Abstract
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
Highlights
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts
Identification of Two Proteins Co-migrating with Bovine ATP Synthase in 1-D Blue Native Gels—Digitonin-solubilized ATP synthase from mammalian mitochondria is a mixture of oligomeric forms [20, 21]
The MLQ protein had been described as 6.8-kDa mitochondrial proteolipid due to its extractability by chloroform/methanol from mitochondrial membranes [29]
Summary
Materials—6-Aminohexanoic acid, imidazole, and digitonin (catalog number 37006, purity Ͼ50%) were obtained from Fluka. Peaks present in almost all MS spectra were defined as contaminants even if the peaks could not be classified as trypsin peaks, matrix clusters, or other laboratory-specific contaminants like keratin These peaks were removed from the mass list prior to database search (background peak list, Supplemental Table S3). MS/MS spectra were processed and searched except that the signal-to-noise threshold for the monoisotopic labeling was 6 and that the maximal number of allowed peaks was 50 (see Supplemental Table S6C). These strict settings for the peak labeling ensure a high quality of the mass lists submitted to the database search. ClustalW version 1.83 (www.expasy. org) was used for direct alignments of protein sequences
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