In yeast, the role for the Elongator complex in tRNA anticodon modification is affected by phosphorylation of Elongator subunit Elp1. Thus, hyperphosphorylation of Elp1 due to inactivation of protein phosphatase Sit4 correlates with Elongator-minus phenotypes including resistance towards zymocin, a tRNase cleaving anticodons of Elongator-dependent tRNAs. Here we show that zymocin resistance of casein kinase hrr25 mutants associates with hypophosphorylation of Elp1 and that nonsense suppression by the Elongator-dependent SUP4 tRNA is abolished in hrr25 or sit4 mutants. Thus changes that perturb the evenly balanced ratio between hyper- and hypophosphorylated Elp1 forms present in wild-type cells lead to Elongator inactivation. Antagonistic roles for Hrr25 and Sit4 in Elongator function are further supported by our data that Sit4 inactivation is capable of restoring both zymocin sensitivity and normal ratios between the two Elp1 forms in hrr25 mutants. Hrr25 binds to Elongator in a fashion dependent on Elongator partner Kti12. Like sit4 mutants, overexpression of Kti12 triggers Elp1 hyperphosphorylation. Intriguingly, this effect of Kti12 is blocked by hrr25 mutations, which also show enhanced binding of Kti12 to Elongator. Collectively, our data suggest that rather than directly targeting Elp1, the Hrr25 kinase indirectly affects Elp1 phosphorylation states through control of Sit4-dependent dephosphorylation of Elp1.
Here we show that Hrr25 interacts with Elongator in a fashion dependent on Kti12 suggesting that the contact between Hrr25 and Elongator is bridged by Kti12. Moreover, hrr25 mutants cause Elp1 hypophosphorylation, a defect opposite to the hyperphosphorylation of Elp1 seen in sit4 phosphatase mutants. Given the biochemical interaction between Hrr25 and Sit4 and genetic interrelations which show that loss of SIT4 suppresses hrr25 phenotypes and Elp1 phosphorylation defects, the Hrr25 kinase is likely to oppose the Sit4 phosphatase. Apparently, this antagonism is important for maintaining well-balanced levels of hyper- and hypophosphorylated Elp1 forms that are critical for Elongator function in tRNA modification and tRNA-related processes. Based on further data that Hrr25 impacts on the interaction between Elongator and Kti12 and that Hrr25 promotes the ability of Kti12 to modulate Elp1 phosphorylation states, the Hrr25 kinase may indirectly affect Elp1 phosphorylation by controlling Sit4-dependent dephosphorylation of Elp1.
Characterization of hrr25 mutants
Consistent with the notion that the HRR25 (KTI14) gene confers zymocin sensitivity, hrr25-3 (kti14-1) or hrr25-4 (kti14-2) mutations that result in N-terminal amino acid substitution (Hrr25-3) or C-terminal truncation (Hrr25-4) (Fig. 1A) cause zymocin resistance (Butler et al., 1994; Mehlgarten and Schaffrath, 2003). His6-tagged versions of wild-type Hrr25 kinase and the mutated Hrr25-3 and Hrr25-4 proteins were purified from E. coli to quantify kinase activities in assays recording CKI-dependent ATP consumption or monitoring casein phosphorylation directly using [γ-32P]-ATP (Fig. 1B). In either assay, Hrr25-3 had no detectable kinase activity and Hrr25-4 showed about threefold reduced activity compared with Hrr25 (Fig. 1B), so Hrr25-3 is kinase-dead in vitro and Hrr25-4 has a significant kinase deficit. Together with previous data showing that the hrr25-3 and hrr25-4 mutants survive growth inhibition by zymocin (Mehlgarten and Schaffrath, 2003), a response typical of Elongator mutants (Frohloff et al., 2001), we conclude that phosphorylation by the Hrr25 kinase is likely to be linked to Elongator function.
