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Title: Identification and initial characterisation of protein methylation sites in Saccharomyces cerevisiae
Author: Plank, Michael
ISNI:       0000 0004 6063 2683
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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The field of protein arginine methylation has seen a surge in the number of known methylation sites in higher eukaryotes over recent years, but our understanding of the functions of these modifications is lagging behind. A better knowledge of arginine methylation in a model organism like Saccharomyces cerevisiae that is relatively amenable to experimental manipulation may inform on arginine methylation also in higher eukaryotes. The proteome-wide identification of methylation sites in S. cerevisiae, based on liquid chromatography tandem mass spectrometry in combination with several enrichment and fractionation strategies and on the use of heavy methyl SILAC is presented here. The majority of sites identified are in good agreement with previous reports of arginine methylation, i.e. occur in glycine-arginine-rich regions, locate to proteins involved in RNA-processing and overlap with known sites. Additionally, the data obtained suggest a bias for localisation of arginine methylation in unstructured protein regions and asparagine-rich contexts. It is further shown that the use of heavy methyl SILAC is useful for confident site identification. To overcome challenges in the global approach, the detection of methylation sites on overexpressed candidate proteins was also employed. This strategy yielded methylation sites on more than half of the 38 successfully purified proteins, but also brought to light challenges in the localisation of sites. Subsequently, it was investigated which methyltransferases are responsible for methylating mapped sites on two of these proteins. These experiments strongly indicate that Lsm4 is neither exclusively methylated by Hmt1 nor Hsl7, while Hmt1 appears to be the sole methyltransferase responsible for methylating Scd6. It was further shown that the methylation of the glycine-arginine-rich region of Scd6 is reduced upon prolonged sodium azide stress which may have potential implications in stress-induced translational repression. Overall, these results both inform on methods for the assignment of arginine methylation and improve its biological characterisation.
Supervisor: Schofield, Christopher ; Kessler, Benedikt ; Pears, Catherine Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available