Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase.
Succinate dehydrogenase (EC 1.3.99.1) is an intrinsic bacterial or inner mitochondrial membrane protein that catalyses the oxidation of succinate and donates electrons to the respiratory chain via quinone acceptors. It is a heterotetramer composed of a flavoprotein, an iron-sulfur, and two hydrophobic subunits. We purified succinate dehydrogenase by blue native gel electrophoresis, determined the amino-terminal sequence of the Sdh4p subunit and used this information to clone the SDH4 gene. It encodes a precursor protein of 181 amino acids that is converted to the 150-amino acid mature Sdh4p protein with a mass of 16,638 Da. Hydrophobicity analysis predicts that Sdh4p forms three transmembrane alpha-helices. We have constructed an SDH4 mutant by targeted gene disruption; it retains the ability to grow on rich glycerol medium. Western blot analysis of SDH4 disruption mutant membrane fractions indicates that membrane attachment of the flavoprotein and iron-sulfur subunits is impaired but not abolished. This membrane-bound enzyme is able to reduce ubiquinone, although less efficiently than the wild-type enzyme. These findings indicate that Sdh4p contributes both to the membrane attachment of the catalytic flavoprotein and iron-sulfur subunits and to electron transfer to ubiquinone.
Bullis
B L
BL
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Lemire
B D
BD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000201:enzymology; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013385:Succinate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D004271:DNA, Fungal; D046911:Macromolecular Substances; D013385:Succinate Dehydrogenase