA)₆) were probed with 3F10 (anti-HA) and Q5 to detect Tot1-HA, Tot2-HA, and phosphoforms () of Tot1 and Tot1-HA. The truncated (∼20 kDa) Tot1-HA form is indicated (^*) (also see Figure 2B). (C) Phosphorylation of Tot1 is supported by Elongator partner protein Kti11. 9E10 (anti-c-Myc) immunoprecipitates from the strains FFY3/2dt (KTI11) and DJY3/2-d11 (kti11Δ) coexpressing epitope-tagged Tot2 and Tot3 were probed with anti-Elp1/Tot1, Q5 and Q7 antibodies. Truncated Tot1 is indicated (^*). Numbers (A and B) refer to protein marker sizes in kilodaltons (Invitrogen).

Figure 4.

Sit4·Sap185/190 is specific for Tot1 dephosphorylation. (A) sit4Δ cells suppress Tot1 dephosphorylation. 9E10 (anti-c-Myc) Elongator immunoprecipitates from strains DJY110 (SIT4 TOT5-(cmyc)₃ TOT4-(HA)₆) and DJY109 (sit4Δ TOT5-(c-myc)₃ TOT4-(HA)₆) were probed with anti-Elp1/Tot1 and Q5 antibodies. The positions of hypo-(Tot1) and hyperphosphorylated Tot1 () forms are shown. sit4Δ cells accumulate the up-shifted Tot1- form. (B) sit4Δ or multicopy SAP155 cells suppress Tot1 dephosphorylation. Protein extracts from strains DJY103 (sit4Δ TOT1-(HA)₆), DJY102 (SIT4 TOT1-(HA)₆), and DJY102 carrying the indicated multicopy SAP constellations were standardized by anti-Pfk1 (see Figure 2B) and probed with 3F10 (anti-HA). (C) Suppressed Tot1 dephosphorylation by multicopy SAP155 is antagonized by excess Sap185/190. Protein extracts from indicated TOT-(HA)₆ expressors were probed with 3F10 (anti-HA) to monitor Tot1-HA migration dependent on SAP copy number. (D) sap185Δsap190Δ cells phenocopy high phospho-Tot1 levels of sit4Δ cells. Strains DJY102 (SIT4), DJY103 (sit4Δ), DJY115 (sap4Δsap155Δ), and DJY116 (sap185Δsap190Δ) expressing TOT1-(HA)₆ were analyzed as described above (B and C). Non- and phosphorylated forms () of Tot1-HA (B-D) are shown by arrows; vector control (B and C) denotes empty YEp24 vector used to clone the SAP genes in multicopy (Luke et al., 1996).

Figure 6.

Balanced de-/phospho-Tot1 ratios involve opposing effects of Tot4 and Sit4. (A) tot4Δ cells enhance Tot1 dephosphorylation. Standardized protein extracts from indicated TOT1-(HA)₆ expressing strains DJY102 (SIT4 TOT4), DJY103 (sit4Δ), DJY104 (tot4Δ), and DJY105 (sit4Δtot4Δ) were probed with 3F10 (anti-HA). The positions of non- and phosphorylated () Tot1-HA forms are shown. (B) Excess Tot4 shifts electrophoretic mobility of Tot1, a situation antagonized by excess Sit4·Sap190. Standardized protein extracts from strain DJY102 (SIT4 TOT4) expressing TOT1-(HA)₆ and maintaining multicopy (mc) TOT4, SIT4, and SAP190 genes as indicated were probed with 3F10 (anti-HA). As a control served DJY104 (tot4Δ) (see A). (C) mcTOT4 intensifies Tot1 phosphorylation. 9E10 (anti-c-Myc) immunoprecipitates from strain FFY2/1dt (TOT1-(HA)₆ TOT2-(c-myc)₃) maintaining single copy or mcTOT4 were probed with 3F10 (anti-HA), 9E10 (antic-Myc), Q5 (anti-phosphoserine) and Q7 (anti-phosphothreonine) antibodies to detect Tot1-HA, Tot2-c-Myc and phospho-Tot1-HA (). Before immunoprecipitation, extracts were standardized by anti-Pfk1 to follow content of Pfk1 α and β subunits (Figure 2B). Tot1-HA truncation is indicated (^*). For zymocin read-outs (A and C), see Figure 1A.