Hrr25•Elongator interaction requires holo-Elongator and Kti12
Although tandem affinity purification (TAP) has shown Elongator to copurify with Hrr25-TAP (Gavin et al., 2006; Schäfer et al., 2006), the C-terminal TAP-tag affects Hrr25 function and copies hrr25-like resistance to zymocin (Fig. S1). Given that N-terminal tags support zymocin sensitivity (Fig. S1), we resorted to TAP- or HA-HRR25 strains for further analyses. Similar to the pattern of an HA-Hrr25Deg variant (Kafadar et al., 2003), Western blots revealed that HA-HRR25 and HA-hrr25-3 cells expressed two HA-responsive bands (Fig. 2A, lane 2 and 3) implying that full-length Hrr25 is subject to proteolysis or some other modification. As predicted, HA-hrr25-4 cells produced a unique band corresponding to the Hrr25-4 truncation (Fig. 2A, lane 4). This suggests that the P/Q-rich motif may affect the modification of the Hrr25 kinase. Intriguingly, like wild-type Hrr25 (Fig. 2A, lane 2), Hrr25-3 and Hrr25-4 co-precipitated Elp1 (Fig. 2A, lanes 3 and 4) and Elp3 (not shown) meaning that Elongator association does not depend on the kinase activity of Hrr25 or its P/Q-rich motif.
Studying the Hrr25•Elongator interaction further, we found that while clearly present in the wild-type control (Fig. 2B, lane 2), precipitation of Elp1 and Elp3 (not shown) by Hrr25 was absent in Elongator (elp1Δ, elp3Δ, elp5Δ) and kti12Δ mutants (Fig. 2B, lanes 3–6) and greatly reduced in multicopy KTI12 cells (Fig. 2B, lane 7). Hence, Hrr25•Elongator interaction requires not only an intact Elongator complex but also Kti12. Our finding that Hrr25•Elongator interaction appears to be sensitive to KTI12 dosage is intriguing because Kti12 itself partners with the Elongator complex and excess levels of Kti12 suppress zymocin sensitivity (Butler et al., 1994; Frohloff et al., 2001; Fichtner et al., 2002).
Hrr25 interacts with Elongator partner Kti12
To study potential Hrr25•Kti12 interaction in vivo, we used immune precipitation combined with the TAP technique (Rigaut et al., 1999). Following isolation of TAP-tagged Hrr25 from cells coexpressing KTI12-c-myc, Western blots with anti-c-Myc antibodies revealed copurification of a protein corresponding to Kti12-c-Myc that is absent in purifications from cells lacking the TAP bait (Fig. 3A). In addition to Hrr25•Elongator interaction (Fig. 2A), this suggests that Hrr25 also interacts with Kti12. To test whether this interaction depends on Elongator, we performed immune precipitations on wild-type and Elongator-minus cells expressing KTI12-c-myc and HA-HRR25. The corresponding Western blots confirmed Hrr25•Kti12 interaction in wild-type cells (Fig. 3B, lane 3) and showed that the interaction was absent in elp1Δ and elp5Δ mutants (Fig. 3B, lane 4 and 5). In sum, Hrr25 and Kti12 copurify with one another in a fashion that requires the Elongator complex indicating mutual dependencies of these physical interactions.
hrr25 mutants accumulate hypophosphorylated forms of Elp1
As Elp1 dephosphorylation requires the Sit4 phosphatase we examined if Elp1 phosphorylation was influenced by the Hrr25 kinase using mobility shifts in Western blots as described by Jablonowski et al. (2004). Figure 4A shows anti-HA Western blots of Elp1-HA from wild-type, hrr25-4 and sit4Δ cells before and after λ-phosphatase treatment. The wild-type displayed a balanced ratio between two forms of Elp1 (Fig. 4A, lane 1) as described previously (Jablonowski et al., 2004). Treatment with λ-phosphatase caused an electrophoretic downshift of the slower migrating Elp1 form to the position of the faster migrating one, while the position of the latter was unchanged (Fig. 4A, lane 2). So clearly phosphorylation is responsible for the distinct electrophoretic behaviour of the two forms and we will refer to the slower and faster migrating species as hyper- and hypophosphorylated Elp1 forms respectively. sit4Δ mutants accumulated mainly the hyperphosphorylated Elp1 form (Fig. 4A, lane 5) treatment of which by λ-phosphatase caused a shift to the hypophosphorylated Elp1 form (Fig. 4A, lane 6 and 7).
In contrast, hrr25-4 cells accumulated hypophosphorylated Elp1 forms that were insensitive to λ-phosphatase (Fig. 4A, lanes 3 and 4). Elp1 hypophosphorylation was also observed in the kinase-dead mutant hrr25-3 (Fig. 4B) stressing that the Hrr25 kinase activity is required to modulate Elp1 phosphorylation states. In line with a link between these hrr25 defects and zymocin resistance, transformation of hrr25-3 and hrr25-4 cells with a wild-type copy of the HRR25 gene restored Elp1 phosphorylation and zymocin sensitivity (Fig. 4B). In further support for a causal relationship between the kinase activity of Hrr25 and Elongator function, destruction of Hrr25 in a GAL-HRR25Deg strain (Kafadar et al., 2003) was found to rapidly trigger Elp1 hypophosphorylation and zymocin resistance (not shown). In sum, hrr25 defects cause Elp1 hypophosphorylation which is opposite to Elp1 hyperphosphorylation in the sit4Δ mutant but like the latter is also associated with zymocin resistance.
