Abstract
Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.
Highlights
Ndi1 participates in a CIII2CIV2 respiratory supercomplex. Min8 promotes assembly of Cox12 into an intermediate complex IV
Mitochondria were lysed, proteins digested, and cross-linked peptides enriched by peptide size exclusion chromatography, peptide SEC fractions containing cross-links were analyzed by LC-Mass spectrometry (MS)/MS and searched against a dedicated database with pLink 1 [60, 61]
The majority of quantified peptide residue pairs remained unchanged, we found that ϳ72% of cross-linked proteins with quantified residue pairs showing a fold change Ն 2 belonged either to the inner membrane (IM) or to the matrix, regardless of the cross-linker
Summary
Protein interactions in mitochondria isolated from S. cerevisiae grown on glycerol or glucose medium were analyzed by XLMS. We cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. Mass spectrometry (MS)-based proteomics have allowed the identification, localization, and quantitation of mitochondrial proteins in various species [7,8,9,10,11,12,13] and revealed their post-translational modifications [14, 15] Based on these studies, mitochondria contain ϳ1000 proteins in yeast [16] and up to 1500 in mammals [17]. In a very recent study of yeast mitochondria an enrichable, MS-cleavable and stable-isotope labeled cross-linker was used to investigate protein-protein interactions [50]. We identified the so far uncharacterized protein Min as a new constituent of CIV
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