Reproductive Fitness and Quinone Content of Caenorhabditis elegans clk-1 Mutants Fed Coenzyme Q Isoforms of Varying Length
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m308760200
ISSN1083-351X
AutoresTanya Jonassen, Diana Davis, Pamela L. Larsen, Catherine F. Clarke,
Tópico(s)ATP Synthase and ATPases Research
ResumoCaenorhabditis 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. 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. Coenzyme Q, also known as ubiquinone or Q, 1The abbreviations used are: Qubiquinone (coenzyme Q)DMQdemethoxy-QRQrhodoquinoneHPLChigh performance liquid chromatographyECDelectrochemical detectionERendoplasmic reticulumIBisolation bufferTMREtetramethylrhodamine, ethyl ester. is a redox active lipid synthesized by both prokaryotes and eukaryotes (1Dutton P.L. Ohnishi T. Darrouzet E. Leonard M.A. Sharp R.E. Gibney B.R. Daldal F. Moser C.C. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Inc., Boca Raton, FL2000: 65-82Google Scholar). Q consists of a benzoquinone head group, which is capable of undergoing reversible reduction and oxidation, and a hydrophobic isoprenoid tail. The number of isoprene units in the tail varies between organisms. For instance, Escherichia coli produce a tail with eight isoprene units (Q8), Caenorhabditis elegans contain nine units (Q9), and humans make Q10 (2Crane F.L. J. Am. Coll. Nutr. 2001; 20: 591-598Crossref PubMed Scopus (709) Google Scholar). The hydrophobic tail has long been thought to simply anchor Q to the membrane. It is unknown why different organisms produce quinones with different tail lengths. ubiquinone (coenzyme Q) demethoxy-Q rhodoquinone high performance liquid chromatography electrochemical detection endoplasmic reticulum isolation buffer tetramethylrhodamine, ethyl ester. In eukaryotes, Q is found primarily in the mitochondrial inner membrane, where it serves a critical role in respiration, moving electrons from Complex I or II to Complex III (1Dutton P.L. Ohnishi T. Darrouzet E. Leonard M.A. Sharp R.E. Gibney B.R. Daldal F. Moser C.C. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Inc., Boca Raton, FL2000: 65-82Google Scholar). However, Q is found in other intracellular membranes where it serves a variety of functions, including trans-plasma membrane electron transport (3Santos-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 (55) Google Scholar), as a key cofactor in uridine synthesis (4Nagy M. Lacroute F. Thomas D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8966-8970Crossref PubMed Scopus (151) Google Scholar), and as a lipid soluble antioxidant (5Kagan V.E. Nohl H. Quinn P.J. Cadenas E. Packer L. Handbook of Antioxidants. Marcel Dekker, Inc., New York1996: 157-201Google Scholar). Reduced Q (QH2) can act either directly to scavenge lipid peroxyl radicals or indirectly by regenerating vitamin E. Although Q can be taken as a supplement, most organisms synthesize Q de novo. Much of the Q biosynthetic pathway has been identified using Q-deficient E. coli and Saccharomyces cerevisiae mutants (6Jonassen T. Clarke C.F. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Inc., Boca Raton, FL2000: 185-208Google Scholar). In yeast, the COQ7 gene product was found to be responsible for the penultimate step in Q biosynthesis, the hydroxylation of demethoxy-Q (DMQ) to demethyl-Q (7Proft M. Kotter P. Hedges D. Bojunga N. Entian K.D. EMBO J. 1995; 14: 6116-6126Crossref PubMed Scopus (66) Google Scholar, 8Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 9Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The predicted structure of Coq7p suggests that it is a di-iron carboxylate protein (10Stenmark P. Grünler 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 (106) Google Scholar). C. elegans with mutations in the clk-1 gene, the COQ7 homologue, show slowed adult behaviors, including defecation cycles and pharyngeal pumping, slowed embryonic and post-embryonic development, and decreased brood sizes (11Ewbank J.J. Barnes T.M. Lakowski B. Lussier M. Bussey H. Hekimi S. Science. 1997; 275: 980-983Crossref PubMed Scopus (266) Google Scholar, 12Wong A. Boutis P. Hekimi S. Genetics. 