Elongator roles in tRNA modification depend on normal Elp1 phosphoregulation
Our findings that opposite Elp1 phosphodefects associate with zymocin resistance imply inactivation of Elongator's function in tRNA modification and suggest that Elp1 phosphorylation does not simply act as an Elongator on/off switch. To extend these findings, we analysed the requirement for Hrr25 and Sit4 to promote nonsense suppression by the Elongator-dependent tRNA suppressor SUP4 (Shaw and Olson, 1984; Huang et al., 2005; Jablonowski et al., 2006). hrr25-4, sit4Δ and elp1Δ mutations all abolished SUP4 read-through of the ade2-101 ochre stop codon, yielding adenine auxotrophy and colony pigmentation typical of non-suppressed ade2-101 cells (Fig. S2). Similarly, suppression of the can1-100 ochre mutation by SUP4, which allows the arginine analogue canavanine to kill yeast, was abolished in hrr25-4, sit4Δ and elp1Δ mutants causing canavanine resistance (Fig. 5). Intriguingly, this trait was also seen in a sap185Δsap190Δ mutant (Fig. 5) lacking the Sit4 subunits Sap185 and Sap190, which are jointly required for Sit4-dependent Elp1 dephosphorylation (Luke et al., 1996; Jablonowski et al., 2004). Conversely, removal of Sap4 and Sap155 from sap4Δsap155Δ cells, two Sit4 subunits dispensable for Elp1 dephosphorylation (Jablonowski et al., 2004), failed to affect the tRNA suppressor in either assay (Fig. 5 and not shown). This agrees with recent findings by Huang et al. (2008) that hrr25, sit4Δ and sap185sapΔ190Δ mutants copy tRNA modification defects typical of Elongator mutants. So, influences on Elp1 phosphomodification by the Hrr25 kinase and the Sit4 phosphatase affect the performance of an Elongator-dependent tRNA suppressor and an Elongator-dependent tRNase toxin. Thus our data indicate that Elongator's role in tRNA modification requires both the hypo- and hyperphosphorylated Elp1 forms and that the casein kinase Hrr25 and the phosphatase Sit4 control the relative abundance of the two forms.
Interactions between HRR25 and SIT4
In line with a previous high-throughput study (Ho et al., 2002), we confirmed that Hrr25 and Sit4 physically interact with each other (not shown).
To further analyse interrelations between Hrr25 and Sit4, we wished to combine sit4Δ null-alleles with hrr25 mutations. However, the hrr25 parent strains were found to have ssd1-d status (Fig. S3), so the hrr25 mutants were not expected to survive in the absence of Sit4, because ssd1-d alleles of SSD1 (suppressor of SIT4 deletion) do not tolerate SIT4 deletions while SSD1-v alleles do (Sutton et al., 1991). We therefore constructed a conditional GAL-HA-SIT4 allele to allow the effect of Sit4 depletion on glucose medium to be studied in hrr25 mutants. Surprisingly, compared with the starting hrr25-3 mutant, viability of GAL-HA-SIT4 hrr25-3 cells was hardly affected on glucose (Fig. 6A). Thus the synthetic lethality predicted from combining an ssd1-d allele with Sit4 depletion (Sutton et al., 1991) appears to be suppressed by the hrr25 mutation. This is particularly notable because repression of the SIT4 gene on glucose (Fig. 6B) restored zymocin sensitivity in the hrr25-3 background (Fig. 6A).
