TYMS and DHFRL1 localize to mitochondria. TYMS-GFP fusion proteins (A) and DHFRL1-GFP fusion proteins (B) were expressed in HeLa cells, as well as a mitochondrial marker mito-CFP. (A) TYMS-GFP localizes to the nucleus, cytoplasm, and colocalizes with the mito-CFP mitochondrial marker. (B) DHFRL1-GFP localizes exclusively to mitochondria. (C) TYMS, DHFRL1, and SHMT2 proteins localize to the matrix and inner membrane of fractionated HepG2 cells. Lane 1, mitochondrial extract; lane 2, outer membrane fraction; lane 3, inner membrane space; lane 4, matrix; lane 5, inner membrane. Hsp60 is the inner membrane/matrix marker, Cox I is an inner membrane marker, and Bcl-xS/L is an outer membrane marker.