Ubiquinone Is Necessary for Caenorhabditis elegansDevelopment at Mitochondrial and Non-mitochondrial Sites

Ubiquinone (UQ) is a lipid co-factor that is involved in numerous enzymatic processes and is present in most cellular membranes. In particular, UQ is a crucial electron carrier in the mitochondrial respiratory chain. Recently, it was shown thatclk-1 mutants of the nematode worm Caenorhabditis elegans do not synthesize UQ9 but instead accumulate demethoxyubiquinone (DMQ9), a biosynthetic precursor of UQ9 (the subscript refers to the length of the isoprenoid side chain). DMQ9 is capable of carrying out the function of UQ9 in the respiratory chain, as demonstrated by the functional competence of mitochondria isolated from clk-1mutants, and the ability of DMQ9 to act as a co-factor for respiratory enzymes in vitro. However, despite the presence of functional mitochondria, clk-1 mutant worms fail to complete development when feeding on bacteria that do not produce UQ8. Here we show that clk-1 mutants cannot grow on bacteria producing only DMQ8 and that wormcoq-3 mutants, which produce neither UQ9 nor DMQ9, arrest development even on bacteria producing UQ8. These results indicate that UQ is required for nematode development at mitochondrial and non-mitochondrial sites and that DMQ cannot functionally replace UQ at those non-mitochondrial sites. Ubiquinone (UQ) is a lipid co-factor that is involved in numerous enzymatic processes and is present in most cellular membranes. In particular, UQ is a crucial electron carrier in the mitochondrial respiratory chain. Recently, it was shown thatclk-1 mutants of the nematode worm Caenorhabditis elegans do not synthesize UQ9 but instead accumulate demethoxyubiquinone (DMQ9), a biosynthetic precursor of UQ9 (the subscript refers to the length of the isoprenoid side chain). DMQ9 is capable of carrying out the function of UQ9 in the respiratory chain, as demonstrated by the functional competence of mitochondria isolated from clk-1mutants, and the ability of DMQ9 to act as a co-factor for respiratory enzymes in vitro. However, despite the presence of functional mitochondria, clk-1 mutant worms fail to complete development when feeding on bacteria that do not produce UQ8. Here we show that clk-1 mutants cannot grow on bacteria producing only DMQ8 and that wormcoq-3 mutants, which produce neither UQ9 nor DMQ9, arrest development even on bacteria producing UQ8. These results indicate that UQ is required for nematode development at mitochondrial and non-mitochondrial sites and that DMQ cannot functionally replace UQ at those non-mitochondrial sites. ubiquinone demethoxy- ubiquinone Ubiquinone (UQ)1 is a prenylated benzoquinone that is an essential co-factor in the mitochondrial respiratory chain, where its function is best characterized. UQ is also found in many other locations in the cell, such as the lysosome and Golgi membranes, as well as in nuclear and plasma membranes (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). The exact role of UQ at these extramitochondrial sites is being actively explored (e.g. Refs. 2Santos-Ocana C. Villalba J.M. Cordoba F. Padilla S. Crane F.L. Clarke C.F. Navas P. J. Bioenerg. Biomembr. 1998; 30: 465-475Crossref PubMed Scopus (53) Google Scholar and 3Gille L. Nohl H. Arch Biochem. Biophys. 2000; 375: 347-354Crossref PubMed Scopus (99) Google Scholar).The gene clk-1 of the nematode Caenorhabditis elegans affects many physiological rates, including embryonic and post-embryonic development, rhythmic behaviors, reproduction, and life span (4Branicky R. Benard C. Hekimi S. Bioessays. 2000; 22: 48-56Crossref PubMed Scopus (72) Google Scholar). clk-1 encodes a 187-amino acid protein that is localized in mitochondria (5Felkai S. Ewbank J.J. Lemieux J. Labbe J.C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (217) Google Scholar) and that is homologous to the yeast protein Coq7p, which has been shown to be required for UQ biosynthesis (6Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). clk-1 has also been shown to be necessary for UQ biosynthesis in worms (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar, 8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and in the mouse (9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Indeed, UQ9 is entirely absent from mitochondria purified from worm and mouse clk-1 mutants (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) (the subscript refers to the length of the isoprenoid side