Monitoring checkpoints of metabolism and protein biogenesis in mitochondria by Phos-tag technology.

A role for reversible phosphorylation in regulation of mitochondrial proteins has been neglected for a long time. Particularly, the import machineries that mediate influx of more than 1000 different precursor proteins into the organelle were considered as predominantly constitutively active entities. Only recently, a combination of advanced phosphoproteomic approaches and Phos-tag technology enabled the discovery of several phosphorylation sites at the translocase of the outer membrane TOM and the identification of cellular signalling cascades that allow dynamic adaptation of the protein influx into mitochondria upon changing cellular demands. Here, we present a protocol that allows biochemical and semi-quantitative profiling of intra-mitochondrial protein phosphorylation. We exemplify this with the pyruvate dehydrogenase complex (PDH), which serves as a central metabolic switch in energy metabolism that is based on reversible phosphorylation. Phos-tag technology allows rapid monitoring of the metabolic state via simultaneous detection of phosphorylated and non-phosphorylated species of the PDH core component Pda1. Our protocol can be applied for several further intra-organellar proteins like respiratory chain complexes or protein translocases of the inner membrane. SIGNIFICANCE: Our manuscript describes for the first time how Phos-tag technology can be applied to monitor phosphorylation of intramitochondrial proteins. We exemplify this with the regulation of the pyruvate dehydrogenase complex as central regulatory switch in energy metabolism. We show that our protocol allows a rapid monitoring of the metabolic state of the cell (phosphorylated PDH is inactive while non-phosphorylated PDH is active) and can be applied for rapid profiling of different metabolic conditions as well as for profiling phosphorylation of further intramitochondrial protein (complexes).