Fig. 2
From: Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma
Phe53 substitution with Leu in SDHAF3 leads to impaired function of SDHAF3 in mammalian cells. a SDHAF3 was knocked down in HEK293 cells, using siRNA, to examine effects of SDHAF3 p.Phe53Leu on SDH (succinate hydrogenase) activity. Data shown as mean ± SD (n = 3; **p < 0.05). SDHAF3 interacts with SDHB in vitro. SDHAF3-SDHB complexes pulled down by DDK-tagged SDHAF3, following 24 h co-transfection with GFP-tagged SDHB in HEK293 cells, were immunoblotted for GFP and DDK. Stability of SDHB expression was assessed (whole cell lysates), following 24 h co-transfection with DDK-tagged SDHAF3 and GFP-tagged SDHB in HEK293 cells, when immunoblotted for GFP and GAPDH. b Interaction of wildtype SDHAF3 with wildtype SDHB was evident (Lane 1); complete abrogation of this interaction occurred with SDHB p.Arg242His (Lane 9); reduced interaction was observed for all other mutants (Lanes 3–5, 7–8) with exception of SDHB p.Ile127Ser whose interaction was unaffected. Stable SDHB expression was observed in whole cell lysates. c Introduction of SDHAF3 p.Phe53Leu with wildtype SDHB leads to reduced SDHAF3-SDHB interaction (Lane 1); complete loss of interaction was observed with SDHB p.Arg242His (Lane 9), p.Arg46Gly (Lane 4) and p.Arg46Gln (Lane 5); interaction with SDHB p.Ala43Pro and p.Pro197Arg was unaffected; while binding was enhanced with SDHB p.Ile127Ser and p.Cys101Tyr. Stable SDHB expression was observed in whole cell lysates. d Relative SDH and succinate:quinone oxidoreductase (SQR) activity with Sdh2 R235H in isolated mitochondria (from cells grown until late-log phase in SC media plus 2% raffinose/0.2% glucose). Data shown as mean ± SD (n = 3; **p < 0.05). e Blue native-PAGE analysis of mitochondrial lysates to visualize mature SDH complexes. III2IV2, a supercomplex consisting of complex III and complex IV, as loading control. f Porcine SDH (PDB: 1ZOY). SDHA (gray); B (blue); C (green); D (Brown); Fe-S centers (red and yellow spheres); Ubiquinone in the Q binding site (magenta stick); Arg242, corresponding to Arg235 in yeast (red stick)