Abstract
Protein phosphorylations, as well as phosphate metabolite binding, are well characterized post-translational mechanisms that regulate enzyme activity in the cytosol, but remain poorly defined in mitochondria. Recently extensive matrix protein phosphorylation sites have been discovered but their functional significance is unclear. Herein we describe methods of using 32P labeling of intact mitochondria to determine the dynamic pools of protein phosphorylation as well as phosphate metabolite association. This screening approach may be useful in not only characterizing the dynamics of these pools, but also provide insight into which phosphorylation sites have a functional significance. Using the mitochondrial ATP synthetic capacity under appropriate conditions, inorganic 32P was added to energized mitochondria to generate high specific activity γ-P32-ATP in the matrix. In general, SDS denaturing and gel electrophoresis was used to primarily follow protein phosphorylation, whereas native gel techniques were used to observe weaker metabolite associations since the structure of mitochondrial complexes was minimally affected. The protein phosphorylation and metabolite association within the matrix was found to be extensive using these approaches. 32P labeling in 2D gels was detected in over 40 proteins, including most of the complexes of the cytochrome chain and proteins associated with intermediary metabolism, biosynthetic pathways, membrane transport, and reactive oxygen species metabolism. 32P pulse-chase experiments further revealed the overall dynamics of these processes that included phosphorylation site turnover as well as phosphate-protein pool size alterations. The high sensitivity of 32P resulted in many proteins being intensely labeled, but not identified due to the sensitivity limitations of mass spectrometry. These low concentration proteins may represent signaling proteins within the matrix. These results demonstrate that the mitochondrial matrix phosphoproteome is both extensive and dynamic. The use of this, in situ, labeling approach is extremely valuable in confirming protein phosphorylation sites as well as examining the dynamics of these processes under near physiological conditions.
| Original language | English |
|---|---|
| Pages (from-to) | 63-80 |
| Number of pages | 18 |
| Journal | Methods in Enzymology |
| Volume | 457 |
| Issue number | B |
| DOIs | |
| State | Published - 2009 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
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Chapter 4 32P Labeling of Protein Phosphorylation and Metabolite Association in the Mitochondria Matrix. / Aponte, Angel M.; Phillips, Darci; Harris, Robert; Blinova, Ksenia; French, Stephanie; Johnson, D. Thor; Balaban, Robert S.
In: Methods in Enzymology, Vol. 457, No. B, 2009, p. 63-80.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Chapter 4 32P Labeling of Protein Phosphorylation and Metabolite Association in the Mitochondria Matrix
AU - Aponte, Angel M.
AU - Phillips, Darci
AU - Harris, Robert
AU - Blinova, Ksenia
AU - French, Stephanie
AU - Johnson, D. Thor
AU - Balaban, Robert S.
PY - 2009
Y1 - 2009
N2 - Protein phosphorylations, as well as phosphate metabolite binding, are well characterized post-translational mechanisms that regulate enzyme activity in the cytosol, but remain poorly defined in mitochondria. Recently extensive matrix protein phosphorylation sites have been discovered but their functional significance is unclear. Herein we describe methods of using 32P labeling of intact mitochondria to determine the dynamic pools of protein phosphorylation as well as phosphate metabolite association. This screening approach may be useful in not only characterizing the dynamics of these pools, but also provide insight into which phosphorylation sites have a functional significance. Using the mitochondrial ATP synthetic capacity under appropriate conditions, inorganic 32P was added to energized mitochondria to generate high specific activity γ-P32-ATP in the matrix. In general, SDS denaturing and gel electrophoresis was used to primarily follow protein phosphorylation, whereas native gel techniques were used to observe weaker metabolite associations since the structure of mitochondrial complexes was minimally affected. The protein phosphorylation and metabolite association within the matrix was found to be extensive using these approaches. 32P labeling in 2D gels was detected in over 40 proteins, including most of the complexes of the cytochrome chain and proteins associated with intermediary metabolism, biosynthetic pathways, membrane transport, and reactive oxygen species metabolism. 32P pulse-chase experiments further revealed the overall dynamics of these processes that included phosphorylation site turnover as well as phosphate-protein pool size alterations. The high sensitivity of 32P resulted in many proteins being intensely labeled, but not identified due to the sensitivity limitations of mass spectrometry. These low concentration proteins may represent signaling proteins within the matrix. These results demonstrate that the mitochondrial matrix phosphoproteome is both extensive and dynamic. The use of this, in situ, labeling approach is extremely valuable in confirming protein phosphorylation sites as well as examining the dynamics of these processes under near physiological conditions.
AB - Protein phosphorylations, as well as phosphate metabolite binding, are well characterized post-translational mechanisms that regulate enzyme activity in the cytosol, but remain poorly defined in mitochondria. Recently extensive matrix protein phosphorylation sites have been discovered but their functional significance is unclear. Herein we describe methods of using 32P labeling of intact mitochondria to determine the dynamic pools of protein phosphorylation as well as phosphate metabolite association. This screening approach may be useful in not only characterizing the dynamics of these pools, but also provide insight into which phosphorylation sites have a functional significance. Using the mitochondrial ATP synthetic capacity under appropriate conditions, inorganic 32P was added to energized mitochondria to generate high specific activity γ-P32-ATP in the matrix. In general, SDS denaturing and gel electrophoresis was used to primarily follow protein phosphorylation, whereas native gel techniques were used to observe weaker metabolite associations since the structure of mitochondrial complexes was minimally affected. The protein phosphorylation and metabolite association within the matrix was found to be extensive using these approaches. 32P labeling in 2D gels was detected in over 40 proteins, including most of the complexes of the cytochrome chain and proteins associated with intermediary metabolism, biosynthetic pathways, membrane transport, and reactive oxygen species metabolism. 32P pulse-chase experiments further revealed the overall dynamics of these processes that included phosphorylation site turnover as well as phosphate-protein pool size alterations. The high sensitivity of 32P resulted in many proteins being intensely labeled, but not identified due to the sensitivity limitations of mass spectrometry. These low concentration proteins may represent signaling proteins within the matrix. These results demonstrate that the mitochondrial matrix phosphoproteome is both extensive and dynamic. The use of this, in situ, labeling approach is extremely valuable in confirming protein phosphorylation sites as well as examining the dynamics of these processes under near physiological conditions.
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UR - http://www.scopus.com/inward/citedby.url?scp=65449171986&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(09)05004-6
DO - 10.1016/S0076-6879(09)05004-6
M3 - Article
VL - 457
SP - 63
EP - 80
JO - ImmunoMethods
JF - ImmunoMethods
SN - 1046-2023
IS - B
ER -