Prompted by this lack of synthetic lethality, we generated a chromosomal SIT4 disruption in the hrr25-3 mutant expressing ELP1-HA. Again Sit4 removal sustained viability and suppressed zymocin resistance in the resulting hrr25-3 sit4Δ double mutant (Fig. 6C). Intriguingly, the hrr25-3 sit4Δ double mutant lacking SIT4 function performed significantly weaker (Fig. 6C) in comparison with the hrr25-3 GAL-HA-SIT4 strain when grown on glucose, i.e. under SIT4-repressing conditions (Fig. 6A). Whether leaky SIT4 expression in the latter cells explains this growth difference is not entirely clear, because Sit4 levels were undetectable by Western blot analysis under glucose repression (Fig. 6B). Nonetheless, comparison of Elp1 phosphorylation states by mobility shifts revealed that in contrast to the Elp1 phosphodefect of the original hrr25-3 mutant, the hrr25-3 sit4Δ double mutant displayed a wild-type-like Elp1 phosphorylation pattern with roughly equivalent levels of the two Elp1 forms (Fig. 6D). This shows that Elp1 hypophosphorylation, a defect typical of hrr25 mutants, can be neutralized by concomitant Sit4 inactivation. Consistent with this, multicopy SAP155, which reduces Sit4•Sap185 and Sit4•Sap190 phosphatase activities (Jablonowski et al., 2004), also restored Elongator function in the hrr25-3 mutants as indicated by suppressed zymocin resistance (Fig. 7A) and this multicopy SAP155 effect was broadly comparable to the effect of Sit4 depletion in hrr25-3 cells (Fig. 7B). In sum, loss of Sit4 function is tolerated in hrr25-3 cells independently of SSD1-v and restores balanced Elp1 phosphorylation states.
Hrr25 influences Kti12 and Elongator properties
As for the role of Elp1 phosphorylation, individual Elongator subunit interactions were not affected in hrr25-4 cells (Fig. S4). Likewise Elongator integrity is not perturbed in kti12Δ cells and in both cases Elp1 is hypomodified (Jablonowski et al., 2004). This suggests that Elp1 phosphorylation impacts on Elongator function rather than its structure or assembly. Next we tested whether hrr25 defects may interfere with the interaction between Elongator and its partner Kti12. Immune precipitation showed that compared with an HRR25 control the fraction of HA-tagged Kti12 that associated with c-Myc-tagged Elp1 or Elp5 was drastically enhanced in the hrr25-4 mutant (Fig. 8A). Based on unaltered Kti12 levels in extracts prior to precipitation (Fig. 8A), the enhanced binding of Kti12 to Elongator appears to be specifically linked to the hrr25 defect.
Whether increased levels of Elongator-bound Kti12 influence Elongator function is not known. However, multicopy KTI12 suppresses zymocin sensitivity and increases the proportion of hyperphosphorylated Elp1 forms (Frohloff et al., 2001; Jablonowski et al., 2004). Intriguingly, immune precipitation studies revealed that despite Kti12 overexpression, the amount of Elongator-bound Kti12 is unaffected in multicopy KTI12 cells (Fig. S5). Thus hrr25 mutations lead to an enhanced interaction of Kti12 with Elongator and hypophosphorylated Elp1, while increased KTI12 dosage promotes Elp1 hyperphosphorylation without changing the levels of Elongator-bound Kti12. In sum, the effects of the hrr25-4 allele and multicopy KTI12 appear quite distinct despite the intimate involvement of the two proteins in Elongator phosphorylation and function. Further studies into Elp1 phosphomodulation via Hrr25 and Kti12 revealed that Elp1 hyperphosphorylation due to multicopy KTI12 was eliminated in hrr25-3 cells (Fig. 8B). This suggests that the kinase activity of Hrr25 mediates the multicopy KTI12 effect on upregulated Elp1 phosphorylation and explains why the hrr25-3 kinase-dead mutant is insensitive to increased KTI12 dosage (Fig. 8B).
Hrr25•Elongator interaction and Elongator-minus traits of hrr25 mutants
Although Elp1 phosphomodification involves the Sit4 phosphatase (Jablonowski et al., 2004), the significance of Elp1 phosphorylation and its underlying Elongator kinase (ELK) activity have been elusive. We suspected that Elp1 phosphorylation may require the CKI isoform Hrr25 because hrr25, elp and sit4 mutants all survive zymocin, a tRNase whose ability to cleave tRNAs depends on Elongator's role in tRNA modification (Huang et al., 2005; 2008; Jablonowski et al., 2006; Lu et al., 2005; 2008). The hrr25 mutants studied here are either kinase-dead (hrr25-3) or have reduced (hrr25-4) kinase activity. Despite partial kinase activity towards casein, Hrr25-4 like Hrr25-3 fails to support zymocin toxicity or Elp1 phosphorylation states in vivo. This stresses the importance of phosphorylation by the Hrr25 kinase for zymocin action and Elongator activity.