1995; 139: 1247-1259Crossref PubMed Google Scholar). These animals were found to accumulate DMQ9, the expected biosynthetic intermediate, and a small amount of Q8, incorporated from their E. coli diet (13Miyadera 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 (170) Google Scholar, 14Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (167) Google Scholar). When clk-1 worms are fed GD1, a Q-less E. coli, they arrest at the L2 larval stage if fed from hatching or develop into sterile adults if fed GD1 as dauer larvae (14Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 421-426Crossref PubMed Scopus (167) Google Scholar). The diet-derived Q8 masks a Q auxotrophy in clk-1 animals. Unexpectedly, wild-type animals fed the Q-less E. coli have an extended adult life span (15Larsen P.L. Clarke C.F. Science. 2002; 295: 120-123Crossref PubMed Scopus (213) Google Scholar). This suggests an interesting contrast in the requirements for Q; it is essential for development, yet exogenous Q8 is detrimental to the longevity of adult animals. It is not understood why the clk-1 animals remain abnormal when supplied a Q8-replete diet. The diet-derived Q8 is present in mitochondria isolated from clk-1 mutant adults; however, it is important to note that the amount of Q8 assimilated is far lower than the amount of Q9 produced by N2 (16Jonassen T. Marbois B.N. Faull K.F. Clarke C.F. Larsen P.L. J. Biol. Chem. 2002; 277: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We considered that uptake of the shorter Q8 isoform derived from the diet may contribute to the Clk-1 phenotypes, because the worms normally synthesize the Q9 isoform. To further understand the effects of tail length on Q function and transport, we utilized E. coli strains that had been engineered to produce quinones with different tail lengths (17Okada K. Kamiya Y. Zhu X. Suzuki K. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 5992-5998Crossref PubMed Google Scholar, 18Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). The polyprenyldiphosphate synthase gene ispB was disrupted, and homologues from different organisms were introduced on plasmids, generating E. coli that produced Q7, Q8, Q9, or Q10. We examined the effects of feeding these different Q isoforms on reproductive fitness and progeny viability in both N2 and clk-1 animals. Although N2 animals showed no significant differences in brood size or morphology regardless of the Q fed, we found that clk-1(qm30) animals displayed a sterile phenotype when fed bacteria producing the shortest isoform, Q7. The gonads of these animals were shriveled and disordered. Lipid extracts from both the E. coli food and worms were analyzed to determine the levels of uptake and subcellular distribution of the varying Q isoforms within the worms. Although these animals assimilate Q7, it does not accumulate as efficiently within the mitochondria. Interestingly, the clk-1(e2519) animals have a very similar quinone profile to that of clk-1(qm30) animals, yet they produce viable progeny when fed the Q7-producing food. The gonadal morphology is also less affected. Mitochondria isolated from clk-1(qm30) mutants uniquely separate into two densities when purified on Nycodenz gradients, a functionally normal fraction and a less functional, denser fractions (termed the bottom band). Although this bottom band mitochondrial fraction is not observed in the clk-1(e2519) mutants fed Q7, it is present when this strain is fed a Q-less diet, which results in sterility. Together the data suggest that the Clk phenotypes result from several biochemical defects: a lack of endogenously produced Q, a decrease in bacterially derived mitochondrial Q content, poorly functioning mitochondria, and the specific mutation in the CLK-1 polypeptide itself. Culture Conditions and Strains—Methods for C. elegans were standard (19Sulston J.E. Hodgkin J. Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 587-606Google Scholar). N2 (Bristol strain) was used as wild type, and the clk-1 alleles used in this study were e2519 and qm30 (12Wong A. Boutis P. Hekimi S. Genetics. 1995; 139: 1247-1259Crossref PubMed Google Scholar). E. coli strains used in this study are listed in Table I.Table IE. coli food sourcesPredominant Q isoformE. coli strainGenotypeispB homologue sourceSourceNoneGD1HW272, ubiG::KanrNot applicableRef. 38Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google ScholarQ8OP50uraNot applicableRef. 39Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google ScholarQ7K0229:pMN18FS1576, ispB::CmrH. influenzaeRef. 17Okada K. Kamiya Y. Zhu X. Suzuki K. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 5992-5998Crossref PubMed Google ScholarQ8K0229:pKA3FS1576, ispB::CmrE. coliRef. 17Okada K. Kamiya Y. Zhu X. Suzuki K. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 5992-5998Crossref PubMed Google ScholarQ9K0229:pSN18FS1576, ispB::CmrSynechocystis sp. strain PCC6803Ref. 17Okada K. Kamiya Y. Zhu X. Suzuki K. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 5992-5998Crossref PubMed Google ScholarQ10K0229:pLD23FS1576, ispB::CmrG. suboxydansM. Kawamukai, Shimane University Open table in a new tab Brood Size Determination—The number of progeny was determined for animals fed the designated E. coli from the L1 stage and from the dauer larval stage. To obtain synchronous L1 larvae, gravid adult animals were treated with alkaline hypochlorite to isolate eggs as per Sulston and Hodgkin (19Sulston J.E. Hodgkin J. Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 587-606Google Scholar). The eggs were incubated in S Medium overnight (N2) or over two nights (clk-1) without E. coli to obtain synchronous starved L1 larvae. Worms were placed individually onto nematode growth medium plates with a thin lawn of E. coli and were moved once or twice daily to fresh plates during the egg laying period. Eggs and freshly hatched L1 larvae were counted (egg count). Plates were retained and observed until the larvae reached L4, as determined by the developing vulval morphology, or for 2 weeks. Larvae that reached L4 were removed and counted (progeny count). Isolation and Quantification of Quinones—For the determination of E. coli quinone content, cultures were grown in Luria Broth at 37 °C overnight and collected by centrifugation. Nematodes were cultured at 20 °C in S Medium in the presence of OP50 until dauer larvae were obtained. To obtain large quantities of gravid adults, dauer larvae were isolated with 1% SDS treatment and allowed to recover in S Medium with OP50. Eggs were obtained from the gravid adults as above and were allowed to hatch overnight in S Medium without food. The synchronous L1 larvae were then fed the E. coli ispB mutants engineered to produce either Q7 or Q8. When the animals had developed to young adults, as determined by vulval morphology, they were collected by centrifugation, separated from bacteria and debris by sucrose floatation, and allowed to clear bacteria from the gut for a minimum of 30 min in M9. Following another sucrose floatation, the samples were stored at -80 °C until use. Prior to the lipid extraction, all samples were resuspended in ∼12 ml of water, and three 1-ml aliquots were moved to preweighed Eppendorf tubes. The small aliquots were centrifuged at maximum speed, and the wet weights of the pellets were determined. These samples were dried overnight using a heated speedvac, and the dry pellet weights were determined. The dried pellets were then resuspended in 1 n NaOH using ∼1 ml per 2 mg dry weight. The samples were assayed for protein concentration by the bicinchoninic acid assay (Pierce). This allowed for direct comparisons between wet weight, dry weight, and amount protein. The remaining 9-ml samples were pelleted in tared glass tubes, and the wet pellet weights were determined. In general, ∼0.55-g wet weight nematodes and 0.35-g wet weight E. coli was used per extraction. The pellets were resuspended in a small volume of water, and the internal standards (in ethanol) were added at a concentration of 20-30 pmol per μl of final resuspension volume. Q10 was used as an internal standard for the nematode extractions and for the Q7-, Q8-, and Q9-producing bacterial strains. Q6 was the internal standard for the Q10-producing bacteria. 