chain). Instead, these mitochondria accumulate demethoxyubiquinone (DMQ9), which is an intermediate in the synthesis of UQ9 (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Consistently, recent evidence suggests that clk-1 encodes a DMQ hydroxylase (10Stenmark P. Grunler J. Mattsson J. Sindelar P.J. Nordlund P. Berthold D.A. J. Biol. Chem. 2001; 276: 33297-33300Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), which converts DMQ to ubiquinol. In Escherichia coli, DMQ8 is able to sustain respiration in isolated membranes although at a lower rate than Q8 (11Wallace B.J. Young I.G. Biochim. Biophys. Acta. 1977; 461: 75-83Crossref PubMed Scopus (43) Google Scholar). Similarly, DMQ9 is capable of sustaining electron transport in eukaryotic mitochondria, as the function of purified mitochondria (5Felkai S. Ewbank J.J. Lemieux J. Labbe J.C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (217) Google Scholar), and mitochondrial enzymes (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), from clk-1 worm mutants appears to be almost intact compared with the wild type. In addition, synthetic DMQ2 has been shown to function in vitro as a co-factor for electron transport from worm complex I and, albeit more poorly, from complex II (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Finally, it was found that in the absence of any exogenous UQ the oxygen consumption of mouse embryonic stem cells with a deleted mclk1 gene is only reduced by 35% (9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).Recently, it has been found that clk-1 mutants are unable to grow on a UQ-deficient bacterial strain despite the presence and the activity of DMQ9 (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar). Although, dietary UQ is generally not capable of reaching mitochondria (reviewed in Ref. 1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar), this has been interpreted to suggest that DMQ9 is insufficient for normal mitochondrial function and that dietary bacterial UQ8 can reach the mitochondria and function there in trace amounts (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar).To resolve these issues, we have generated a strain carrying a knockout mutation in the nematode gene coq-3, which encodes a methyltransferase required for UQ synthesis, and whose inactivation does not lead to DMQ accumulation in yeast (12Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar). We find thatcoq-3 worms are not able to complete development even on bacteria that contain UQ8. These results indicate that 1) dietary UQ cannot complement a UQ deficiency in the absence of DMQ, 2) the growth impairment of clk-1 mutants without a dietary supply of UQ is due to a non-mitochondrial requirement for UQ, and 3) DMQ cannot functionally replace UQ at the non-mitochondrial sites.DISCUSSIONOur results suggest that UQ is necessary for C. elegansgrowth and development at different subcellular locations, in particular it appears to be necessary at sites distinct from the mitochondrial respiratory chain (Fig. 3). Indeed, for its respiratory function in the mitochondria, endogenous DMQ9 can functionally replace endogenous UQ9, as indicated by the observation that clk-1 mutant mitochondria do not appear to contain UQ9 but are functionally competent (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). On the other hand, coq-3mutants, in which a failure to manufacture UQ9 and DMQ9 is expected, display a much more severe phenotype thanclk-1 mutants. Thus, at some still unknown site or sites, distinct from the respiratory chain, endogenous DMQ9, or dietary DMQ8, cannot functionally replace endogenous UQ9, while dietary UQ8 can. In fact,clk-1 mutants, which have functional mitochondria and make DMQ9, cannot develop and grow without dietary UQ8, even in the presence of dietary DMQ8 fromubiF bacteria.This model is consistent with the findings by numerous studies on UQ uptake and metabolism in other systems, such as rodents (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). What has been found is that dietary UQ appears to be taken up only poorly (2–3% of the initially ingested ubiquinone), and the majority is then distributed to the plasma membrane, the lysosomes, and the Golgi, with only minute quantities, if any at all, appearing in the mitochondria. Given that every cell endogenously produces UQ, it is possible that no active uptake system exists to assimilate this rather complex lipid.Our studies clarify the roles of