Use of 32P to study dynamics of the mitochondrial phosphoproteome

Angel M. Aponte, Darci Phillips, Rachel K. Hopper, D. Thor Johnson, Robert A. Harris, Ksenia Blinova, Emily S. Boja, Stephanie French, Robert S. Balaban

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

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 32P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The 32P 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 32P labeling. The high sensitivity of 32P 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 32P labeling in the intact mitochondria, and revealed 32P-incorporation for the α, β, γ, 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.

Original languageEnglish (US)
Pages (from-to)2679-2695
Number of pages17
JournalJournal of Proteome Research
Volume8
Issue number6
DOIs
StatePublished - Jun 5 2009

Keywords

  • 2D gel electrophoresis
  • P
  • Complex I
  • Complex V
  • Mitochondria
  • Phosphate-metabolite association
  • Protein phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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    Aponte, A. M., Phillips, D., Hopper, R. K., Johnson, D. T., Harris, R. A., Blinova, K., Boja, E. S., French, S., & Balaban, R. S. (2009). Use of 32P to study dynamics of the mitochondrial phosphoproteome. Journal of Proteome Research, 8(6), 2679-2695. https://doi.org/10.1021/pr800913j