Figure 6.
Bcp1 triggers the release of Rkm1 from methylated uL14. (A) GST-Rkm1/uL14 or GST-Rkm1(Y273F)/uL14 complex was immobilized on the glutathione beads. Buffer alone, containing 0.5 mM SAM or purified Bcp1, was added and incubated at 30°C for 80 min. After gently spinning, the supernatants and beads were collected separately. TCA addition precipitated the proteins from supernatants (Sup). The glutathione beads were washed three times, and proteins were eluted in 1× SDS sample buffer. Proteins were analyzed by Coomassie blue staining and Western blotting with α-methylation (α-Me), α-uL14, and α-Bcp1 antibodies. (B) Bcp1-His6 in the supernatants from reactions 7 and 8 in Fig. 6 A was applied for Ni-NTA purifications. The bound proteins on the NTA beads are shown as lanes 1 and 2, respectively. (C) uL14 was reacted with Rkm1 and SAM in vitro (Fig. 6 A, lane 2), and the in-gel digestion was performed with AspN. The intensities of methylated and unmethylated peptides were calculated, and the ratios were shown. (D and E) A complex of GST-Rkm1 with uL14, uL14(K106R), uL14(K110R), or uL14(RR) was immobilized on the glutathione beads. SAM (AdoMet) or with the purified Bcp1 were added (D). A complex of GST-Rkm1 with uL14 (+) or uL14∆loop (∆) was immobilized on the glutathione beads. The purified Bcp1 (+) or bcp1∆N20 (∆) with SAM were added (E). The in vitro methylation assays proceeded as described above. Proteins that remained on the beads were shown in the Coomassie blue staining gel or detected by western blotting. (F) Rkm1-HA or rkm1(Y273F)-HA was immunoprecipitated and the associated proteins were detected by western blotting. (G) uL14-HA or uL14(RR)-HA was immunoprecipitated and the associated proteins were detected by western blotting. (H) WT or bcp1ts strains containing RKM1, or rkm1(Y273F) on 2 μ plasmids were normalized and serially diluted. Equal amounts of cells were spotted on the plates and incubated at the temperature indicated on the blot. (I) The growth tests of bcp1ts strain containing RPL23 or rpl23Δ(RR) plasmids. Source data are available for this figure: SourceData F6.

Bcp1 triggers the release of Rkm1 from methylated uL14. (A) GST-Rkm1/uL14 or GST-Rkm1(Y273F)/uL14 complex was immobilized on the glutathione beads. Buffer alone, containing 0.5 mM SAM or purified Bcp1, was added and incubated at 30°C for 80 min. After gently spinning, the supernatants and beads were collected separately. TCA addition precipitated the proteins from supernatants (Sup). The glutathione beads were washed three times, and proteins were eluted in 1× SDS sample buffer. Proteins were analyzed by Coomassie blue staining and Western blotting with α-methylation (α-Me), α-uL14, and α-Bcp1 antibodies. (B) Bcp1-His6 in the supernatants from reactions 7 and 8 in Fig. 6 A was applied for Ni-NTA purifications. The bound proteins on the NTA beads are shown as lanes 1 and 2, respectively. (C) uL14 was reacted with Rkm1 and SAM in vitro (Fig. 6 A, lane 2), and the in-gel digestion was performed with AspN. The intensities of methylated and unmethylated peptides were calculated, and the ratios were shown. (D and E) A complex of GST-Rkm1 with uL14, uL14(K106R), uL14(K110R), or uL14(RR) was immobilized on the glutathione beads. SAM (AdoMet) or with the purified Bcp1 were added (D). A complex of GST-Rkm1 with uL14 (+) or uL14∆loop (∆) was immobilized on the glutathione beads. The purified Bcp1 (+) or bcp1∆N20 (∆) with SAM were added (E). The in vitro methylation assays proceeded as described above. Proteins that remained on the beads were shown in the Coomassie blue staining gel or detected by western blotting. (F) Rkm1-HA or rkm1(Y273F)-HA was immunoprecipitated and the associated proteins were detected by western blotting. (G) uL14-HA or uL14(RR)-HA was immunoprecipitated and the associated proteins were detected by western blotting. (H) WT or bcp1ts strains containing RKM1, or rkm1(Y273F) on 2 μ plasmids were normalized and serially diluted. Equal amounts of cells were spotted on the plates and incubated at the temperature indicated on the blot. (I) The growth tests of bcp1ts strain containing RPL23 or rpl23Δ(RR) plasmids. Source data are available for this figure: SourceData F6.

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