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Aponte AM, Phillips D, Hopper RK, Johnson DT, Harris RA, Blinova K, Boja ES, French S, Balaban RS: Use of (32) P to study dynamics of the mitochondrial phosphoproteome. J Proteome Res. 2009 Jun;8(6):2679-95.
Protein phosphorylation is a well-characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of (32) P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The (32) P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with (32) P labeling. The high sensitivity of (32) P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using nondisruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after (32) P labeling in the intact mitochondria, and revealed (32) P-incorporation for the alpha, beta, gamma, OSCP, and d subunits in Complex V and the 75, 51, 42, 23, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver. |
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