9 ml of MeOH and 6 ml of petroleum ether was added to each tube, and the extraction proceeded with shaking overnight in the dark at 4 °C. The tubes were centrifuged at 2000 rpm, 4 °C for 10 min. The top petroleum ether layers were removed, and a second extraction was done with 4 ml of petroleum ether for one more hour. The petroleum ether extracts were combined and dried under nitrogen. The dried lipids were finally resuspended in 9:1 MeOH/EtOH. The nematode lipids were resuspended in 100 μl, and the bacterial lipids were resuspended in 2 ml of 9:1 MeOH/EtOH. Quantification by HPLC linked with an electrochemical detector (ECD) was performed as described previously (16Jonassen T. Marbois B.N. Faull K.F. Clarke C.F. Larsen P.L. J. Biol. Chem. 2002; 277: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Mitochondrial Isolation—Nematode subcellular fractionation and crude mitochondrial isolation were done as described previously (16Jonassen T. Marbois B.N. Faull K.F. Clarke C.F. Larsen P.L. J. Biol. Chem. 2002; 277: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) except a protease inhibitor mixture was added (catalog number 1873580; Roche Applied Science) to the isolation buffer (IB). The crude mitochondria were further purified over a linear Nycodenz gradient as described by Glick and Pon (20Glick P.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar) with the following adjustments. The step gradient was prepared from a 60% Nycodenz stock in IB diluted to 30, 25, 20, 15, and 10% in IB and layered. The brown band at the 20-25% region contained the purified mitochondria, which were isolated as by Glick and Pon (20Glick P.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar). The purified mitochondrial pellets were resuspended in a volume of IB similar to that originally loaded on the gradient. A lower, more dense band was seen in mitochondrial preparations from clk-1(qm30) animals at the 25-30% region. This band was also present in mitochondrial preparations of clk-1(e2519) animals fed GD1. These bands were separately extracted and identified as bottom bands. Protein concentrations were determined by the bicinchoninic acid assay. Lipid extractions and electrochemical detection were performed as above. Enzyme Assays—Mannosidase II (Golgi) activity assays were adapted from Ren et al. (21Ren J. Castellino F.J. Bretthauer R.K. Biochem. J. 1997; 324: 951-956Crossref PubMed Scopus (27) Google Scholar) and were performed in a total volume of 0.2 ml containing 0.1 m sodium acetate, pH 6.0, 4 mmp-nitrophenyl-α-d-mannopyranoside as substrate, and 100-250 μg of protein per assay. Glucose 6-phosphatase (ER) activity and 5′-nucleotidase (plasma membrane) activity were assayed as by Stephenson and Clarke (22Stephenson R.C. Clarke S. J. Biol. Chem. 1992; 267: 13314-13319Abstract Full Text PDF PubMed Google Scholar) except the incubations took place at 30 °C. Succinate-cytochrome c reductase activity was measured using 50 μg of mitochondrial protein per assay in 40 mm sodium phosphate, pH 7.4, 20 mm sodium succinate, 500 μm EDTA, pH 7.4, and 250 μm potassium cyanide. Samples were incubated in this assay buffer for 15 min at 30 °C. The reaction was initiated with the addition of 50 μm horse heart cytochrome c and monitored for 3.5 min via spectrophoto-metric measurements of absorbance at 550 nm minus 540 nm. The reduction rate of cytochrome c was calculated using the extinction coefficient 18.5 mm-1 cm-1. Western Blotting—Western analysis utilizing the antibody against the β subunit of the F1-ATPase was done as by Jonassen et al. (16Jonassen T. Marbois B.N. Faull K.F. Clarke C.F. Larsen P.L. J. Biol. Chem. 2002; 277: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Similar methods were used with the following primary antibodies: hexokinase (1:1000 dilution) and actin (1:1000 dilution) except that 80 μg of protein were loaded in each lane, and horseradish peroxidase-linked secondary antibodies to rabbit IgG were used at a 1:1000 dilution. Image Acquisition—Dauer larvae were moved to nematode growth medium plates containing 1 μg/ml tetramethylrhodamine, ethyl ester (TMRE; Molecular Probes) inoculated with either OP50 or the Q7-producing strain. The animals were allowed to develop to adulthood. The adults were picked to small droplets of 10 μg/ml Hoechst 33342 (Sigma) on a 2% agarose pad and decapitated just below the pharyngeal bulb, allowing exposure and extension of the gonadal arm. Fluorescent images and DIC images were acquired using an Olympus Fluoview 300 confocal microscope with a 40× oil objective (numerical aperture = 1.35) and Cy3 filter, performed at the Optical Imaging