Our findings that wild-type Hrr25 and the mutated proteins Hrr25-3 and Hrr25-4 associate with Elp1 and Elp3 show that Elongator interaction does not require either the kinase activity or the P/Q-rich motif of Hrr25. However, the interaction depends on Elongator partner Kti12 and holo-Elongator suggesting that Hrr25 operates in zymocin sensitivity when associated with Kti12 and the Elongator complex. In support of this, Kti12 removal (kti12Δ) or Hrr25 inactivation (hrr25-3) each trigger Elp1 hypophosphorylation (Jablonowski et al., 2004). Moreover, based on the findings that Kti12 and Hrr25 interact with each other and with the Elongator complex and importantly, that excess Kti12 suppresses Hrr25•Elongator interaction, the Hrr25 kinase appears to be bridged to Elongator via Elongator-bound Kti12. We therefore propose that Kti12 promotes Hrr25•Elongator interaction and that both proteins, Hrr25 and Kti12, are likely to act in a common pathway that modulates Elp1 phosphorylation states. Consistently, multicopy KTI12 triggers Elp1 hyperphosphorylation and suppresses zymocin sensitivity (Butler et al., 1994; Frohloff et al., 2001; Jablonowski et al., 2004). With the latter Elp1 phosphodefect being eliminated by the kinase-dead hrr25-3 allele, the ability of Kti12 to alter Elp1 phosphorylation states apparently depends on the kinase activity of Hrr25.
Antagonism between Hrr25 and Sit4
In spite of carrying the ssd1-d allele, a condition usually synthetically lethal with sit4Δ null-mutations (Sutton et al., 1991), hrr25 mutants survive deletion of SIT4. Conversely, Sit4 removal from the hrr25-3 mutant reverses its zymocin resistance and restores a wild-type-like balance between hyper- and hypophosphorylated Elp1 forms. The reappearance of Elp1 phosphoforms in the kinase-dead hrr25-3 sit4Δ double mutant implies that Hrr25 is unlikely to qualify as an ELK or an ELK-activating kinase. To explain the opposing effects of Hrr25 and Sit4 on Elongator phosphoregulation and the role that Kti12 plays in modulating Elp1 phosphorylation states, we propose two possible scenarios. In the first, Kti12 may directly phosphorylate Elp1 as an ELK. In support of this option, Kti12 carries a kinase-related P-loop motif potentially involved in NTP binding and a kti12Δ mutant confers Elp1 hypophosphorylation, a defect one may predict for an ELK-minus condition (Butler et al., 1994; Jablonowski et al., 2004). However, this is in contrast to Elp1 hyperphosphorylation observed in a sit4Δkti12Δ double mutant (Jablonowski et al., 2004) and demonstrates that hyperphosphorylated forms of Elp1 can be found in the absence of Kti12. So, Kti12 is unlikely to be the sole Elp1 kinase.
In the second scenario, Hrr25 and Kti12 are proposed to control Sit4 and affect Elp1 phosphorylation states indirectly by countering Elp1 dephosphorylation. In favour of such inhibition, Hrr25 not only partners with Kti12 and Elongator but also copurifies by means of TAP with the Sit4•Sap185 and Sit4•Sap190 phosphatases known to promote Elp1 dephosphorylation (Ho et al., 2002; Jablonowski et al., 2004). Also, Elp1 phosphorylation states are particularly sensitive to KTI12 dosage, with hrr25-like hypophosphorylation and sit4Δ-like hyperphosphorylation of Elp1 seen in kti12Δ and multicopy KTI12 cells respectively (Jablonowski et al., 2004). Strikingly, the multicopy KTI12 effect is antagonized by SIT4 overexpression (Jablonowski et al., 2004) suggesting that upregulated Sit4 phosphatase activity may bypass the negative Kti12 effect. In line with the dependence of Kti12-mediated inhibition on Hrr25, Elp1 hyperphosphorylation due to multicopy KTI12 is eliminated in the kinase-dead hrr25-3 mutant. This strongly suggests that Hrr25 and Kti12 act in a common pathway and consistent with this notion, we observed that Elp1 hypophosphorylation typical of the kti12Δ mutant was insensitive to HRR25 gene dosage and remained unaltered in multicopy HRR25 cells (not shown). In future, it will be crucial to identify whether Hrr25 may phosphorylate Kti12 or Elongator-related factors including Elongator subunits other than Elp1.
