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Phosphorylation of ribosomal proteins in cytoplasm and nucleus of Arabidopsis thaliana
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Ribosome assembly is a complicated process that takes place in both the cell cytosol and nucleus. Shuttling between these compartments of both ribosomal proteins and ribosome subunits is necessary for the formation of active ribosomes. The goal of this work was to make a comparison of ribosomal proteins from nucleus and cytosol of Arabidopsis thaliana. We made separate preparations of ribosomes from cytosol and nucleus. Our proteomic analysis of ribosomes isolated from Arabidopsis thaliana leaves detected 146 ribosomal proteins from cytosol and 135 ribosomal proteins from the cell nucleus. Phosphopeptides from these preparations were enriched using TiO2 and analyzed using nanoLC-MS/MS. This method allowed us to identify 13 phosphopeptides from 11 ribosomal proteins: S2-3, S6-1, S6-2, L13-1, L13-3, L29-1, P0-2, P0-3, P1 (P1-1, P1-2, P1-3), P2 (2-1, 2-2, 2-3), P3 (3-1, 3-2, 3-3) and additionally two phosphopeptides from two ribosomal associated proteins: Nascent polypeptide-associated complex subunit alpha-like protein 1 and 3.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-77236OAI: oai:DiVA.org:liu-77236DiVA: diva2:525700
Available from: 2012-05-09 Created: 2012-05-09 Last updated: 2012-05-09Bibliographically approved
In thesis
1. Phosphoproteomic analysis of Arabidopsis thaliana ribosomes
Open this publication in new window or tab >>Phosphoproteomic analysis of Arabidopsis thaliana ribosomes
2012 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 30 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 121
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77237 (URN)978-91-7519-920-7 (ISBN)
Presentation
2012-05-29, Linden, ingång 65, plan 9, Campus US, Linköpings universitet, Linköipng, 09:00 (Swedish)
Supervisors
Available from: 2012-05-09 Created: 2012-05-09 Last updated: 2012-05-09Bibliographically approved

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Klang Årstrand, HannaVener, Alexander V.

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