Fig 2: Cytosol-stabilized MMADHC increases MTR protein and activity(A) Scatter plot of fold change in protein levels of MMADHC KO (sg1, sg2 combined) vs control (sgOR7G3 CRISPR cutting control) cells treated with antimycin and oligomycin (anti+oligo) for three days and analyzed by TMT proteomics. Average fold from two replicates for control (sgOR7G3) and four replicates across two separate sgRNAs for MMADHC are plotted.(B) Bar plots of MMADHC and MTR levels from the experiment shown in (A)(C) Schematic of MTR activity assay in cell extracts. MTR specific activity measured in extracts of K562 and HEK293 cells treated with or without antimycin and oligomycin for three days. Error bars represent standard deviation across three biological replicates.(D) Schematic of deuterated formate tracing. Quantification of MTR substrate homocysteine and product methionine (unlabeled and deuterated) by mass spectrometry in K562 cells treated with or without antimycin and oligomycin for three days and labelled with fully deuterated formate in the presence or absence of vitamin B12 for 9 hours. Error bars represent standard deviation across four biological replicates. (E) Western blot analysis of MMADHC in total lysate, cytosolic, and mitochondrial fractions of HEK293 cells transiently over-expressing MMADHC lacking the first 38 a.a. (MMADHCΔN38). Blot is representative of three biological replicates.(F) Western blot analysis of MMADHC and MTR in HEK293 cells stably over-expressing empty vector or MMADHCΔN38. Total protein is stained with Ponceau for loading control. Bar plots on the right show western blot quantification, error bars represent standard deviation across five biological replicates.(G) MTR specific activity measured as in ‘C’ in HEK293 cells transiently over-expressing empty vector or MMADHCΔN38. Error bars represent standard deviation across three biological replicates.

Fig 3: Model for the regulation of MMADHC by the mitochondrial proton motive force and LONP1(A) Context-dependent MMADHC abundance and localization: MMADHC is present at very low steady state levels in cells with healthy mitochondria but accumulates in mitochondria with LONP1 inhibition and in the cytosol with mitochondrial depolarization.(B) MMADHC is stabilized earlier than PINK1 upon PMF collapse and accumulates in the cytosol where it boosts the levels and activity of the B12-dependent MTR.