Mitochondria! participation in cell signaling via reversible phosphorylation cascades is an understudied, yet emerging area. We will capitalize on our mitochondrial expertise to assist in the discovery and characterization of participants of kinase signaling cascades both at the mitochondrial surface (at the outer mitochondrial membrane), as well as within its multiple subcompartments (the intermembrane space, the inner mitochondrial membrane, and the matrix. The mitochondrial core (Core D)will serve the interrelated projects of this application in the following ways: A) provision of purified mitochondria and mitochondrial fractions (outer membrane, intermembrane space, inner membrane, and matrix). These will be probed by Western blotting and used for EM by the Microscopy Core to identify the mitochondrial compartment in which the signaling proteins reside. As well, these fractions can be used as starting materials in further separation protocols for mass spectrometry-based identification by the Beta Cell Proteomics Core of novel kinase-interating proteins. B) assessment of mitochondrial bioenergetic and physiological function in cells and mitochondria with elevated/decreased levels of expression of target proteins. Endpoint measures will include: 1) rates of respiration (maximal rates of phosphorylating and uncoupler-stimulated respiration, levels of uncoupling) on oxidizable substrates that feed into different complexes of the electron transport chain) which can be done on permeabilized cells or isolated mitochondria 2) levels of cellular ATP and ADP, rates of mitochondrial ATP synthesis 3) rates of production of reactive oxygen species in cells or isolated mitochondria
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