Mitochondrial depolarization stabilizes the vitamin B₁₂ chaperone MMADHC in the cytosol to increase MTR activity

Of the ~1,100 mitochondrial proteins, only a handful like PINK1 and ATFS-1 are known to stabilize and relocalize upon collapse of the proton motive force (PMF) to execute signaling roles. To systematically identify genes that increase exclusively at the protein level upon PMF collapse, we performed a joint proteomic and RNA-seq screen. The screen revealed 10 candidates (six mitochondrial), including two genes in vitamin B₁₂ metabolism - the B₁₂ chaperone MMADHC and cytosolic B₁₂-dependent 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR). MMADHC is short-lived across cell types and we show that its levels increase with PMF collapse. MMADHC stabilization precedes PINK1 activation in a time course of increasing mtDNA depletion, suggesting greater sensitivity to PMF collapse. MMADHC accumulates in mitochondria with LONP1 inhibition but in the cytosol upon PMF collapse, likely due to mitochondrial import failure. Cytosol-stabilized MMADHC increases MTR levels and activity. Altogether, the mitochondrial PMF regulates the cytosolic B₁₂-dependent MTR, integral to one-carbon metabolism, by controlling the stability and compartmentalization of the B₁₂ chaperone MMADHC.