MGARP is ultrastructurally located in the inner faces of mitochondrial membranes

Mitochondria, the centers of energy production, are highly organized with inner membranes, cristae and outer membranes. The mitochondrial architecture determines their functions in all cellular processes. Changes in the mitochondrial ultrastructure are tightly related to a wide variety of diseases. MGARP, a mitochondria-localized protein, was predicted by bioinformatics and confirmed by cellular and biochemical methods to be located in mitochondria, but there is no direct and clear evidence for its precise location. This report demonstrates the precise ultrastructural location of MGARP within mitochondria by the ascorbate peroxidase 2 (APEX2) system in combination with electron microscopy (EM). EM revealed that more MGARP is located in the inner/cristae membranes, with its C-terminus at the inner faces of the intramembrane spaces, than in the outer membranes. MGARP overexpression caused both mitochondrial remodeling and cristae shaping, leading to the collapse of the mitochondrial network. The mitochondrial morphologies in MGARP-overexpressing cells were diverse; the cells became round or short, and their cristae were deformed and became discontinuous or circular. An engineered MGARP mutant deficient in its transmembrane domain no longer localized to the mitochondria and lost its effects on mitochondrial structure, confirming that the localization of MGARP in the mitochondria depends on its structural integrity. Collectively, our findings define the location of MGARP within the mitochondria, which is associated with its functional implications for the architecture and organization of mitochondria.