endogenous and dietary UQ in the worm's biology. Also, we demonstrate for the first time the functional importance of UQ at non-mitochondrial locations for an organism's viability. Action of dietary UQ at non-mitochondrial sites could underlie the beneficial effects of dietary UQ for patients with mitochondrial diseases (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). For example, UQ has been found to participate in reactions that regulate the redox state of the cell at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). Disease states that arise from deficient mitochondria are often found to increase cellular oxidative stress, and dietary UQ could stimulate a protective function at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). In addition, in bacteria, quinones have recently been found to act as the primary signal of the redox state of the cell (29Georgellis D. Kwon O. Lin E.C. Science. 2001; 292: 2314-2316Crossref PubMed Scopus (374) Google Scholar). InE. coli, UQ negatively modulates the phosphorylation status and function of ArcB, an important global regulator of gene expression. The eventual discovery of additional roles for UQ in eukaryotes, and in particular as a signaling cue, will help to better understand the pleiotropic effects of mutations in genes that affect UQ, includingclk-1.Finally, we note that the coq-3 and clk-1 mutant strains provide genetic models to identify compounds that could selectively replace ubiquinone at the mitochondria and/or at non-mitochondrial sites. The development of such bio-available ubiquinone mimetics could be of great medical interest. Ubiquinone (UQ)1 is a prenylated benzoquinone that is an essential co-factor in the mitochondrial respiratory chain, where its function is best characterized. UQ is also found in many other locations in the cell, such as the lysosome and Golgi membranes, as well as in nuclear and plasma membranes (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). The exact role of UQ at these extramitochondrial sites is being actively explored (e.g. Refs. 2Santos-Ocana C. Villalba J.M. Cordoba F. Padilla S. Crane F.L. Clarke C.F. Navas P. J. Bioenerg. Biomembr. 1998; 30: 465-475Crossref PubMed Scopus (53) Google Scholar and 3Gille L. Nohl H. Arch Biochem. Biophys. 2000; 375: 347-354Crossref PubMed Scopus (99) Google Scholar). The gene clk-1 of the nematode Caenorhabditis elegans affects many physiological rates, including embryonic and post-embryonic development, rhythmic behaviors, reproduction, and life span (4Branicky R. Benard C. Hekimi S. Bioessays. 2000; 22: 48-56Crossref PubMed Scopus (72) Google Scholar). clk-1 encodes a 187-amino acid protein that is localized in mitochondria (5Felkai S. Ewbank J.J. Lemieux J. Labbe J.C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (217) Google Scholar) and that is homologous to the yeast protein Coq7p, which has been shown to be required for UQ biosynthesis (6Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). clk-1 has also been shown to be necessary for UQ biosynthesis in worms (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar, 8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and in the mouse (9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Indeed, UQ9 is entirely absent from mitochondria purified from worm and mouse clk-1 mutants (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) (the subscript refers to the length of the isoprenoid side chain). Instead, these mitochondria accumulate demethoxyubiquinone (DMQ9), which is an intermediate in the synthesis of UQ9 (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Consistently, recent evidence suggests that clk-1 encodes a DMQ hydroxylase (10Stenmark P. Grunler J. Mattsson J. Sindelar P.J. Nordlund P. Berthold D.A. J. Biol. Chem. 2001; 276: 33297-33300Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), which converts DMQ to ubiquinol. In Escherichia coli, DMQ8 is able to sustain respiration in isolated membranes although at a lower rate than Q8 (11Wallace B.J. Young I.G. Biochim. Biophys. Acta. 1977; 461: 75-83Crossref PubMed Scopus (43) Google Scholar). Similarly, DMQ9 is capable of sustaining electron transport in eukaryotic mitochondria, as the function of purified mitochondria (5Felkai S. Ewbank J.J. Lemieux J. Labbe J.C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (217) Google Scholar), and mitochondrial enzymes (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), from clk-1 worm mutants appears to be almost intact compared with the wild type. In addition, synthetic DMQ2 has been shown to function in vitro as a co-factor for electron transport from worm complex I and, albeit more poorly, from complex II (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Finally, it was found that in the absence of any exogenous UQ the oxygen consumption of mouse embryonic stem cells with a deleted mclk1 gene is only reduced by 35% (9Levavasseur F. Miyadera H. Sirois J. Tremblay M. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Recently, it has been found that clk-1 mutants are unable to grow on a UQ-deficient bacterial strain despite the presence and the activity of DMQ9 (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar). Although, dietary UQ is generally not capable of reaching mitochondria (reviewed in Ref. 1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar), this has been interpreted to suggest that DMQ9 is insufficient for normal mitochondrial function and that dietary bacterial UQ8 can reach the mitochondria and function there in trace amounts (7Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (161) Google Scholar). To resolve these issues, we have generated a strain carrying a knockout mutation in the nematode gene coq-3, which encodes a methyltransferase required for UQ synthesis, and whose inactivation does not lead to DMQ accumulation in yeast (12Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar). We find thatcoq-3 worms are not able to complete development even on bacteria that contain UQ8. These results indicate that 1) dietary UQ cannot complement a UQ deficiency in the absence of DMQ, 2) the growth impairment of clk-1 mutants without a dietary supply of UQ is due to a non-mitochondrial requirement for UQ, and 3) DMQ cannot functionally replace UQ at the non-mitochondrial sites. DISCUSSIONOur results suggest that UQ is necessary for C. elegansgrowth and development at different subcellular locations, in particular it appears to be necessary at sites distinct from the mitochondrial respiratory chain (Fig. 3). Indeed, for its respiratory function in the mitochondria, endogenous DMQ9 can functionally replace endogenous UQ9, as indicated by the observation that clk-1 mutant mitochondria do not appear to contain UQ9 but are functionally competent (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). On the other hand, coq-3mutants, in which a failure to manufacture UQ9 and DMQ9 is expected, display a much more severe phenotype thanclk-1 mutants. Thus, at some still unknown site or sites, distinct from the respiratory chain, endogenous DMQ9, or dietary DMQ8, cannot functionally replace endogenous UQ9, while dietary UQ8 can. In fact,clk-1 mutants, which have functional mitochondria and make DMQ9, cannot develop and grow without dietary UQ8, even in the presence of dietary DMQ8 fromubiF bacteria.This model is consistent with the findings by numerous studies on UQ uptake and metabolism in other systems, such as rodents (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). What has been found is that dietary UQ appears to be taken up only poorly (2–3% of the initially ingested ubiquinone), and the majority is then distributed to the plasma membrane, the lysosomes, and the Golgi, with only minute quantities, if any at all, appearing in the mitochondria. Given that every cell endogenously produces UQ, it is possible that no active uptake system exists to assimilate this rather complex lipid.Our studies clarify the roles of endogenous and dietary UQ in the worm's biology. Also, we demonstrate for the first time the functional importance of UQ at non-mitochondrial locations for an organism's viability. Action of dietary UQ at non-mitochondrial sites could underlie the beneficial effects of dietary UQ for patients with mitochondrial diseases (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). For example, UQ has been found to participate in reactions that regulate the redox state of the cell at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). Disease states that arise from deficient mitochondria are often found to increase cellular oxidative stress, and dietary UQ could stimulate a protective function at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). In addition, in bacteria, quinones have recently been found to act as the primary signal of the redox state of the cell (29Georgellis D. Kwon O. Lin E.C. Science. 