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 (32)P 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 (32)P was added to energized mitochondria to generate high specific activity gamma-P(32)-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. (32)P 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. (32)P 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 (32)P 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.