Key points are not available for this paper at this time.
A mutation in the gene gas-1 alters sensitivity to volatile anesthetics, fecundity, and life span in the nematode Caenorhabditis elegans. gas-1 encodes a close homologue of the 49-kDa iron protein subunit of Complex I of the mitochondrial electron transport chain from bovine heart.gas-1 is widely expressed in the nematode neuromuscular system and in a subcellular pattern consistent with that of a mitochondrial protein. Pharmacological studies indicate thatgas-1 functions partially via presynaptic effects. In addition, a mutation in the gas-1 gene profoundly decreases Complex I-dependent metabolism in mitochondria as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in mitochondria from gas-1 animals. There is no apparent alteration in physical structure in mitochondria from gas-1nematodes compared with those from wild type. These data indicate thatgas-1 is the major 49-kDa protein of complex I and that the GAS-1 protein is critical to mitochondrial function in C. elegans. They also reveal the importance of mitochondrial function in determining not only aging and life span, but also anesthetic sensitivity, in this model organism. A mutation in the gene gas-1 alters sensitivity to volatile anesthetics, fecundity, and life span in the nematode Caenorhabditis elegans. gas-1 encodes a close homologue of the 49-kDa iron protein subunit of Complex I of the mitochondrial electron transport chain from bovine heart.gas-1 is widely expressed in the nematode neuromuscular system and in a subcellular pattern consistent with that of a mitochondrial protein. Pharmacological studies indicate thatgas-1 functions partially via presynaptic effects. In addition, a mutation in the gas-1 gene profoundly decreases Complex I-dependent metabolism in mitochondria as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in mitochondria from gas-1 animals. There is no apparent alteration in physical structure in mitochondria from gas-1nematodes compared with those from wild type. These data indicate thatgas-1 is the major 49-kDa protein of complex I and that the GAS-1 protein is critical to mitochondrial function in C. elegans. They also reveal the importance of mitochondrial function in determining not only aging and life span, but also anesthetic sensitivity, in this model organism. general anesthetic-sensitive gene the first protein complex of the electron transport chain in mitochondria polymerase chain reaction base pair(s) green fluorescence protein enhanced GFP 4-morpholinepropanesulfonic acid bovine serum albumin number of ADP molecules converted to ATP per oxygen atom respired electron transport chain kilobase(s) succinate dehydrogenase neuromuscular junction acetylcholine NADH-ferricyanide reductase flavoprotein iron protein thenoyltrifluoroacetone, an inhibitor of electron transport through Complex II Volatile anesthetics are compounds that are used extensively to produce reversible unconsciousness and relief of pain. It is quite remarkable that their mechanism of action is not understood (1Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1637) Google Scholar, 2Koblin D.D. Miller R.D. Anesthesia. Churchill Livingstone, New York1990: 51-83Google Scholar). Our laboratory exploits a very simple animal model, the nematodeCaenorhabditis elegans, to investigate the molecular mechanism of volatile anesthetic action. We have established that the interactions of multiple genes are crucial in controlling the behavior of C. elegans in volatile anesthetics (3Morgan P.G. Sedensky M.M. Meneely P.M. Proc. Nat. Acad. Sci. 1990; 87: 2965-2968Crossref PubMed Scopus (96) Google Scholar, 4Morgan P.G. Sedensky M.M. Anesthesiology. 1994; 81: 888-898Crossref PubMed Scopus (57) Google Scholar). At least seven genes interact to control the response of C. elegans to volatile anesthetics (3Morgan P.G. Sedensky M.M. Meneely P.M. Proc. Nat. Acad. Sci. 1990; 87: 2965-2968Crossref PubMed Scopus (96) Google Scholar, 4Morgan P.G. Sedensky M.M. Anesthesiology. 1994; 81: 888-898Crossref PubMed Scopus (57) Google Scholar). Mutations in one gene,gas-1 (for generalanesthetic-sensitive)1cause hypersensitivity to all inhalation anesthetics tested as well as to ethanol. gas-1 overrides the effects of the other genes on sensitivity to volatile anesthetics. Nematodes with this mutation are also temperature-sensitive embryonic lethals, have a reduced life span, slow growth rates, and an increased sensitivity to the deleterious effects of free radicals and hyperoxia (5Hartman, P. S., Ishii, N., Kayser, E.-B., Morgan, P. G. and Sedensky, M. M. (2000) Mech. Ageing Dev., in press.Google Scholar). However, they move quite normally in air, indicating a functional neuromuscular system. Previously, we cloned the gas-1 gene and identified a point mutation in the allele fc21 (6Kayser E.-B. Morgan P.G. Sedensky M.M. Anesthesiology. 1999; 90: 545-554Crossref PubMed Scopus (135) Google Scholar). Sequence comparison strongly suggested that gas-1 encoded a homologue of the bovine 49-kDa(IP) subunit of NADH-ubiquinone oxidoreductase (Complex I), the first protein complex of the mitochondrial electron transport chain. Previous studies from other investigators indicated that Complex I was the most sensitive complex to inhibition by volatile anesthetics (7Cohen P.J. Anesthesiology. 1973; 39: 153-164Crossref PubMed Scopus (121) Google Scholar, 8Harris R.A. Munroe J. Farmer B. Kim K.C. Jenkins P. Arch. Biochem. Biophys. 