Facility at University of Texas Health Science Center, San Antonio. Gonadal length was determined using Image J (National Institutes of Health: rsb.info.nih.gov/ij/). Profile of Quinones in Engineered E. coli—Before determining the effects of feeding the engineered E. coli ispB mutant strains to the nematodes, the quinone content of these bacterial strains was quantified with a sensitive electrochemical detection system linked with HPLC. Although the bacteria primarily produced the designated Q isoform, each strain also produced other isoforms (Fig. 1). The total amounts of the primary isoforms for the Q8-producing strains were highest, at 6371 ± 176 pmol Q8 per mg protein for OP50 and 5207 ± 124 pmol Q8 per mg protein for K0229:pKA3 (Q8 diet). K0229:pMN18 (Q7 diet) accumulated 4733 ± 120 pmol Q7 per mg protein, but it also produced a significant amount of Q6, at 1205 ± 44 pmol Q6 per mg protein. The total quinone levels in the longer chain Q9 and Q10-producing strains were 57 and 35% of OP50 levels, respectively. K0229:pSN18 (Q9 diet) accumulated 3024 ± 63 pmol Q9 per mg protein, and K0229:pLD23 (Q10 diet) only accumulated 1733 ± 8 pmol Q10 per mg protein. Interestingly, unlike OP50, the engineered ispB mutants did not contain detectable levels of menaquinone (MK8, vitamin K2), an isoprenoid naphthoquinone required by some prokaryotic electron transport chains (23Gennis R.B. Stewart V. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 217-261Google Scholar). Brood Size of clk-1 Mutants Is Affected by the Distinct Qn Diets—Once the lipid profiles of the bacterial foods had been analyzed, the effects of the distinct Qn isoforms on the reproductive fitness of N2 and clk-1 animals were determined. N2 animals fed OP50 laid ∼300 eggs per individual (Fig. 2A). There was no significant difference in the number of eggs laid by N2 animals fed the different strains of E. coli containing predominantly Q7, Q8, or Q10. There was a statistically significant increase in the number of eggs laid when the animals were fed the Q9 diet; however, the number of progeny that developed to L4 larvae did not vary significantly on any food source tested (Fig. 2B). The clk-1(e2519) animals showed a 40% decrease in egg laying when fed the Q7 diet as compared with the Q8-replete diets. These mutants displayed a statistically significant increase in egg production when fed Q9- or Q10-producing E. coli, with 25-30% more viable progeny than those hermaphrodites fed OP50. The worms fed the Q7-containing food developed slightly more slowly than their siblings fed the Q8-producing food; their generation time, from egg to first egg laid, was about one and a half days longer. The generation time of mutants fed the Q9- or Q10-producing bacteria was about half a day faster than those fed the Q8 diet. clk-1(qm30) animals exhibited a dramatic phenotype when fed the Q7-producing bacteria from the L1 larval stage; some of these worms were completely sterile, whereas the rest laid very few eggs, with a range of 0 to 32 eggs per hermaphrodite. The average number of eggs was 14, but the average number of viable larvae produced from these eggs was less than one. The length of time it took for these eggs to hatch and develop was also variable and extended. It took up to 1 week for these hatchlings to develop to the L4 larval stage, and much of this time was spent in embryonic development. After 2 weeks, all remaining eggs were considered nonviable. The generation time, from egg to first egg laid, was 10 days for mutants fed the Q7 diet, as compared with 6 days when the animals were fed OP50. When fed E. coli producing Q8, Q9, or Q10, the clk-1(qm30) nematodes produced similar numbers of eggs as when fed OP50. Although the number of eggs laid when the animals were fed the Q9- and Q10-producing bacteria was similar to that laid with Q8-replete food, far fewer eggs developed into L4 larvae. The dead eggs were examined, revealing embryos at different stages of development. Thus, it appears that there is a mild embryonic lethality associated with the clk-1(qm30) mutant and the Q9 and Q10 diets. When clk-1(qm30) dauer larvae were recovered on the Q7 diet, the brood sizes were significantly lowered, but the effect was not as striking as when the