Phosphorylation of Elp1 by Hrr25 is required for elongator-dependent tRNA modification in yeast

Abstract

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.

Figure 1. Identification of functionally important sites of phosphorylation in Elp1.

(A) Cartoon showing Elp1 indicating mapped sites of phosphorylation and structural elements predicted using PSIPRED , with predicted α-helix shown in black and β-sheet in grey. (B) Western blot of cell lysate prepared from cells expressing ELP1-6HA (ELP1) or elp1-S1209A-6HA showing recognition of Elp1 by a phosphospecific antibody raised against the phosphopeptide TSTQE(pS)FFTRY that recognizes Elp1 phosphorylated on Ser-1209; blots with anti-HA and anti-Cdc28 antibodies control for levels of Elp1 and total protein loading respectively. (C) Eclipse assays showing the zymocin phenotype of wild-type and a range of single or multiple elp1 phosphorylation site mutants. Lack of growth inhibition around the K. lactis colony placed at the edge of each patch of cells being tested indicates zymocin resistance and hence loss of Elongator function. (D) Conservation of the phosphorylation sites identified in the Elp1 C-terminal domain. The full sequences were first aligned using T-Coffee but only the relevant detail from the alignment is shown. NCBI database accessions for the aligned sequences are Saccharomyces cerevisiae (NP_013488), Ashbya gossypii (NP_984907), Candida albicans (XP_710040), Mus musculus (NP_080355), Homo sapiens (NP_003631) Schizosaccharomyces pombe (NP_595335), Arabidopsis thaliana (NP_196872). (E) C-terminal phosphorylated region of S. cerevisiae Elp1 showing the downstream region with a repeating pattern of serine and threonine residues. Mapped phosphorylation sites are underlined and shown in bold, potential phosphorylation sites downstream of the mapped sites are underlined.

Figure 2. Effect on Elongator function of phosphorylation site mutations in the C-terminal domain of Elp1.

All strains were based on WAY034 (elp1Δ; panel B), YRDS119 (elp1Δ [pAE1]; panel C) or SBY138 (elp1Δ his3Δ1::pSB3; panel D) transformed with YCplac111, YCplac111-ELP1-6HA (wild-type) or mutant derivatives carrying mutations in ELP1 as shown. (A) Key to substitutions present in the different alleles tested (wild-type sequence at the top). (B) Zymocin sensitivity measured by eclipse assay (see Fig. 1 legend), with strains being tested at either 1.0 or 0.1 OD₆₀₀/ml following growth in SCD-Leu medium. (C) Zymocin sensitivity monitored by intracellular expression of the zymocin γ subunit. Cells were grown on SCD-Leu-Ura to select for both plasmids, diluted to 1.0 OD₆₀₀/ml and then equivalent 10-fold dilutions plated on YPD agar (to control for cell viability) and YP agar containing 2% raffinose and 2% galactose to induce expression of zymocin γ-toxin from pAE1. (D) Cells were grown in SCD-Leu (to select for YCplac111 and its derivatives), diluted to 1.0 OD₆₀₀/ml and then equivalent 10-fold dilutions plated on SCD-Leu (to control for cell viability) and SCD-Leu-Ura (to assess efficiency of SUP4-dependent ura3^oc22 suppression.

Figure 3. Quantitation of modified U34 nucleosides in tRNA from selected elp1 mutants.

Content of ncm⁵U and mcm⁵U in small RNA samples from the indicated yeast strains. In each case, A (modified U)/A (U) indicates that the modified nucleoside signal was normalized using the total uridine content to allow comparison of the different samples. Inset: chemical structures of uridine and its Elongator-dependent mcm⁵U and ncm⁵U derivatives.

Figure 4. Elongator assembly is unaffected by a range of phosphorylation site mutations in Elp1.

elp1Δ yeast strains expressing myc-tagged ELP2 and either HA-tagged ELP3 (A) or ELP5 (B) were transformed with either empty vector or plasmids expressing the indicated ELP1 alleles (Table S2). Elp2-myc was immunoprecipitated from extracts and immunoprecipitates were examined by Western blotting with anti-myc and anti-HA antibodies to detect co-immunoprecipitated Elp1, Elp3 and Elp5 as indicated. Note that Elp1 runs as a doublet as observed previously .

