Mitochondria are central to energy homeostasis and numerous signaling pathways. Thus, it is no surprise that mitochondrial dysfunction has severe consequences, contributing to a wide range of aging-related pathologies, including insulin resistance, diabetes and neurodegenerative syndromes. Determining the basic cellular and molecular mechanisms that regulate mitochondrial function is pivotal in understanding and eventually treating diseases related to mitochondrial dysfunction. Expression patterns, knockout phenotypes and recently identified gene targets implicate Estrogen-Related Receptor (ERR) in the regulation of mitochondrial function. As an orphan receptor with no known ligand, resolving the physiological significance of ERR requires in-depth understanding of the signals and processes regulating its activity. The long-term goals of this project are to elucidate the pathways and mechanisms that regulate the activity of ERRp and determine how this regulation relates to mitochondrial function. Specifically, this project will seek to identify proteins and pathways involved in the activation of ERR, as well as the basic mechanisms of this activation. To this end, the specific aims of this project are: 1) elucidation of the molecules and pathways involved in regulation of ERRP activity and 2) identification of the protein modifications contributing to this regulation. To maximize the chances of discovering significant regulators, both an unbiased cDNA genome-wide library plus a pool of potential co-regulators will be screened in an ERRP activity assay. To identify post-translational modifications that regulate ERR, and in particular phosphorylations, mass spectrometry will be used in combination with point mutagenesis to develop a comprehensive phosphorylation map and to determine the sites that affect ERR activity. Determining the molecular mechanisms by which ERR is regulated will provide insight into the specific physiological role of this orphan nuclear receptor as it relates to mitochondrial function and dysfunction. Relevance: Aging-related pathologies, including insulin resistance, diabetes and neuromuscular degenerative syndromes affect a huge proportion of the population and therefore significantly impact public health. Defects in mitochondrial function are thought to contribute to these pathologies. Understanding the basic biological mechanisms regulating mitochondrial function is essential for understanding how dysfunction arises, how this translates to disease, and how dysfunction can be treated and ultimately prevented.