Abstract 16664: Cyclophilin D Binds to Tufm and Suppresses Mitochondrial Translation
Objectives: Cyclophilin D (CypD) has been considered a pivotal regulator of the mitochondrial permeability transition pore (mPTP) and also affects protein structure through its peptidyl prolyl isomerase activity. Despite its importance, the CypD protein interactome has not been well characterized. The purpose of this study is to find unequivocal CypD interaction partners and explore the biological implications of the interactions.
Methods: Mitochondrial lysate from WT and CypD KO mice was separated on blue native PAGE (BNP). CyPD-specific and cyclosporin A (CsA)-sensitive complexes on BNP were then sequenced on LC-MS. The identified candidates were overexpressed with tag in HEK cells and co-immunoprecipitation (Co-IP) was used for cross confirmation.
Results: The majority of the CyPD-specific, cyclosporin A (CsA)-sensitive complexes migrated near 400kD on BNP. LC-MS identified Tufm, HSP60, and stress 70 as the main partners in the CypD Complex. Co-IP employing tagged CypD overexpression in HEK cells successfully pulled down all three partners; however, only the Tufm interaction was sensitive to CsA treatment in isolated mitochondria. Immunoblots showed that CypD overexpression significantly suppressed the translation of mitochondrial proteins, as indicated by a decreased abundance of mitochondrially-encoded proteins such as ND6, COXI, COXII and ATP8. This suppression could be reversed by CypD inhibitor, CsA (200 nM) treatment. Tufm was associated with a very large complex on BNP after in vitro treatment of isolated mitochondria with Ca2+.
Conclusions: CypD binds to Tufm and suppresses mitochondrial translation. CypD is actively involved in the regulation of mitochondria respiratory chain function.
Author Disclosures: S. Wang: None. A. Wei: None. Z. Fu: None. J. Ruker: None. B.D. Foster: None. B. O’Rourke: None. J.E. Van Eyk: None.
- © 2014 by American Heart Association, Inc.