Disease relevance of RIP1

• Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation [1].

High impact information on RIP1

• BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae [2].
• By using biolistic transformation, we have relocated the nuclear RIP1 gene into mitochondria [3].
• The Rieske FeS protein, an essential catalytic subunit of the mitochondrial cytochrome bc1 complex, is encoded in yeast by the nuclear gene RIP1, whose deletion leads to a respiratory-deficient phenotype [3].
• Immunodetection experiments demonstrate that the mitochondrial genome containing RIP1m is able to produce the Rip1 protein in lower steady-state amounts than the wild type but still sufficient to maintain a functional cytochrome bc1 complex and respiratory competence to a RIP1-deleted strain [3].
• These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein [4].

Biological context of RIP1

• The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth [5].
• DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1 [5].
• The nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome bc1 complex of the mitochondrial respiratory chain has been isolated and characterized from Saccharomyces cerevisiae [6].
• Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region [7].
• Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex [8].

Anatomical context of RIP1

• Western analysis of mitochondrial membranes from this disruption strain indicates core protein 1 of the cytochrome bc1 complex is present in normal amounts, while cytochrome c1, the Rieske iron-sulfur protein, subunit 6, and subunit 7 were absent or present in very low amounts [9].
• EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present [10].
• The yeast gene for the Rieske iron-sulfur protein of the cytochrome b.c1 complex was subcloned into the expression vector, pSP64, then transcribed and translated in vitro in a reticulocyte lysate in the presence of [35S]methionine [11].
• The Rieske iron-sulfur protein of the cytochrome bc1 complex is synthesized in the cytosol as a precursor with an additional 30 amino acids at the amino terminus [12].
• The mode of membrane attachment of the Rieske iron-sulfur proteins from cytochrome b6f complex of pea thylakoids and from cytochrome bc1 complex of yeast mitochondria has been studied using biochemical approaches [13].

Associations of RIP1 with chemical compounds

• Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center [4].
• Processing of the presequence of the Schizosaccharomyces pombe Rieske iron-sulfur protein occurs in a single step and can be converted to two-step processing by mutation of a single proline to serine in the presequence [14].
• Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster [15].
• Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal [15].
• Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein [16].

Enzymatic interactions of RIP1

• Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene [5].

Other interactions of RIP1

• We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination [5].
• In addition, ubiquinol-cytochrome c oxidoreductase activity was reduced 40% in mitochondrial membranes from the QCR10 deletion strain, and the Rieske iron-sulfur protein was lost when the cytochrome bc1 complex lacking the 8.5-kDa protein was purified [17].
• Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b [10].

Analytical, diagnostic and therapeutic context of RIP1

• We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines [18].
• The assembly of six deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated by immunoprecipitation from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the intact cytochrome bc1 complex [19].

References