EBP1 Regulates the Capacity of Protein Translation

Arabidopsis pgEBP1-YFP line expressing EBP1-YFP under the EBP1 promoter were grown in 3 repeats alongside with the same number of repeats of the control 35CaMVS:GFP (ref) for 7 days at 16/8 light and 150-200 seedlings were frozen in liquid N2 and total proteins were extracted in 1 mL of lysis buffer(150 mM NaCl, 1% Igepal CA-630, 50 mM Tris HCl (pH 8.0), 1 mM DTT, 3 mM PMSF, protease inhibitor cocktail, 3mM pNPP 10 uM MG132). The total protein extracts (4mg/IP) were cleared by centrifugation at 13,000g and 4°C for 20 min to remove remaining membrane and DNA, and the supernatant was incubated with 50 µl magnetic beads coupled to monoclonal mouse anti-GFP antibody (Miltenyi Biotec) for 15 min on ice. Magnetic columns were equilibrated using 250 µl lysis buffer. Cell lysates were added to the column after incubation and washed two times with 1 ml ice-cold lysis buffer, five times with 2 ml of 1xPBS (Dulbecco’s Phosphate Buffered Saline), and 2 times 1 ml 25 mM ABC (Ammonium bicarbonate), pH 7.5. Purified proteins were predigested by adding 20 µl 25 mM ABC , pH 7.5, 1 mM DTT, and 150 ng trypsin (Promega) for label-free experiments. After in-column digestion for 30 min at room temperature, proteins were eluted by adding two times 50 µl 25 mM ABC, pH 7.5, and 2.2 mM Iodoacetamide. Proteins were digested overnight at room temperature. The digestion was stopped by adding 5 µl 10 % formic acid, and peptides of each experiment were split and purified on two C18 Stage Tips and stored at 4°C.<br><br>The peptide mixture was analyzed by LC-MS/MS using a nanoflow RP-HPLC (LC program: linear gradient of 3-40 % B in 100 min, solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile) on-line coupled to a linear ion trap-Orbitrap (Orbitrap-Fusion-Lumos, Thermo Fisher Scientific) mass spectrometer operating in positive ion mode.<br><br>o determine bait specific interactors, MaxQuant proteomics software version 1.6.6.0 (Cox and 131Mann 2008)based label-free quantification analysis was carried out on the corresponding MS data 132files (.raw) representing 6 repeats of each pull down against the Arabidopsis thaliana protein and 133common laboratory contaminant databases, concatenated with the reverse sequences. Standard 134parameters were kept unchanged with false discovery rates (FDRs) of 0.01 for peptide and protein 135identification, and the minimum ratio set at 1. Unique and razor peptides were used for quantification 136with “match between runs” option enabled to permit for peptide identification transfer between the 137corresponding LC-MS raw files.