RESUMEN
Coenzyme Q (ubiquinone or Q) functions in the respiratory electron transport chain and serves as a lipophilic antioxidant. In the budding yeast Saccharomyces cerevisiae, Q biosynthesis requires nine Coq proteins (Coq1-Coq9). Previous work suggests both an enzymatic activity and a structural role for the yeast Coq7 protein. To define the functional roles of yeast Coq7p we test whether Escherichia coli ubiF can functionally substitute for yeast COQ7. The ubiF gene encodes a flavin-dependent monooxygenase that shares no homology to the Coq7 protein and is required for the final monooxygenase step of Q biosynthesis in E. coli. The ubiF gene expressed at low copy restores growth of a coq7 point mutant (E194K) on medium containing a non-fermentable carbon source, but fails to rescue a coq7 null mutant. However, expression of ubiF from a multicopy vector restores growth and Q synthesis for both mutants, although with a higher efficiency in the point mutant. We attribute the more efficient rescue of the coq7 point mutant to higher steady state levels of the Coq3, Coq4, and Coq6 proteins and to the presence of demethoxyubiquinone, the substrate of UbiF. Coq7p co-migrates with the Coq3 and Coq4 polypeptides as a high molecular mass complex. Here we show that addition of Q to the growth media also stabilizes the Coq3 and Coq4 polypeptides in the coq7 null mutant. The data suggest that Coq7p, and the lipid quinones (demethoxyubiquinone and Q) function to stabilize other Coq polypeptides.
Asunto(s)
Proteínas de Escherichia coli/genética , Oxigenasas de Función Mixta/genética , Mutación , Saccharomyces cerevisiae/genética , Ubiquinona/química , Secuencia de Aminoácidos , Electroquímica , Prueba de Complementación Genética , Vectores Genéticos , Lípidos , Mitocondrias/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Mutación Puntual , Homología de Secuencia de AminoácidoRESUMEN
Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q(9) and instead accumulate demethoxy-Q(9) (DMQ(9)). DMQ(9) has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ(6) in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q(6) and producing solely DMQ were respiratory deficient and unable to support (6)either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ(6) failed to protect cells against oxidative stress generated by H(2)O(2) or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.
Asunto(s)
Antioxidantes/farmacología , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biosíntesis , Ubiquinona/química , Ubiquinona/genética , Alelos , Antioxidantes/metabolismo , Benzoquinonas/metabolismo , Sitios de Unión , Western Blotting , Cromatografía Líquida de Alta Presión , Transporte de Electrón , Citometría de Flujo , Glicerol/metabolismo , Peróxido de Hidrógeno/farmacología , Espectrometría de Masas , Mitocondrias/metabolismo , Modelos Químicos , Mutación , NADH Deshidrogenasa/metabolismo , Estrés Oxidativo , Consumo de Oxígeno , Péptidos/química , Plásmidos/metabolismo , Superóxidos/metabolismo , Factores de Tiempo , Ácido alfa-Linolénico/metabolismo , Ácido alfa-Linolénico/farmacologíaRESUMEN
Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63-65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Protones , Saccharomyces cerevisiae/metabolismo , Ubiquinona/fisiología , Canales Iónicos , Transporte Iónico , Proteínas Mitocondriales , Mutación , Saccharomyces cerevisiae/genética , Transformación Genética , Proteína Desacopladora 1RESUMEN
Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.
Asunto(s)
Benzoquinonas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Ubiquinona/metabolismo , Animales , Benzoquinonas/análisis , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Mitocondrias/metabolismo , Mutación , Fenotipo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Reproducción , Relación Estructura-Actividad , Ubiquinona/químicaRESUMEN
Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.
Asunto(s)
Oxigenasas de Función Mixta/química , Saccharomyces cerevisiae/genética , Ubiquinona/biosíntesis , Ubiquinona/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Carbono/metabolismo , División Celular , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/metabolismo , Flavinas/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Genotipo , Hidroxibenzoatos/metabolismo , Immunoblotting , Potenciales de la Membrana , Mitocondrias/metabolismo , Oxigenasas de Función Mixta/metabolismo , Modelos Químicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Triterpenos/metabolismoRESUMEN
The Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ(9) are high in the clk-1(qm30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E. coli. Furthermore, this E. coli-specific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.