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. 2011 Jan;10(1):M110.000299.
doi: 10.1074/mcp.M110.000299. Epub 2010 Sep 10.

Phosphoproteome Analysis of Functional Mitochondria Isolated From Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

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Free PMC article

Phosphoproteome Analysis of Functional Mitochondria Isolated From Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Xiaolu Zhao et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.

Figures

Fig. 1.
Fig. 1.
Flow chart for analysis of human in vivo skeletal muscle mitochondrial proteome and phosphoproteome.
Fig. 2.
Fig. 2.
Venn diagram of identified non-redundant mitochondrial phosphopeptides (A) and distinct phosphorylated sites (B) from five subjects by four different phosphopeptide enrichment approaches. b, batch mode; m, microcolumn mode.
Fig. 3.
Fig. 3.
Examples of multiply phosphorylated mitochondrial proteins with indication of distinct identifications by four different phosphopeptide enrichment approaches.
Fig. 4.
Fig. 4.
Functional classification of all identified human mitochondrial phosphoproteins.
Fig. 5.
Fig. 5.
Schematic of mitochondrial respiration chain and TCA cycle and identified phosphoproteins involved in these two processes.“●” indicates the number of site-specific phosphorylations.
Fig. 6.
Fig. 6.
Prediction of kinase family for all identified distinct phosphorylation sites by NetworKIN. CKI, casein kinase I; EGFR, EGF receptor.

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