Provided the second scenario held true and Sit4 was kept in check by Hrr25, reducing the activity of the Sit4•Sap185 and Sit4•Sap190 phosphatases ought to make up for defects in hrr25 kinase mutants. This is a prediction confirmed by our suppression data involving hrr25-3 cells that carry a SIT4 deletion, are depleted of Sit4 by repression of GAL-SIT4 or maintain high-copy SAP155. All these conditions, which remove Sit4 activity or suppress Sit4•Sap185 and Sit4•Sap190 phosphatase formation by excess levels of Sap155 (Luke et al., 1996; Jablonowski et al., 2004), restored wild-type-like Elp1 phosphorylation states and zymocin sensitivity in the hrr25-3 mutant. This strongly suggests that the Elp1 phosphodefect in the hrr25-3 mutant results from enhanced Sit4 activity rather than abolished Elp1 phosphorylation. We therefore favour the second model and consider it likely that Hrr25 negatively regulates Sit4 and thus Elp1 dephosphorylation.
Elp1 phosphorylation defects interfere with Elongator and Kti12 functions
Although our data suggest antagonism between Hrr25 and Sit4, future studies addressing ELK identification, Elp1 phosphorylation site mapping and mutagenesis may help to determine the significance of Elp1 phosphorylation for Elongator function. However, our data imply that Elp1 hypophosphorylation due to defective Hrr25 kinase alters the ability of Elongator to interact with Kti12, whose homologue DRL1 is a candidate Elongator regulator in plants (Nelissen et al., 2003; 2005). With the abilities of Kti12 to bind Elongator and to affect the function of this acetylase complex (Fichtner et al., 2002; Jablonowski et al., 2004; Petrakis et al., 2005), reversible Elp1 phosphorylation may therefore provide a means to regulate Elongator by controling Kti12•Elongator association and dissociation. In addition, Elp1 phosphorylation may modulate the acetylase activity of Elongator and control substrate modification (Gardiner et al., 2007). By analogy, differential and compartmentalized substrate deacetylation by the SirT2 deacetylase has been shown to involve phosphoregulation (Vaquero et al., 2006). In summary, the correct balance between hypo- and hyperphosphorylated Elp1 forms is critical for Elongator activity and Elongator loss of function associated with the presence of exclusively one or the other Elp1 form implies that Elongator regulation involves dynamic dephosphorylation and phosphorylation cycles. So rather than being constitutive, Elongator functions are likely to be regulated in response to signal transduction and phosphorylation.
Yeast strains, media and Kluyveromyces lactis zymocin methods
All yeast strains used or generated in this study are described in Table 1. Yeast strains were grown in routine yeast extract, peptone, dextrose (YPD) or galactose (YPG) rich media or synthetic complete (SC) medium (Sherman, 2002). ade2-1 and can1-100 ochre stop codon suppression by the tRNATyr gene SUP4 involved previously described SUP4 strains (Huang et al., 2005) or SUP4 plasmid pTC3 (Shaw and Olson, 1984). For zymocin sensitivity tests of S. cerevisiae by killer eclipse bioassays (Kishida et al., 1996) or YPD plate assays containing 40–65% (v/v) partially purified zymocin (Jablonowski et al., 2004) growth was monitored after 2–3 days at 30°C. K. lactis killer strain AWJ137 (Table 1) was used as the zymocin producer. Yeast transformations with plasmid DNA or polymerase chain reaction (PCR) products used the lithium-acetate method (Gietz et al., 1992).
Table 1. Yeast strains used and generated throughout this study.
