Abstract
Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.
Highlights
Protein import into the mitochondria has been an area of intense study, relatively little is known regarding their cofactor insertion and oligomerization into multisubunit complexes
To reconstruct the entire SDH1 gene, pSDH1 was digested with NdeI, the ends were blunted, and it was cut with EcoRI, and the NdeI/EcoRI fragment encoding the Fp carboxyl terminus was cloned into EcoRI- and SmaI-digested pSfR1
The amount of FAD-modified Fp can be expressed as the amount of Fp immunoprecipitated by the anti-FAD serum divided by the amount of Fp immunoprecipitated by the anti-Fp serum
Summary
Protein import into the mitochondria has been an area of intense study, relatively little is known regarding their cofactor insertion and oligomerization into multisubunit complexes. Most SDH and fumarate reductase enzymes are composed of four nonidentical subunits: a flavoprotein (Fp) of about 70 kDa, an iron-sulfur protein (Ip) of about 30 kDa, and two hydrophobic anchoring subunits of 7–17 kDa. The Fp contains the active site and the unusual cofactor, an 8␣-N(3)histidyl-FAD linked at a conserved histidine residue. The SDH subunits are translated in the cytoplasm, targeted to mitochondria by cleavable amino-terminal presequences, translocated across both mitochondrial membranes, and assembled with each other and their respective co-factors into a functional complex. The relationships between the attachment of covalent cofactors to mitochondrial proteins and their import has been examined for several proteins. Our results are consistent with flavinylation being a post-translocational process that occurs during or after mature Fp folding and prior to its assembly
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