animals were fed this diet commencing as L1 larvae. The average number of eggs laid was 91 ± 38 per individual; however, most of these eggs were nonviable. The average number of progeny developing to the L4 stage was 30 ± 25. The numbers of eggs produced when the animals were fed the Q8, Q9, or Q10 diets were similar to those fed OP50, although the viability of the eggs laid when the nematodes were fed Q9 or Q10 diets was lower. Therefore, with all diets, the clk-1(qm30) animals produced more eggs and more viable progeny when developing from the dauer stage than when developing from L1 larvae on the designated food. Assimilation of Dietary Q7 and Q8 by Nematodes—Considering the lack of viable progeny from the clk-1(qm30) animals fed the Q7-producing strain, it was important to determine whether these worms could assimilate this shorter isoform. N2, clk-1(e2519), and clk-1(qm30) animals were fed the Q7-producing bacteria or the isogenic Q8-producing strain from starved L1 larvae until adulthood. The lipids were extracted from these animals, and the quinones were quantified. There was no significant difference in the number of pmol quinones per mg protein of endogenous Q9 or RQ9 produced in the N2 animals whether they were fed the Q7-orQ8-producing bacteria (Fig. 3). There was slightly more Q8 than Q7 assimilated from the diet in the N2 animals, 101 ± 6 pmol Q8 per mg protein versus 65 ± 5 pmol Q7 per mg protein. The mutant strains clk-1(e2519) and clk-1(qm30) similarly accumulated Q7 and Q8 from their respective diets, in addition to the endogenously synthesized DMQ9 and RQ9. In three separate experiments, the levels of DMQ9 did not show a consistent trend when the animals were fed either food. Characterization of Subcellular Fractions—Although Q7 could be assimilated by all strains tested, the question remained whether it could be successfully transported to the mitochondria where it could function in mitochondrial respiration. Therefore, purified mitochondria were isolated from N2, clk-1(e2519), and clk-1(qm30) adults that had been fed different E. coli foods as starved L1 larvae. In addition to the expected purified mitochondrial band in the Nycodenz gradient, a denser band at the 25-30% region of the gradient was observed in all mitochondrial preparations from clk-1(qm30) animals. This band was also identifiable in mitochondrial preparations of clk-1(e2519) animals fed the Q-less E. coli strain GD1 (data not shown). These bands were separately extracted and identified as bottom bands. Western analysis was used to verify that mitochondrial components were enriched in the purified mitochondrial fractions and to assist in identifying the bottom band fraction. The β subunit of the F1-ATPase, a marker of the inner mitochondrial membrane, was found to be clearly present in the crude and purified mitochondrial fractions from both N2 and clk-1 animals and in the bottom band fraction from clk-1(qm30) animals (data not shown). Additionally, Western analysis verified the lack of hexokinase, a cytosolic marker, in both crude and purified mitochondrial fractions, and the absence of actin, a cytoskeletal marker, in purified mitochondria (data not shown). Unfortunately, antibodies generated against polypeptides localized to various other membranous organelles in other species failed to cross-react with expected homologues in C. elegans. Therefore, to determine the extent of contamination of mitochondria by other organelles, mannosidase II (Golgi), glucose 6-phosphatase (ER), and 5′-nucleotidase (plasma membrane) activities were each assayed in all subcellular fractions of clk-1(e2519) (Table II). Although the crude mitochondrial fractions showed an enrichment of the Golgi and ER membrane marker enzymes per mg protein compared with the post-nuclear supernatants, the purified mitochondrial fractions showed a substantial decrease in mannosidase II and glucose 6-phosphatase activities and an elimination of 5′-nucleotidase activity.Table IIEnzyme assaysclk-1 (e25 19) subcellular fractionMannosidase II activity (Golgi)Glucose 6-phosphatase activity (ER)5′-Nucleotidase activity (plasma membrane)nmol para-nitrophenol produced/hr/mg proteinnmol phosphate produced/min/mg proteinPost-nuclear supernatant8.27.71.5
Referência(s)