MATαleu2 trp1[k1+ k2+] killer and zymocin producer
CY4029 but sap4Δ::LEU2 sap155Δ::HIS3 + pTC3 (SUP4)
CY4029 but sap185Δ::ADE2 sap190Δ::TRP1 + pTC3 (SUP4)
CY4029 but sit4Δ::HIS3 + pTC3 (SUP4)
AY925 but sit4Δ::HIS3+ pSSD1-v (CEN URA3 SSD1-v)
LL20 but sit4Δ::HIS3+ pSSD1-v (CEN URA3 SSD1-v) ura3
KY117 but sit4Δ::HIS3+ pSSD1-v (CEN URA3 SSD1-v)
W303-1a but MATαSUP4
UMY2893 but elp1Δ::KAN
UMY2893 but HIS3-GAL1p-HA-HRR25 + pCM16.2 (hrr25-4)
MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 HRR25-TAP-HIS3
Yeast genetic manipulations
PCR-mediated construction of elp1Δ, elp3Δ, elp5Δ and kti12Δ null-alleles used plasmid templates YDp-KlL (LEU2), YDp-SpH (HIS3) or YDp-KlU (URA3) and knockout primers previously described (Frohloff et al., 2001; Jablonowski et al., 2001c). Strains carrying pCM12.2 (HRR25), pCM15.2 (hrr25-3) or pCM16.2 (hrr25-4) were selected for HRR25 deletions using PCR with knockout primers koHRR25-FW (5′-ATT GGC AGG AAG ATT GGG AGT GGT TCC TTT GGT GAC ATT TAC CAC GGC ACC GAC GGC CAG TGA ATT CCC GG-3′) and koHRR25-RV (5′-GTT GCT TAC AAC CAA ATT GAC TGG CCA GCT GGT TTA TCT TGA GGC GGC TGA GCT TGG CTG CAG GTC GAC GG-3′). Deletions were verified by diagnostic PCR using primers HRR25-FW and HRR25-RV (see below). Substitution of the SIT4 promoter by the GAL1 promoter and simultaneous HA-tagging of Sit4 was as described (Jablonowski et al., 2004). HRR25 promoter swaps used PCR with plasmid pFA6a-HIS3-pGAL1-HA (Longtine et al., 1998) and primers F4-HRR25 (5′-GAG AAG AAA GGT TCG ACA CTC GAG GAA AGC ATT TGG TGG TGA AAA CAC ATG AAT TCG AGC TCG TTT AAA C-3′) and R3-HRR25 (5′-CTC CCA ATC TTC CTG CCA ATA CGA AAT TTC CTT CCT ACT CTT AAG TCC ATG CAC TGA GCA GCG TAA TCT g-3′). This technique allowed N-terminal HA-tagging of Hrr25, which was verified in Western blots (see below). N-terminal TAP tagging of HRR25 involved PCR with plasmid pBS1461 (Rigaut et al., 1999) and primers up-HRR25 (5′-GAG AAG AAA GGT TCG ACA CTC GAG GAA AGC ATT TGG TGG TGA AAA CAC ATG AAC AAA AGC TGG AGC TCA T-3′) and down-HRR25 (5′-CTC CCA ATC TTC CTG CCA ATA CGA AAT TTC CTT CCT ACT CTT AAG TCC ATC TTA TCG TCA TCA TCA AGT G-3′). PCR-mediated C-terminal tagging with HA and c-Myc epitopes used pYM1-pYM5 plasmids and S3/S2-primers as described (Knop et al., 1999; Frohloff et al., 2001). Generation of sit4Δ single and hrr25-3 sit4Δ double mutants used sit4::LEU2 and sit4::HIS3 cartridges (Jablonowski et al., 2004). All genetic manipulations including those that led to epitope tagging were verified by diagnostic PCR and Western blots using anti-HA or anti-c-Myc antibodies and by killer biosassays (Kishida et al., 1996) to validate that tagged proteins conferred unaltered zymocin sensitivity.
Constructions and use of additional vectors
Analysis of KTI12 dosage effects utilized YEplac-based (Gietz and Sugino, 1988) high-copy vectors pJHW27 (LEU2) and pDJ41 (URA3) carrying the wild-type KTI12 gene (Butler et al., 1994) and pDJ20 (LEU2) carrying a KTI12-HA allele (Jablonowski et al., 2004). pDJ20 was constructed by in vivo gap repair using strain DJY114 (Jablonowski et al., 2004). Testing SSD1-v versus ssd1-d allelism involved transformation with pSSD1-v (CEN URA3) prior to sit4::LEU2 disruption (Jablonowski et al., 2004) and counterselection of pSSD1-v by 5-FOA (Sikorski and Boeke, 1991) to distinguish SSD1-v from ssd1-d status. SAP155 gene dosage studies involved plasmid pCB2643, a YEp24 (2 μURA3) derivative carrying the SAP155 gene (Luke et al., 1996). To generate pCM12.2, pCM15.2 and pCM16.2 (see above), wild-type and mutant alleles of HRR25 were amplified from genomic DNA of strains LL20 (HRR25), ARB97 (hrr25-3) and ARBK106 (hrr25-4) by PCR using primers HRR25-FW (5′-TGT GAA GAG TTA CTG CAA CTC TCGC-3′) and HRR25-RV (5′-GTG CGT TTT GAG CAA TAT ATG TTGC-3′) and cloned into pCR2.1-TOPO (Invitrogen). Next, a 1.8 kb HindIII/XbaI-fragment was subcloned into YCplac33 (Gietz and Sugino, 1988) yielding pCM12.2 (HRR25), pCM15.2 (hrr25-3) and pCM16.2 (hrr25-4). E. coli expression of His6-tagged Hrr25, Hrr25-3 and Hrr25-4 and Ni2+-affinity purification used the ‘pTrcHIS TOPO-TA Expression Kit’ (Invitrogen). Cloning involved PCR amplification of the HRR25 and hrr25-3 alleles using DNA from strains LL20 (HRR25) and ARB97 (hrr25-3) and primers FW-HRR25-rec (5′-GGC TCT AGA GAG ATG GAC TTA AGA GTA GGA AGG A-3′) and RV-HRR25-rec (5′-GGC GGA TCC GTT GCT TAC AAC CAA ATT GAC TG-3′). PCR on strain ARBK106 (hrr25-4) with primers FW-HRR25-rec (see above) and RV-hrr25-4-rec (5′-GGC GGA TCC TTA GTT TTC CAT ACC TTT ATC TAG TGC G-3′) eventually yielded the hrr25-4 expression clone.
