Phosphoproteomic analysis of Arabidopsis thaliana ribosomes

• Ribosomes serve as the site of protein synthesis in all living cells. Ribosomes were discovered in 1955 by George E. Palade when he was studying the endoplasmic reticulum which is covered by ribosomes. He received the Nobel Prize in Physiology or Medicine in 1974 for this discovery. Ribosomes are large protein and rRNA complexes which are made up from one small and one large subunit that work together to translate mRNA into a protein chain. Eukaryotic translation is mainly controlled during the initiation, which involves protein phosphorylation. In plants there is a general increase of protein synthesis during the day in order to synthesize proteins needed for photosynthesis. Phosphorylation can alter protein function and localization and is reversibly added and removed by kinases and phosphatases, respectively.The aim of the studies in this thesis was to elucidate the phosphorylation status of ribosomal proteins in the Arabidopsis thaliana 80S ribosome. I have focused on comparing ribosomal protein phosphorylation between different conditions and sub cellular locations, namely day/night conditions and cytosol/nucleus location.By using Fe3+IMAC to enrich phosphorylated peptides from cytosolic ribosomes followed by mass spectrometric analysis eight serine residues in six ribosomal proteins were found to be phosphorylated. Among these was a novel phosphorylation site in 40S ribosomal protein S6 at Serine 231. By using quantification with stable isotope labeling and mass spectrometry this phosphorylated residue and three other ribosomal phosphopeptides were found to have increased phosphorylation levels during day as compared to night ranging from 2 to 4 times. This phosphorylation increase can in turn effect the modulation of the diurnal protein synthesis in Arabidopis thaliana.Ribosome biogenesis involves shuttling of proteins and ribosomal subunits between the cell nucleus and cytoplasm. By purifying ribosomal proteins from these two cellular compartments and enriching for phosphopeptides using TiO2 affinity chromatography combined with mass spectrometry I was able to analyze their phosphorylation status. This method identified 13 phosphopeptides derived from 11 ribosomal proteins as well as phosphopeptides from two ribosomal associated proteins. 40S ribosomal protein S2-3 was found phosphorylated only in the cytoplasmic samples while 60S ribosomal protein L13-1 and the two ribosomal associated proteins were found only in the nuclear enriched samples.

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