Protein Replacement Therapy for Mitochondrial-Membrane proteins: Phosphate Carrier (mPiC) as a model system

The mitochondrial phosphate carrier (mPiC) encoded by the nuclear gene SLC25A3, is synthesized with an N-terminus mitochondrial targeting sequence (MTS), thus enabling its import and processing into mature active protein within the mitochondrial inner membrane (mIM).

PiC, as a component of the ATP Synthasome, imports inorganic phosphate (P_i) to the mitochondrial matrix for two main roles: 1) Producing ATP, 2) As a regulatory factor for mitochondrial Ca²⁺ uptake. PiC also imports copper (Cu), crucial to COX subunit holoenzyme assembly.

Mitochondrial phosphate carrier deficiency (MPCD) is a rare disease. Patients with MPCD die usually within the first year of life.

Medicine nowadays offers no cure for mitochondrial disorders. Our aim is to test if PiC can be delivered to cells in the form of a TAT-fusion protein. The TAT peptide is used for the delivery of proteins across biological membranes.

We designed, produced and partially purified the TAT-MTS-PiC fusion protein. The fused protein is delivered into the mitochondria and localizes within the mIM, its natural cellular location, and as a processed protein.

Treatment of KD-PiC cells (siRNA-PiC) with TAT-MTS-PiC fusion protein increased cell growth and improves bioenergentic capabilities as measured by oxygen consumption rate, ATP production and reduction in lactate secretion. Most importantly, TAT-MTS-PiC restores Pi and Cu transport into the mitochondrial matrix.

This is the first mitochondrial-membrane protein that is designed and delivered into cells, using the TAT-fusion protein system. TAT-mediated replenishment of PiC could be an early treatment strategy in newborns reported vulnerability to crises when PiC is deficient.