Partition and turnover of glutathione reductase in Saccharomyces cerevisiae: a proteomic approach.

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Abstract

The main work presented in this thesis describes proteomics strategies applied to study the trafficking and turnover of glutathione reductase (Glr1) isoforms in the cytosol and mitochondria of Saccharomyces cerevisiae. Additional work was performed in order to understand mass spectrometric response factors and how they can affect peptides representation in the mass spectra. The opportunity to study two sub proteomes involved in biofilm formation of Pseudomonas aeruginosa PAO1 arose during my PhD and their analysis is also presented.Glr1 is a low abundant protein involved in the defence mechanisms against reactive oxygen species, which are sources of many diseases. Because of its biological relevance, considerable effort has been made in order to understand its functional role in the cell. This protein has been studied using biochemical strategies. In yeast, the cytosolic and mitochondrial forms of glutathione reductase are expressed by the same gene, GLR1, using alternative start codons. Translation from the first AUG codon generates the mitochondrial form incorporating a transit peptide necessary for import into the mitochondria. If the translation starts at the second AUG codon, the cytosolic counterpart is generated. Biochemical approaches show that the first AUG codon is not in favourable context and it has been suggested that leaky scanning accounts for the abundance of the cytosolic protein.The analysis of Glr1 forms by mass spectrometry was demanded because only the N-terminal region is informative about similarities and differences between cytosolic and mitochondrial forms. The protein is also of low abundance in both cytosol and mitochondrial compartments. A genetically modified strain, over-expressing this protein was, therefore, used throughout this work in order to analyse glutathione reductase in the mitochondria. This was not possible with the wild-type strain.Because the first AUG codon is now in context, the over-producing strain (MORF) yields both cytosolic and full length mitochondrial isoforms in the cytosol. Analysis of the mitochondrial protein shows that the cleavage of the pre-sequence is not specific. Three different forms of the mitochondrial N-terminal peptide were detected. Some attention was also devoted to glutathione reductase turnover in both cytosol and mitochondrial compartments using the genetically modified strain. Both N-terminal peptides generated from translation starting in the first and second AUG codon as well as mid-chain peptides from the cytosol fraction and one mid-chain peptide from the mitochondrial fraction, were used to calculate relative turnover measurements. My results illustrate that the mitochondrial protein is in faster turnover than the cytosolic counterpart. Moreover, the long and short forms observed in the cytosol also show slightly different turnover rates, the long form presenting faster turnover than the short form. Rapid turnover therefore maintains the level of glutathione reductase in the mitochondria.Despite the exquisite sensitivity of mass spectrometry, its restricted dynamic range compared with the dynamic range of the entire proteome is limiting for such studies. Peptides have different responses in the mass spectrometry and factors such as hydrophobicity and gas-phase basicity, can also contribute to low detectability of some peptides. To maximise the mass spectrometric response of peptides especially the ones derived from low abundant proteins, is extremely important. My thesis therefore includes a study of mass spectrometric response of peptides generated by different enzymes. Applying the kinetic method, the importance of the position of basic residues on gas-phase basicity and thus on the mass spectrometric response was demonstrated.In addition, the opportunity to carry out a related study on the proteome analysis of membrane vesicles and matrix within biofilms of Pseudomonas aeruginosa PAO1 has arisen and results of this study were presented in the final results chapter. It is the first time that two-dimensional chromatography was applied to analyse these sub-proteomes. Moreover, previous studies were mostly limited to the planktonic population; here the proteomes of membrane vesicles and extracellular matrix with the biofilm were addressed.

Keyword(s)

Mass Spectrometry; Proteomics

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