2001; 292: 2314-2316Crossref PubMed Scopus (374) Google Scholar). InE. coli, UQ negatively modulates the phosphorylation status and function of ArcB, an important global regulator of gene expression. The eventual discovery of additional roles for UQ in eukaryotes, and in particular as a signaling cue, will help to better understand the pleiotropic effects of mutations in genes that affect UQ, includingclk-1.Finally, we note that the coq-3 and clk-1 mutant strains provide genetic models to identify compounds that could selectively replace ubiquinone at the mitochondria and/or at non-mitochondrial sites. The development of such bio-available ubiquinone mimetics could be of great medical interest. Our results suggest that UQ is necessary for C. elegansgrowth and development at different subcellular locations, in particular it appears to be necessary at sites distinct from the mitochondrial respiratory chain (Fig. 3). Indeed, for its respiratory function in the mitochondria, endogenous DMQ9 can functionally replace endogenous UQ9, as indicated by the observation that clk-1 mutant mitochondria do not appear to contain UQ9 but are functionally competent (8Miyadera H. Amino H. Hiraishi A. Taka H. Murayama K. Miyoshi H. Sakamoto K. Ishii N. Hekimi S. Kita K. J. Biol. Chem. 2001; 276: 7713-7716Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). On the other hand, coq-3mutants, in which a failure to manufacture UQ9 and DMQ9 is expected, display a much more severe phenotype thanclk-1 mutants. Thus, at some still unknown site or sites, distinct from the respiratory chain, endogenous DMQ9, or dietary DMQ8, cannot functionally replace endogenous UQ9, while dietary UQ8 can. In fact,clk-1 mutants, which have functional mitochondria and make DMQ9, cannot develop and grow without dietary UQ8, even in the presence of dietary DMQ8 fromubiF bacteria. This model is consistent with the findings by numerous studies on UQ uptake and metabolism in other systems, such as rodents (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). What has been found is that dietary UQ appears to be taken up only poorly (2–3% of the initially ingested ubiquinone), and the majority is then distributed to the plasma membrane, the lysosomes, and the Golgi, with only minute quantities, if any at all, appearing in the mitochondria. Given that every cell endogenously produces UQ, it is possible that no active uptake system exists to assimilate this rather complex lipid. Our studies clarify the roles of endogenous and dietary UQ in the worm's biology. Also, we demonstrate for the first time the functional importance of UQ at non-mitochondrial locations for an organism's viability. Action of dietary UQ at non-mitochondrial sites could underlie the beneficial effects of dietary UQ for patients with mitochondrial diseases (1Dallner G. Sindelar P.J. Free Radic. Biol. Med. 2000; 29: 285-294Crossref PubMed Scopus (178) Google Scholar). For example, UQ has been found to participate in reactions that regulate the redox state of the cell at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). Disease states that arise from deficient mitochondria are often found to increase cellular oxidative stress, and dietary UQ could stimulate a protective function at the plasma membrane (28Morre D.M. Lenaz G. Morre D.J. J. Exp. Biol. 2000; 203: 1513-1521PubMed Google Scholar). In addition, in bacteria, quinones have recently been found to act as the primary signal of the redox state of the cell (29Georgellis D. Kwon O. Lin E.C. Science. 2001; 292: 2314-2316Crossref PubMed Scopus (374) Google Scholar). InE. coli, UQ negatively modulates the phosphorylation status and function of ArcB, an important global regulator of gene expression. The eventual discovery of additional roles for UQ in eukaryotes, and in particular as a signaling cue, will help to better understand the pleiotropic effects of mutations in genes that affect UQ, includingclk-1. Finally, we note that the coq-3 and clk-1 mutant strains provide genetic models to identify compounds that could selectively replace ubiquinone at the mitochondria and/or at non-mitochondrial sites. The development of such bio-available ubiquinone mimetics could be of great medical interest. We thank Claire Bénard and Robyn Branicky for careful reading of the manuscript and Robert Poole, Georges Javor, Philip Rather, Catherine Clarke, David Clark, Bernard Lemire, and the E. coli stock center at Yale for sharing bacterial clones.

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