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

Of the [~]1100 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 the vitamin B12 chaperone MMADHC and cytosolic B12-dependent methionine synthase (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. Significance StatementHumans have only two vitamin B12-dependent enzymes - mitochondrial MMUT and cytosolic MTR - and both require a common B12 chaperone MMADHC. We discover that MMADHC is a low abundant, short-lived protein that is continuously imported and degraded by energized mitochondria. Upon collapse of the mitochondrial proton motive force, MMADHC accumulates in the cytosol and increases the levels and activity of MTR, critical for one-carbon metabolism. This PMF-dependent regulation of MMADHC stability and localization is important for understanding cofactor rationing and spatiotemporal compartmentalization of B12 metabolism.