Figure 5. Phosphorylation site mutations in Elp1 lead to changes in Hrr25 association with Elongator.

elp1Δ ELP2-myc₃ KTI12-HA₆ yeast strains were transformed with either empty vector or plasmids expressing the indicated HA₆-tagged ELP1 alleles (Table S2). Elp2-myc was immunoprecipitated from extracts and immunoprecipitates were examined by Western blotting with anti-myc (to detect Elp2), anti-HA antibodies (to detect co-immunoprecipitated Elp1) and anti-Hrr25 (to detect co-immunoprecipitated Hrr25) as indicated. Western blots of the extracts before immunoprecipitation (lower panels) confirm similar levels of each protein in the different strains.

Figure 6. Phosphorylation site mutations in Elp1 lead to changes in Kti12 association with Elongator.

(A) elp1Δ ELP2-myc₃ KTI12-HA₆ yeast strains were transformed with either empty vector or plasmids expressing the indicated HA₆-tagged ELP1 alleles (Table S2). Elp2-myc was immunoprecipitated from extracts and immunoprecipitates were examined by Western blotting with anti-myc (to detect Elp2) and anti-HA antibodies (to detect co-immunoprecipitated Elp1 and Kti12) as indicated. Western blots of the extracts before immunoprecipitation (lower panels) confirm similar levels of each protein in the different strains. (B) Co-immune precipitation of HA-tagged Kti12 with GFP-tagged Elp1. Extracts from strains expressing GFP-tagged wild-type or mutant Elp1 from its genomic locus together with HA-tagged Kti12 were subjected to immune precipitation using GFP-trap and then the immune precipitates examined by Western blotting with anti-GFP (to detect Elp1) and anti-HA antibodies (to detect co-immunoprecipitated Kti12) as indicated. elp1-KR9A, a tRNA binding domain mutation that reduces Kti12 association , is shown as a control. (C) Quantification of co-immunoprecipitation efficiency shown in (B). Immunoprecipitation of HA-tagged Kti12 was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analyzed using a one-way ANOVA, showing a statistically significant, two-fold increase in the association of Kti12 with Elp1-S1209A.

Figure 7. Purified Elongator shows Hrr25-dependendent phosphorylation that requires Ser-1198 and Ser-1202.

(A) Elongator was purified using TAP-tagged Elp3 from either a HRR25 wild-type or a hrr25-I82G analog-sensitive mutant strain and supplemented with [γ-³²P]ATP to examine endogenous Elp1 phosphorylation in the presence of DMSO (drug vehicle), 1NM-PP1 or 3MB-PP1. Phosphorylated Elp1 was visualized by autoradiography following SDS-PAGE. (B) Equivalent 10-fold serial dilutions of HRR25 ELP1, hrr25-I82G ELP1 and HRR25 elp1Δ yeast strains were plated out on YPAD agar with or without 1% (v/v) zymocin in the presence or absence of 10 µM 1NM-PP1 or an equivalent volume of DMSO (drug vehicle) as indicated and photographed after 2 days' growth at 30°C. (C) Elongator was prepared as in (A) from elp1Δ ELP3-TAP strains carrying plasmids encoding the indicated ELP1 alleles and incubated with [γ-³²P]ATP in the presence or absence of added recombinant Hrr25 (100 ng). Phosphorylated Elp1 was visualized by autoradiography following SDS-PAGE. Western blotting to detect Elp3-TAP and Elp1-6HA demonstrates equivalent recovery of Elongator from each strain.

Figure 8. In vitro phosphorylation of peptides corresponding to the Elp1 C-terminal region by Hrr25.

(A) Sequences of synthetic peptides used as substrates shown in alignment with Elp1 residues 1191-1230. Lower-case bold letters represent alanine and/or glutamate substitutions relative to the native Elp1 sequence. (B) Time course of Hrr25-dependent incorporation of ³²P from [γ-³²P]ATP into Peptides 89 and 92. (C-E) Incorporation of ³²P from [γ-³²P]ATP into peptides as assayed by SDS-PAGE and autoradiography. Note that in (D) and (E) two panels are shown in each case because some unnecessary intervening lanes were removed when preparing the figure. * indicates autophosphorylated recombinant Hrr25.