Mutational analysis of the role of conserved histidine residues of the SDHC subunit of Complex II

Background : SDHC protein is a critical component of the succinate-ubiquinone oxidoreductase also known as respiratory chain Complex II. Because SDHC mutations are associated with several human diseases, a detailed understanding of the role of SDHC in Complex II and overall cellular physiology is important. In this study, we have tested the functional complementation of SDHC-null cells with different variants of the SDHC proteins fused with GFP at the C-terminus. Specifically, we determined the role of four conserved histidines (H55, H71, H127, H134) in SDHC on Complex II function and mitochondrial morphology. Methods : The SDHC-deficient Chinese hamster cell line CCL16-B9 was transfected with the plasmid constructs expressing the SDHC variants (SDHC, SDHC∆, SDHC-H55N, SDHC-H71N, SDHC-H127N, SDHC-H134N) fused with either GFP or HA-GFP at the C-terminus. Restoration of Complex II function in the transfected CCL16-B9 cells by the SDHC variants was tested by galactose selection, biochemical assays, Western blotting, and the assessment of mitochondrial morphology by fluorescence microscopy. Results : The key findings of this study are: (1) The GFP at the C-terminus does not interfere with SDHC import, orientation, and function. (2) Wild type and mutant SDHCs can deliver GFP or HA-GFP into the IMS. (3) The third transmembrane domain is essential for SDHC function. (4) While H55, and H71are non-essential, the H127 and H134 are essential for SDHC function. The H127N and H134N mutations impair Complex II function and alter mitochondrial morphology. The most detrimental effects are due to H127N mutation. (5) SDHC can serve as a carrier for delivering the proteins of interest into IMS. Conclusions : This study advances our understanding of the role of SDHC subunit in Complex II, mitochondrial morphology, and the overall cellular physiology.