Figure 5.

Zymocicity strictly depends on Sit4·Sap185/190 and high copy TOT4 zymocin resistance is suppressed by Sit4·Sap190. (A) K. lactis killer eclipse assays (see Figure 1A) involved S. cerevisiae strains LFY5 (tot3Δ), AY925 (wild-type (wt)), and phosphatase mutants CY3938 (sit4Δ), CY5224 (sap185Δsap190Δ), DEY132-1C (pph21Δ), DEY10-2B (pph22Δ), DEY132-2C (pph21Δpph22Δ), EDN75 (ppz1Δ), JA103 (ppz2Δ), EDN76 (ppz1Δppz2Δ), MMY09 (cna1Δcna2Δ), and YJN519 (cnb1Δ). (B) Eclipse assays involving resistant control DYJ100 (tot4Δ) and wild-type TOT4 strain CY4029 carrying multicopy (mc) TOT4, SIT4, and/or SAP genes. (C) Eclipse assays involving mcSIT4/SAP genes maintained in strain DYJ100 (tot4Δ) and compared with wild-type TOT4 CY4029 cells. (D) Eclipse assays of zymocin-resistant strain DYJ100 strain (tot4Δ) and wild-type CY4029 (TOT4) cells carrying the indicated mcSIT4/SAP genes. As a high copy control served mcTOT4. For phenotypic zymocin readouts, see Figure 1A. Vector controls (B-D) refer to empty YEplac181, YEp24, and YEplac112 plasmids used to clone the SIT4, SAP, and TOT4 genes, respectively (Butler et al., 1994; Luke et al., 1996; Jablonowski et al., 2001c).

Figure 7.

Sit4, Tot4, and Tot1 cofractionate and Tot4 is retained in sit4Δ-nuclei. (A) Cell fractionation. Tot4-HA, HA-Sit4, and Tot1-HA were detected in sucrose-gradient fractions (lanes 1-9 from a total of 14) from strains DJY114 (TOT4-(HA)₆), DJYHS4 ((HA)₃-SIT4), and DJY8A-1H3 (TOT1-(HA)₃) by using 3F10 (anti-HA). Their positions are indicated by arrows. The truncated Tot1-HA form (see Figure 3B) is indicated (^*). (B) Tot4 is retained in sit4Δ-nuclei. Fractions of DJY114 (SIT4) or DJY113 (sit4Δ) cells expressing TOT4-(HA)₆ were probed using 3F10 (anti-HA) and compared with nuclear and cytoplasmic marker proteins Nop1 and Cdc19, respectively (see Figure 1C). Their positions are indicated by arrows.

Figure 8.

Model for the role of Sit4 in the cell cycle arrest imposed by K. lactis zymocin's γ-toxin subunit. In the absence of γ-toxin (A), the SSD1-v pathway () and Sit4-dependent dephosphorylation of the toxin-target (TOT) substrate () activate a key cell cycle regulator (□) required for processes preSTART. In the presence of γ-toxin (B), the critical protein can be sequestered and inactivated in combination with Sit4's dephosphorylated TOT substrate (•) to induce the G1 arrest (Tot⁺), irrespective of whether the alternative, Sit4-dispensable SSD1-v pathway would be sufficient. Failure to dephosphorylate TOT (C) no longer permits inactivation of the crucial regulator and causes sit4Δ cells to resist γ-toxicity (Tot^-) at the expense of being G1 cell cycle delayed. The as yet elusive Elongator-specific kinase is marked by?.