Ribosomal protein uS7/Rps5 serine-223 in protein kinase-mediated phosphorylation and ribosomal small subunit maturation

Cellular translation should be precisely controlled in response to extracellular cues. However, knowledge is limited concerning signal transduction-regulated translation. In the present study, phosphorylation was identified in the 40S small subunit ribosomal protein uS7 (Yjr123w/previously called as Rps5) by Ypk1 and Pkc1, AGC family protein kinases in yeast Saccharomyces cerevisiae. Serine residue 223 (Ser223) of uS7 in the conserved C-terminal region was crucial for this phosphorylation event. S223A mutant uS7 caused severe reduction of small ribosomal subunit production, likely due to compromised interaction with Rio2, resulting in both reduced translation and reduced cellular proliferation. Contrary to optimal culture conditions, heat stressed S223A mutant cells exhibited increased heat resistance and induced heat shock proteins. Taken together, an intracellular signal transduction pathway involving Ypk1/Pkc1 seemed to play an important role in ribosome biogenesis and subsequent cellular translation, utilizing uS7 as a substrate.


Supplemental Table S1
Screening of proteins affected in ypk1∆ strain.
Logarithmically growing yeast cells were harvested at OD 600 =0.6 and cytosolic protein fractions were prepared. Samples were first separated with nonequlibrium pH gel electrophoresis and then SDS-PAGE. Gels were then stained with SYPRO Ruby and Pro-Q Diamond reagents to visualize levels of protein abundance and phosphorylation, respectively. Thirteen different spots picked up for mass spectrometry analysis from 2D gels resulted in candidate peptide identification. This table shows the candidate molecules responsible for each spot. Increase and decrease of abundance was indicated as "+" and "-", respectively. Spot ID 16 further confirmed that uS7 is the protein responsible by the following experiments in this manuscript.

Supplemental Table S4
Oligonucleotide sequence of probes and primers used in this study

Legends for Supplemental Figures
Supplemental Figure S1. Proteomic identification protein affected in ypk1∆ strain Cytosolic proteins extracted from WT and ypk1∆ strain were separated by 2D-gel.
Protein spots were visualized with SYPRO Ruby (for protein abundance) and Pro-Q Diamond (for protein phosphorylation). From each strain, corresponding pI value and molecular weight was indicated. Magnified image (boxed) is available in Fig 1. Supplemental Figure S2. Structure of yeast ribosome complex uS7 localization within the head of the yeast 80S ribosome complex. PDB file 4V88 was used for modeling. Image was produced using PyMOL software. The small subunit proteins are depicted in blue, and the large subunit proteins in green. uS7 was shown in yellow and its 223th Ser (arrow) was highlighted in red.

Supplemental Figure S3. Kinase activity toward uS7 in kinase assay
A. Ypk1 activity toward uS7.
Protein kinase activity of WT Ypk1, immunoprecipitated with anti-Ypk1 and protein G-Sepharose, toward uS7 was measured with kinase reaction buffer in the presence of [γ 32 P]-ATP. BAS-2500 radioactive imager (Fuji film) was used to measure 32 P incorporation after SDS-PAGE gel was CBB stained and dried. Presence of Ypk1 and uS7 was confirmed with Western blotting and CBB staining, respectively. Although presence of radioactive signals was detectable in the controls without anti-Ypk1 (left three lanes), enhancement of radioactivity was found with Ypk1 (right three lanes).
Only a mild increase was found with incremental uS7 addition to the assay, indicating that Ypk1 activity was detected at a rate similar to the Vmax. Without addition of immunoprecipitated material, no radioactivity was found in the size of uS7, indicating that uS7 fraction does not have protein kinase activity toward uS7.
To understand the radioactivity found in the control condition, different conditions of Ypk1 immunoprecipitation were tried. Control WT strain without GST-tagged YPK1 vector was GST-precipitated with glutathione-Sepharose. Radioactivity was measured as in A. Consistent background radioactivity was detected. Again, additive effect was observed in the condition with GST-YPK1 plasmid. Therefore, this assay background is caused by the co-precipitated kinase protein with Sepharose resin. Ypk1 activity could be measured as additive effect from the background. GST-uS7 protein used as substrate was not stable more than a week, thus GST-uS7 protein was freshly prepared for each kinase assay. Apparent signal strength varied by the [γ 32 P]-ATP, thus this reagent was used within one half-life of 32 P.

Supplemental Figure S4. Phospho-mimetic mutation
Alanine, aspartate or glutamate mutation was introduced to uS7-S223 site. Mutant uS7s were expressed from plasmid vector pRS415 with own promoter in the condition endogenous uS7 was turned off utilizing Tet-OFF system with 10 µg/mL doxycycline.

5-fold serial dilutions of indicated yeast cells were spotted onto SD plate and incubated
at 30˚C as in Fig 2C. Supplemental Figure S5. Cellular uptake of radiolabeled methionine-cysteine