Abstract
Despite their importance, membrane proteins have traditionally been underrepresented in proteomics studies due to their incompatibility with common methods used in this field. Therefore, new methods have to be developed for studying this class of proteins. In this thesis, new approaches are described, which were applied to the mitochondrial membranes
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of the yeast Saccharomyces cerevisiae. Because these membranes have been used extensively in the past as a platform for the development of proteomic approaches, the results could be easily compared to results obtained previously. First, interactions between lipids en proteins were searched by incorporating lipid probes containing a photoactivatable moiety in yeast mitochondrial membranes to perform photo-crosslinking. To detect cross-linked proteins, either the intrinsic absorbance of the probe was used or a reporter molecule was attached to the cross-link products via click chemistry. After addition of biotin as a reporter molecule, cross-linked proteins could be purified and subsequently identified using state-of-the-art mass spectrometry-based protein identification methods. Known interactions partners of phospholipids that were analogues to the lipid probes were found, as well as proteins that were previously not described as membrane proteins or as interactions partners of these phospholipids. Second, the stability of membrane protein complexes in yeast mitochondria was studied using a 2D gel electrophoresis approach for the identification of complexes that are stable in SDS but dissociate in the presence of fluorinated alcohols such as trifluoroethanol. Surprisingly, yeast mitochondrial membrane protein complexes were found to be less stable in than membrane protein complexes in E. coli inner membranes, despite their evolutionary relationship. This finding was explained by the reduced hydrophobicity of mitochondrial transmembrane proteins as compared to their E. coli counterparts. Finally, the influence of the level of phosphatidylcholine, a common phospholipid, on mitochondrial protein levels was investigated. Upon depletion of the cellular PC content, a limited number of mitochondrial proteins was found to become more abundant, including proteins involved in oxidative stress. In addition, non-mitochondrial proteins were enriched in isolated PC-depleted mitochondria, indicating stronger association of mitochondria with other organellar membranes under these conditions. In conclusion, the new methods enabled us to study yeast mitochondrial membrane organization on a proteome-wide scale and provided new insights in how these membranes are structured.
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