Criticality of the Phosphate Carrier SLC25A3 for Mitochondrial Inorganic Phosphate Uptake to Sustain Striated Muscle Function
Technical Report,30 Sep 2017,29 Sep 2018
Thomas Jefferson University Philadelphia United States
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This project investigates the criticality of the mitochondrial phosphate carrier PiC for oxidative phosphorylation oxphos Aim 1 and buffering of mitochondrial matrix Ca2 Aim 2. Aim 3 focuses on the generation of TAT fusion proteins for the PiC and their ability to rescue phenotypes induced by PiC depletion. During this first year of the project there were accomplishments for each Aim. An accomplishment benefitting all Aims is the development of a new model, namely HEK293T cells with CRSIPR-cas9-mediated PiC knockout. The main accomplishments for Aim 1 were 1 finding that a hypothesized alternative Pi uptake pathway, namely the dicarboxylate transporter DiC, does not compensate for PiC loss in HEK cells, 2 developing a tool to study the role of the DiC in isolated mouse skeletal muscle mitochondria, and 3 that, in opposition to a recent report in the literature showing indirect evidence that PiC is required for copper transport into mitochondria, we find that the PiC is not required for copper transport. Accomplishments towards Aim 2 are 1 the development of a robust experimental design to simultaneously measure mitochondrial matrix free Ca2 as well as Ca2 external to mitochondria in the HEK model and in isolated skeletal muscle mitochondria, and 2 the finding that matrix free Ca2 concentration is elevated when PiC is depleted, supporting an important role for the PiC in matrix Ca2 buffering. Finally, for Aim 3, TAT-PiCA has been generated and will soon be ready for testing in cells. Overall, this project is expected to 1 advance our basic knowledge about a fundamental process, namely how Pi is supplied to mitochondria for certain critical functions, and 2 to generate a potential therapeutic tool that might be useful in the management of a subset of human myopathies.
- Medicine and Medical Research
- Anatomy and Physiology