Abstract
Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.
Highlights
Succinate dehydrogenase is a tetrameric, mitochondrial membrane protein needed for respiratory energy generation
Comparison of Paralogs with Wild Type Subunits—Shh3p shows high sequence identity (57%) with Sdh3p (Fig. 1A), including the presence of three transmembrane segments predicted by TopPred [32] and the presence of a mitochondrial targeting sequence predicted by MitoProt II [33]
Three Sdh4p residues that we have previously proposed to be part of a distal quinone-binding site, Phe-100, Ser-102, and Lys-163, are conserved in Shh4p, but only the Ser and the Lys are found in Tim18p [27]
Summary
Succinate dehydrogenase is a tetrameric, mitochondrial membrane protein needed for respiratory energy generation. Results: Yeast succinate dehydrogenase containing Sdh3p and Sdh4p paralogs is catalytically active. Significance: At least four functional succinate dehydrogenase isoenzymes containing zero, one, or two paralogs can be expressed in yeast mitochondria. Succinate dehydrogenase (SDH), known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). The Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex
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