Prohibitin controls MRS2-Mediated mitochondrial Mg²⁺ uptake to Shape bioenergetics

Mitochondrial magnesium (_mMg²⁺) is essential for cellular metabolism and bioenergetics, yet the mechanisms governing its transport remain poorly understood. Although MRS2 constitutes the pore of the _mMg²⁺ channel, the molecular machinery regulating its function is unknown. Here, unbiased proteomics identified the prohibitin (PHB) complex as a prominent MRS2-interacting partner. Integrated biochemical and functional analyses demonstrate that the conserved coiled-coil domain mediates MRS2 homo-oligomerization, whereas the C-terminal region of MRS2 interacts with PHB1 to promote channel activity. Quantitative calibration of mitochondria-targeted MagFRET sensors revealed maximal _mMg²⁺ uptake (∼15 mM), which was markedly reduced in Phb1-deficient hepatocytes. Complementary loss- and gain-of-function studies establish PHB1 as a positive regulator of MRS2-mediated _mMg²⁺ uptake without affecting MCU-dependent Ca²⁺ transport. In vivo, hepatic Phb1 deletion attenuated _mMg²⁺ uptake and enhanced cellular bioenergetics. These findings identify PHB1 as an activator of the MRS2, advancing our understanding of _mMg²⁺ uptake machinery and its role in metabolic regulation.