Figure 1.
Bcp1 and Rkm1 form a complex with uL14 and maintain the stability of uL14. (A) Wild-type, bcp1ts, rkm1∆, and bcp1tsrkm1∆ were normalized and spotted on the YPD plates. The plates were incubated at the temperatures indicated in the figure for 2–3 days. The doubling time was estimated from three independent samples cultured in liquid YPD medium at 35°C (Ave ± SD). (B) The size exclusion chromatography (left). The result of Coomassie blue gels (right). The y-axis is the normalized intensity of UV280 nm. The x-axis is the column volume of the Superdex 200 column. The peaks indicated with blue arrows were collected and analyzed in SDS-PAGE. (C and D) Bcp1-myc was immunoprecipitated from wild-type and rkm1Δ (C). Bcp1-myc was immunoprecipitated from wild-type and GAL::RPL23 strain. Cells were cultured in Leu− medium containing 2% galactose to OD 0.2–0.3, and 2% glucose was added for another 4 h (D). The associated proteins were detected by Western blotting. (E) The normalized cell lysates were prepared from the strains above and spun at 80,000 rpm for 1 h to separate free proteins (supernatant) and ribosomal subunits (pellet). The supernatants were precipitated with TCA and analyzed by Western blotting. (F) Various amounts of trypsin, as indicated in the figure, were added to purified uL14 or the purified complexes of Rkm1/uL14, Bcp1/uL14, and Rkm1/Bcp1/uL14, followed by incubation at 37°C for 30 min. The samples were subsequently analyzed using SDS-PAGE with Coomassie blue staining or western blotting. The intensity of uL14 was quantified using Image J, and the relative amounts were calculated compared with the control (no trypsin). Three independent replicates were conducted, and the Student’s t test was performed against uL14 alone to assess statistical significance (*P < 0.05; **P < 0.01). Source data are available for this figure: SourceData F1.

Bcp1 and Rkm1 form a complex with uL14 and maintain the stability of uL14. (A) Wild-type, bcp1ts, rkm1∆, and bcp1tsrkm1∆ were normalized and spotted on the YPD plates. The plates were incubated at the temperatures indicated in the figure for 2–3 days. The doubling time was estimated from three independent samples cultured in liquid YPD medium at 35°C (Ave ± SD). (B) The size exclusion chromatography (left). The result of Coomassie blue gels (right). The y-axis is the normalized intensity of UV280 nm. The x-axis is the column volume of the Superdex 200 column. The peaks indicated with blue arrows were collected and analyzed in SDS-PAGE. (C and D) Bcp1-myc was immunoprecipitated from wild-type and rkm1Δ (C). Bcp1-myc was immunoprecipitated from wild-type and GAL::RPL23 strain. Cells were cultured in Leu− medium containing 2% galactose to OD 0.2–0.3, and 2% glucose was added for another 4 h (D). The associated proteins were detected by Western blotting. (E) The normalized cell lysates were prepared from the strains above and spun at 80,000 rpm for 1 h to separate free proteins (supernatant) and ribosomal subunits (pellet). The supernatants were precipitated with TCA and analyzed by Western blotting. (F) Various amounts of trypsin, as indicated in the figure, were added to purified uL14 or the purified complexes of Rkm1/uL14, Bcp1/uL14, and Rkm1/Bcp1/uL14, followed by incubation at 37°C for 30 min. The samples were subsequently analyzed using SDS-PAGE with Coomassie blue staining or western blotting. The intensity of uL14 was quantified using Image J, and the relative amounts were calculated compared with the control (no trypsin). Three independent replicates were conducted, and the Student’s t test was performed against uL14 alone to assess statistical significance (*P < 0.05; **P < 0.01). Source data are available for this figure: SourceData F1.

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