The Carboxyl Terminus of the Saccharomyces cerevisiaeSuccinate Dehydrogenase Membrane Subunit, SDH4p, Is Necessary for Ubiquinone Reduction and Enzyme Stability

Kayode S Oyedotun,Bernard D Lemire

doi:10.1074/jbc.272.50.31382

Abstract

The succinate dehydrogenase (SDH) of Saccharomyces cerevisiae is composed of four nonidentical subunits encoded by the nuclear genes SDH1, SDH2, SDH3, and SDH4. The hydrophilic subunits, SDH1p and SDH2p, comprise the catalytic domain involved in succinate oxidation. They are anchored to the inner mitochondrial membrane by two small, hydrophobic subunits, SDH3p and SDH4p, which are required for electron transfer and ubiquinone reduction. Comparison of the deduced primary sequence of the yeast SDH4p subunit to SDH4p subunits from other species reveals the presence of an unusual 25-30 amino acid carboxyl-terminal extension following the last predicted transmembrane domain. The extension is predicted to be on the cytoplasmic side of the inner mitochondrial membrane. To investigate the extension's function, three truncations were created and characterized. The results reveal that the carboxyl-terminal extension is necessary for respiration and growth on nonfermentable carbon sources, for ubiquinone reduction, and for enzyme stability. Combined with inhibitor studies using a ubiquinone analog, our results suggest that the extension and more specifically, residues 128-135 are involved in the formation of a ubiquinone binding site. Our findings support a two-ubiquinone binding site model for the S. cerevisiae SDH.

Highlights

Succinate dehydrogenase (SDH)1 and fumarate reductase (FRD) form a family of highly conserved and functionally related respiratory chain proteins
In Vivo Characterization of SDH4 Carboxyl-terminal Deletion Mutants—The presence of the unusual C-terminal extension in the yeast SDH4p sequence (Fig. 1) prompted us to determine whether this extension is required for proper enzyme function
These data indicate that the SDH4 carboxyl-terminal extension is essential for SDH function in vivo

Summary

EXPERIMENTAL PROCEDURES

Strains and Media—The parental yeast strain, MH125 and the Escherichia coli strain, DH5␣, have been described previously [12]. sdh4W2 (MH125; SDH4⌬-1::TRP1) was constructed by replacing the 0.8-kilobase XhoI to SpeI fragment containing the entire SDH4 open reading frame with the TRP1 gene by targeted gene disruption in MH125. For the preparation of mitochondria, cultures were grown in a semisynthetic galactose to late logarithmic phase (A600 about 3), harvested, and lysed enzymatically as described [10]. This paper is available on line at http://www.jbc.org plasmid pSDH4 –17 as template [10], and the following oligonucleotides, 5Ј-TACTTCTAGACGTCGACACCATCATTCTCGGTTTC-3Ј, 5Ј-TACTTCTAGACCCATAGACTTTTTACTAAACC-3Ј, and 5Ј-TACTTCTAGACTTTCTCGGAAGAATCCC-3Ј, stop codons, encoded within the XbaI restriction sites (underlined), were introduced into the SDH4 gene following the Val-127, Trp-135, and Lys-140 codons (Fig. 1) to produce the SDH4⌬C23, SDH4⌬C15, and SDH4⌬C10 constructs, respectively. When the secondary plots were linear, as for the SDH4⌬C23 enzyme, suggesting simple, linear, non-competitive inhibition, the hypothesis was confirmed by performing a non-linear least squares fit to Equation 1,. Miscellaneous Methods—Measurements of covalently bound flavin have been described [10]

RESULTS

TABLE I Respiratory chain activities of mitochondrial membranes

TABLE II Apparent Michaelis constants for quinone reduction

TABLE III Apparent inhibition constants for quinone reduction

DISCUSSION