Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondria are dynamically modified organelles that perform several vital functions in the eukaryotic cell. In addition to their role in energy metabolism, mitochondria are also involved in intermediary metabolism, biochemical synthesis, calcium signaling, redox regulation, and apoptosis 1,2. The complexity of mitochondria requires a sophisticated system of intracellular communication, capable of responding rapidly to changes in cellular energy-metabolism as well as many other processes. In recent years, reversible protein phosphorylation has emerged as a potential ubiquitous regulatory mechanism in mitochondria. Several proteomic screening studies, using a combination of SDS gel electrophoresis and mass spectrometry, have revealed an extensive network of phosphoproteins in mitochondria 3–10. Additionally, our lab recently coupled 32P-labeling in intact mitochondria with 2D gel electrophoresis to identify dozens of mitochondrial proteins with potential regulatory (i.e., 32P-turnover) phosphorylation sites 3,5. While data continues to accumulate for the functionality of the mitochondrial phosphoproteome 5,11,12, the exact mechanisms governing reversible phosphorylation remain poorly defined for a majority of mitochondrial proteins. In fact, unlike pyruvate dehydrogenase (PDH) and branched chain α-ketoacid dehydrogenase (BCKDH) complexes, which have their own matrix-localized kinases 13,14, most mitochondrial phosphoproteins have not been linked to specific kinases. To date, no experimental studies have directly examined the localization of active kinases to mitochondria. protein complexes outside of the PDH complex. Thus, identifying the matrix kinases remains an important challenge for resolving the mechanism and regulation of mitochondrial phosphorylation events. The relatively low abundance of mitochondrial kinases in complex mixtures limits the usefulness of mass spectrometry and gel electrophoresis approaches for detecting these proteins. That is, the ratio of target protein to kinase is on the order of 10 to 100 or even greater, resulting in a very low concentration of kinase protein. Furthermore, current techniques are unable to distinguish mitochondrial kinases from co-purifying contaminants (i.e., cytosolic proteins associated with the mitochondrial outer-membrane). This is especially problematical when a kinase is present in both the cytosol and mitochondrial matrix, as this makes enhancement via mitochondrial purification difficult to interpret 15, 16. For instance, more than ~30% of the heart’s cellular protein is mitochondrial, which means that the enhancement ratio can only approach 4 under the best of conditions. To better screen for active mitochondrial kinases, we coupled direct ATP-dependent 32P-labeling in isolated protein complexes with blue native gel electrophoresis (BN)-PAGE. Specifically, the non-denaturing conditions of BN-PAGE were used to resolve mitochondrial enzyme complexes in their near native conformation 17 in the pig and rat heart mitochondria. To screen for protein phosphorylation events, these mitochondrial complexes were then incubated in vitro with γ-32P-ATP and resolved into individual protein subunits by SDS gel electrophoresis (2D BN/SDS-PAGE). Autoradiography of the 2D BN/SDS-PAGEs directly evaluated protein phosphorylation and other covalent modifications, since the presence of SDS removed a majority of weak metabolite associations. This study revealed dozens of kinase-mediated phosphorylations for the proteins associated with all five complexes of oxidative phosphorylation However, despite this extensive network of mitochondrial protein phosphorylation, few kinases—with the exception of the well characterized mitochondrial PDH kinases—were detected. These data suggest a significant role for autophosphorylation within the mitochondrial oxidative phosphorylation protein complexes.