1. Moco-dependent localization of SO
Moco-dependent localization of SO. (A,B) Expression of WT-SO in HEK-293 cells (A) and Moco-deficient fibroblasts (B) 48 h after transfection.
Moco-dependent localization of SO. (A,B) Expression of WT-SO in HEK-293 cells (A) and Moco-deficient fibroblasts (B) 48 h after transfection. Cells were stained for SO with an anti-SO antibody (green) and examined by confocal laser scanning microscopy. Mitochondria were stained with MitoTracker Red CMXRos. Representative images of three independent experiments are shown. Scale bars: 10 µm. Cartoons illustrate the status of cofactor saturation and oligomerization: L, leader sequence; H, heme; Mo, Moco; DD, dimerization domain. (C) Quantification of Moco saturation in 100 pmol of purified WT-SO and SO-R367H-K380R variant using HPLC FormA analysis. Error bars represent standard deviations of three independent experiments. (D,E) Size exclusion chromatography of purified WT-SO (D) and SO-R367H-K380R (E) variant eluting mainly as a dimer and monomer, respectively. Red lines illustrate heme absorption at 413 nm. Representative profiles of four independent experiments are shown. (F) Expression of SO-R367H-K380R variant in HEK-293 cells. Transfection and staining was conducted as described for A and B. Scale bar: 10 µm. (G) Pearson correlation coefficients were determined by means of Perkin Elmer's VOLOCITY software. Five images were separately analysed; average and standard deviations are shown. (H) WT and SO-R367H-K380R were expressed in HEK-293 cells for 48 hours. Cytosolic (C) and mitochondrial (M) fractions were separated from each other and total amounts of protein were determined in both fractions. Representative amounts of mitochondrial and cytosolic protein were loaded on a 12% SDS gel for western blotting. Gels were stained for VDAC as a mitochondrial and for gephyrin (GEPH) as a cytosolic marker. Myc-tagged, recombinantly expressed SO was stained using anti-myc antibody. Similar results were obtained in three independent experiments.
Julian M. Klein, and Guenter Schwarz J Cell Sci 2012;125:
© Published by The Company of Biologists Ltd