Fig. 2: COASY, the CoA biosynthesis enzyme, is primarily cytosolic.
a, Schematic of putative pathways supporting the mitochondrial CoA pool: COASY-mediated de novo biosynthesis or transporter-mediated mitochondrial CoA uptake. b, Western blot of COASY from whole-cell lysate (0.1 million cells) or mitochondrial fraction (ten million cells, 100× enrichment) from K562 cells expressing HA-MITO tag. c, Anti-FLAG and anti-COASY western blots of ectopically expressed C-terminal FLAG-tagged COASYα, COASYβ and endogenous COASY from whole-cell lysate and mitochondrial fraction (~20× enrichment) from K562 cells expressing HA-MITO tag. SHMT2 and VDAC, mitochondrial markers; GAPDH, cytosolic protein marker. d, Anti-FLAG immunofluorescence staining of HeLa cells expressing 3×HA–EGFP–OMP25 (HA-MITO tag) and FLAG-tagged COASYα. Magnified view of framed areas is shown on the right. Scale bars, 5 μm. e, COASY isoform expression from human tissues with major transcripts (bottom) annotated by translation start site (arrows). Data were obtained from the GTEx portal, GTEx Analysis Release v.10 and dbGaP accession number phs000424.v10.p2. Data in b, c and d are representative of experiments that were independently repeated at least two times with similar results.
