Altered Quinone Biosynthesis in the Long-lived clk-1Mutants of Caenorhabditis elegans

Abstract

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ 9 ) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ 9 . Instead, the UQ 9 biosynthesis intermediate, demethoxyubiquinone (DMQ 9 ), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ 9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ 9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ 2 was chemically synthesized. We find that DMQ 2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ 2 , cannot. The accumulation of DMQ 9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.The understanding of the biological pathways that control life span can be studied in Caenorhabditis elegans through the identification of genes that alter the length of life when mutated (1). For example, mutations in clk-1 are known to cause an extended life span, as well as the slowing of a variety of developmental and physiological events, including the cell cycle, embryogenesis, post-embryonic development, and rhythmic adult behaviors (2, 3). Thus, CLK-1 is expected to play a unique biological role that is necessary to determine the life span and to coordinate these various biological processes. However, the biochemical differences between clk-1 mutants and the wildtype strain, which might indicate the function of CLK-1, have yet to been identified (1, 4 -7).clk-1 encodes a 187-residue polypeptide that is homologous to yeast coq7/cat5 (8). COQ7/CAT5 is located in the inner membrane of yeast mitochondria and is necessary for the biosynthesis of ubiquinone (UQ) 1 in yeast (9, 10). Therefore, yeast coq7/cat5 mutants, which lack UQ 6 , are unable to grow on nonfermentable carbon sources (9). Orthologs of clk-1/coq7/ cat5 have also been reported from mammals, including human (11-13), and appear to be highly conserved among species.Recently, a green fluorescent protein fusion to C. elegans CLK-1 was shown to localize to the mitochondria of all the somatic cells of the worm (14). However, in contrast to the situation in yeast, which is defective in respiratory growth, C. elegans clk-1 mutants are able to respire almost normally. In fact, the metabolic capacities and the A...

A Measurable Increase in Oxidative Damage Due to Reduction in Superoxide Detoxification Fails to Shorten the Life Span of Long-Lived Mitochondrial Mutants of Caenorhabditis elegans

“…Several of the mutants that we have examined (clk-1, isp-1, and mev-1) are closely involved in the process of electron transport Feng et al 2001;Miyadera et al 2001). Thus, it is likely that these mutants have altered ROS metabolism (Feng et al 2001;Senoo-Matsuda et al 2001;Shibata et al 2003), as indicated by the various effects of SOD knockdowns on the mutant phenotypes of the long-lived mutants (Tables 1-4).…”

“…One of the main sources of cellular superoxide production is believed to be the bifurcating transfer of electrons from ubiquinone to the ''Rieske'' ironsulfur protein and the cytochrome b of complex III of the mitochondrial respiratory chain (Raha and Robinson 2000). Two of the three long-lived mutants that we are analyzing are directly involved in this process: isp-1 (qm150) is a point mutation in the iron-sulfur protein (Feng et al 2001), and clk-1 affects the biosynthesis of ubiquinone (Miyadera et al 2001). The third gene, daf-2, encodes an insulin-receptor-like kinase (Kimura et al 1997) and affects a variety of processes in worms, including their resistance to oxidative stress (Honda and Honda 2002).…”

“…We focused on strains carrying one or several mutations in four well-characterized genes whose alteration can result in increased longevity and whose interactions have been previously studied. daf-2 encodes an insulin-receptor-like tyrosine kinase (Kenyon et al 1993); clk-1 encodes a ubiquinone biosynthetic enzyme (Ewbank et al 1997;Stenmark et al 2001) and clk-1 mutants display respiratory defects (Felkai et al 1999;Miyadera et al 2001;Braeckman et al 2002a,b;Kayser et al 2004); isp-1 encodes the ''Rieske'' iron-sulfur protein (Feng et al 2001), a catalytic subunit of mitochondrial complex III; and ctb-1, a gene encoded by the mitochondrial genome, encodes the cytochrome b of complex III (Feng et al 2001). Mutations in daf-2 and clk-1 act synergistically to induce a very long life span (Lakowski and Hekimi 1996).…”

Evidence for only two independent pathways for decreasing senescence in Caenorhabditis elegans

“…In particular, nonnull mutations can give the false impression that pathways are independent when they are not. In this study, the clk-1 mutations tested are functional null mutations (e2519 is a missense mutation and qm30 is a deletion mutation) that both completely suppress the synthesis of ubiquinone (UQ9; Ewbank et al 1997;Miyadera et al 2001). While the daf-2 mutation used is not a null mutation (null mutations prevent the worms from developing into an adult), the worms are phenotypically similar to null mutations (i.e., they form 100% dauers) at our control temperature of 25°C.…”

RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation ofCOQ7

“…21 COQ7 enzyme activity is the only step in CoQ biosynthesis in which the non-reactive intermediate demethoxyubiquinone is accumulated by either nutritional limitations or mutant strains, and it is proposed to be a key step in this pathway. 41,42 Recently, it has been described that Clu1/CluA homolog (CLUH) encodes for mRNA binding protein that bound to hundreds of mRNAs encoding mitochondrial-directed proteins, including COQ3 and COQ6 mRNAs although there is not demonstration of its role in their stability and/or translation. 43 COQ7 mRNA was included as a putative target of HuR, 24 and thus we focused on COQ7 mRNA interaction with RBPs as a novel posttranscriptional regulatory mechanism of CoQ biosynthesis.…”