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Abstract
Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part. Recent studies indicated that the number of mitochondrial phosphoproteins is higher than expected; however, the effect of reversible phosphorylation on mitochondrial structure and function has only been defined in a few cases. It is thus crucial to determine authentic protein phosphorylation sites from highly purified mitochondria in a genetically tractable organism. The yeast Saccharomyces cerevisiae is a major model organism for the analysis of mitochondrial functions. We isolated highly pure yeast mitochondria and performed a systematic analysis of phosphorylation sites by a combination of different enrichment strategies and mass spectrometry. We identified 80 phosphorylation sites in 48 different proteins. These mitochondrial phosphoproteins are involved in critical mitochondrial functions, including energy metabolism, protein biogenesis, fatty acid metabolism, metabolite transport, and redox regulation. By combining yeast genetics and in vitro biochemical analysis, we found that phosphorylation of a serine residue in subunit g (Atp20) regulates dimerization of the mitochondrial ATP synthase. The authentic phosphoproteome of yeast mitochondria will represent a rich source to uncover novel roles of reversible protein phosphorylation.
Highlights
Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part
A limitation of these approaches is the inability to identify the specific phosphorylated amino acid residues in addition to the possibility of nonspecific labeling of proteins. (ii) Proteomics analysis of isolated mitochondria by mass spectrometry revealed the presence of numerous protein kinases and phosphatases (28 –34), implying that reversible protein phosphorylation may be a widespread mechanism of regulating mitochondrial function
Identification of Phosphorylation Sites of Yeast Mitochondrial Proteins—S. cerevisiae mitochondria of very high purity were obtained by differential centrifugation and subsequent sucrose gradient centrifugation [28, 34, 48, 49]
Summary
Isolation of Yeast Mitochondria—The wild-type yeast strain YPH499 was grown on non-fermentable medium (YPG: 1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 3% (w/v) glycerol) at 30 °C to an OD of approximately 1. Cells were homogenized in the presence of phosphatase inhibitor mixtures I and II (Sigma-Aldrich). Crude mitochondria were isolated by differential centrifugation and further purified via a three-step sucrose gradient as described previously [28, 34, 48, 49]. Purified mitochondria were resuspended in SE buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2) and stored in aliquots at Ϫ80 °C. IMAC Enrichment after SDS-PAGE—Highly purified mitochondria (400 g of protein) were separated on a BisTris SDS-PAGE system
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