Elucidation of protein-protein interactions in mitochondria by cross-linking mass spectrometry

Linden, Andreas

dc.contributor.advisor	Urlaub, Henning Prof. Dr.
dc.contributor.author	Linden, Andreas
dc.date.accessioned	2020-08-04T13:14:20Z
dc.date.available	2020-08-04T13:14:20Z
dc.date.issued	2020-08-04
dc.identifier.uri	http://hdl.handle.net/21.11130/00-1735-0000-0005-1446-6
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-8148
dc.language.iso	deu	de
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc	570	de
dc.title	Elucidation of protein-protein interactions in mitochondria by cross-linking mass spectrometry	de
dc.type	doctoralThesis	de
dc.contributor.referee	Urlaub, Henning Prof. Dr.
dc.date.examination	2020-07-06
dc.description.abstracteng	Saccharomyces cerevisiae can generate energy in form of ATP either by fermenting or by respiring nutrition. This depends on the availability of fermentable or non-fermentable carbon sources. The respective energy metabolism of yeast cells affects protein abundances and protein-protein interactions (PPIs). This is also observed in respective mitochondria, organelles in eukaryotic cells that are key players in ATP production under respiratory conditions. Proteins and their abundances can be identified by mass spectrometry (MS). In combination with chemical protein-protein cross-linking, MS is also capable of identifying hundreds of protein-protein cross-links in a single sample (XL-MS). A cross-linker induces a covalent bond between reactive amino acids providing low-resolution information about their proximity to each other under native conditions. In this thesis, protein-protein cross-linking networks in mitochondria isolated from yeast grown either on glucose, a fermentable carbon source, or on glycerol, a non-fermentable carbon source, were elucidated by XL-MS. Mitochondria were treated with the non-cleavable cross-linker BS3 and cross-linked peptides were identified by separate database searches covering the 400 most abundant proteins for each condition. This approach resulted in 386 and 396 uniquely identified protein-protein cross-links in the glucose and the glycerol condition, respectively. Additionally, a quantitative analysis of residue-to-residue cross-links between both conditions was performed by using an isotopically labeled cross-linker. However, differences in protein abundances induced by the carbon sources rather than PPI dynamics were the main driving force for the obtained significant changes. In mitochondria derived from yeast grown on glycerol-containing medium, the internal NADH:ubiquinone oxidoreductase Ndi1 located in the matrix of mitochondria was shown to participate in an Ndi1CIII2CIV2 electron transport chain supercomplex. Also, hitherto undescribed interactions and interactions of uncharacterized proteins were identified. Amongst them, the association of Min8 to the cytochrome c oxidase was revealed. Biochemical experiments further corroborated this interaction and could show that Min8 might be involved in the assembly of Cox12 into an intermediate complex of cytochrome c oxidase. The established cross-linking workflow was also applied to mitoplasts generated from mitochondria of human HEK cells. Here, it could be demonstrated that the use of cross-linkers with different reaction chemistries improves the results by providing complementary protein-protein cross-links.	de
dc.contributor.coReferee	Rehling, Peter Prof. Dr.
dc.subject.eng	Mass spectrometry	de
dc.subject.eng	Cross-linking	de
dc.subject.eng	Protein-protein interactions	de
dc.subject.eng	Yeast	de
dc.subject.eng	Mitochondria	de
dc.identifier.urn	urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-1446-6-0
dc.affiliation.institute	Biologische Fakultät für Biologie und Psychologie	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.identifier.ppn	1726217345