Hrr25 kinase assays
In vitro casein kinase activities of His6-tagged Hrr25, Hrr25-3 and Hrr25-4 enzymes (see above) obtained by Ni2+-NTA affinity purification were measured by incorporation of 32P radiolabel from [γ-32P]-ATP into casein. Assays containing 5 μl Hrr25, Hrr25-3 and Hrr25-4 eluates, 5 μl 5% (w/v) casein (Sigma), 5 μl 5× buffer (0.25 M Tris-HCl pH 7.5, 50 mM MgCl2, 0.5% 2-mercaptoethanol, 0.5 mM EGTA-KOH pH 7.5), and 5 μl [γ-32]-ATP (1 mM, ∼18.5 MBq/mmol) were incubated for 10 min at 30°C. Upon addition of 1 ml 25% (w/v) TCA, precipitates were pelleted, washed three times in 1 ml 25% (w/v) TCA and radioactivity was quantified by Cerenkov counting. Alternatively, in vitro casein kinase activities were measured based on an enzyme-coupled system (Bergmeyer, 1976). In brief, Hrr25-dependent ATP hydrolysis and casein phosphorylation were coupled to the catalysis of pyruvate kinase (generating pyruvate and ATP from phosphoenolpyruvate and ADP) and lactate dehydrogenase (generating lactate and NAD+ from pyruvate and NADH). Hence, ATP consumption by casein kinase activity was determined indirectly by spectrophotometry (OD 340 nm) measuring the decrease in NADH levels. Reactions contained 5 μl recombinant Hrr25, Hrr25-3 and Hrr25-4 eluates, 100 mM Tris-HCl (pH 7.9), 5 mM MgCl2, 10 mM KCl, 300 μg ml−1 BSA, 0.25 mM NADH (Sigma), 2 mM phosphoenolpyruvate, 1 mM ATP, 6.6 U ml−1 pyruvate kinase, 13.5 U ml−1 lactate dehydrogenase and 0.145 mM casein (Sigma).
Detection of tagged proteins used anti-c-Myc and anti-HA antibodies (Roche). Elp1 and Elp3 were detected with anti-Elp1 and anti-Elp3 antibodies (1:3000, kindly provided by Dr J. Svejstrup) as described (Otero et al., 1999; Wittschieben et al., 1999). Protein concentrations were determined (Bradford, 1976) and checked with anti-Pfk1 antibodies recognizing yeast Pfk1 α and β subunits (1:50 000, kindly provided by Dr J. Heinisch) or anti-Cdc19 serum (1:10 000, kindly provided by Dr J. Thorner). Elp1-HA phosphoanalysis involved 6% SDS-PAGE, anti-HA Western blots and electrophoretic mobility shift assays as described (Jablonowski et al., 2004). Immune precipitation, antibody cross-linking to protein A-Sepharose, preparation of protein extracts and TAP were performed as described (Zachariae et al., 1996; Rigaut et al., 1999; Frohloff et al., 2001).
Thanks are due to Drs K. Arndt, A. Byström, B. Cyert, J. Heinisch, J. Svejstrup, J. Thorner and W. Zachariae for yeast strains, plasmids, antibodies and advice. We thank A. Anders, C. Bär, J. E. Täubert and P. Studte for technical assistance. C.M. received a FEBS summer fellowship. K.D.B., M.J.R.S. and R.S. acknowledge grant support through DFG to R.S. (Scha750/2) and K.D.B. and R.S. (SFB648) and through BBSRC to M.J.R.S. (BB/F0191629/1) and to R.S. (BB/F019106/1).