1971; 142: 435-444Crossref PubMed Scopus (68) Google Scholar). The 49-kDa(IP) proteins are common to both the very complicated eukaryotic Complex I (41 different subunits) and to the much simplerParacoccus enzyme (only 15 subunits). Both enzyme complexes catalyze the same reaction, i.e. proton-pumping across the mitochondrial membrane, driven by the transfer of electrons from NADH to a quinone (9Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar, 10Xu X. Matsuno-Yagi A. Yagi T. Biochemistry. 1992; 31: 6925-6932Crossref PubMed Scopus (44) Google Scholar). A knockout mutant of the “49-kDa(IP) gene” inNeurospora completely lacked NADH-dehydrogenase activity, because the “matrix arm” of the enzyme complex failed to assemble (11Preis D. van der Pas J.C. Nehls U. Rohlen D.-A. Sackmann U. Jahnke U. Weiss H. Curr. Genet. 1990; 18: 59-64Crossref PubMed Scopus (38) Google Scholar). All of the Complex I mutants in Neurosporawere reported to have reduced growth rates, and their conidia were less viable. This is reminiscent of the reduced growth rate, life span, and brood size of fc21. The matrix arm of Complex I contains the binding site for NADH as well as all but one of the redox centers. Lastly, the 49-kDa(IP) subunit from Rhodobacter has been implicated in binding the head group of quinones (such as the electron acceptor, ubiquinone) and quinone-like inhibitors of Complex I. Thus, 49-kDa(IP) proteins seem to be essential for the core function of Complex I (10Xu X. Matsuno-Yagi A. Yagi T. Biochemistry. 1992; 31: 6925-6932Crossref PubMed Scopus (44) Google Scholar, 12Anderson W.M. Trgovcich-Zacok D. Biochem. Pharmacol. 1995; 49: 1303-1311Crossref PubMed Scopus (15) Google Scholar). gas-1(fc21) is the first known mutation of this subunit in animals. The fact that, in gas-1(fc21) animals, a strictly conserved amino acid residue is affected in a subunit essential for Complex I function suggests that the activity of the mutant Complex I is decreased or abolished. However, it has not been proven that the GAS-1 protein has a mitochondrial function. In addition, during the sequencing of the genome of C. elegans a second homologue of the 49 kDa(IP) subunit, T26A5.3, was identified (13The C. elegans Sequencing Consortium.Science. 1998; 282: 2012-2018Crossref PubMed Scopus (3634) Google Scholar). Because the relative importance of these two genes in mitochondrial function is not known, we studied their expression and the effects of thegas-1(fc21) mutation on mitochondrial function. In these studies we show that gas-1 is abundantly expressed in multiple tissues, including those most likely to confer anesthetic-induced immobility: neurons and body wall muscle. Expression of the second homologue of the 49-kDa(IP) subunit, T26A5.3, could not be detected via a promoter reporter. Pharmacological studies with aldicarb (14Miller K.G. Alfonso A. Nguyen M. Crowell J.A. Johnson C.D. Rand J.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12593-12598Crossref PubMed Scopus (340) Google Scholar) indicate that the effect of gas-1 on anesthetic sensitivity is partially presynaptic and most consistent with neuronal effects. We also show that isolated mitochondria fromgas-1 animals have reduced Complex I enzymatic activities, as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in gas-1(fc21) animals. These results confirm that GAS-1 is the major isoform of the 49-kDa(IP) subunit in Complex I, and that it is a crucial component of electron transport and oxidative phosphorylation in C. elegans. The conventions for C. elegansnomenclature have been followed throughout (15Horvitz H.R. Brenner S. Hodgkin J. Herman R.K. Mol. Gen. Genet. 1979; 175: 129-133Crossref PubMed Scopus (218) Google Scholar). Gene names are italicized 3-letter abbreviations followed by a hyphen and a number,e.g. gas-1, the gene. This designation can also specify worms homozygous for a mutation in this gene, e.g. gas-1, the mutant worm. The wild type allele is indicated by a superscript plus following the gene name, e.g. gas-1 +. Individual allele names are represented by a combination of one or two letters and a number, either alone or added in parentheses, e.g. fc21 or ingas-1(fc21). Non-italicized, all capital letters indicate the protein, e.g. the protein GAS-1. Brackets indicate transgenic constructs. For example, gas-1 + are animals carrying the wild type gas-1 gene as a result of microinjection into a mutant gas-1 background. The wild type C. elegans, N2, as well as the mutants mnDp1; unc-3(e151) andunc-7(e5) were from the in gas-1(fc21) was isolated in a for worms in P.G. Sedensky M.M. Anesthesiology. 1994; 81: 888-898Crossref PubMed Scopus (57) Google Scholar) of with is by were used for and of C. elegans and for gas-1 S. PubMed Google Scholar, R.K. of C. elegans. The Caenorhabditis elegans. Scholar). and were added to on an been as in studies (14Miller K.G. Alfonso A. Nguyen M. Crowell J.A. Johnson C.D. Rand J.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12593-12598Crossref PubMed Scopus (340) Google Scholar, M. Alfonso A. Johnson C.D. Rand J.B. 1995; PubMed Google Scholar). of the were on the Nematodes were through a for to the during a response been They were as no was seen for were as (3Morgan P.G. Sedensky M.M. Meneely P.M. Proc. Nat. Acad. Sci. 1990; 87: 2965-2968Crossref PubMed Scopus (96) Google Scholar, 4Morgan P.G. Sedensky M.M. Anesthesiology. 1994; 81: 888-898Crossref PubMed Scopus (57) Google Scholar). At least animals were of and the were from the to different in the gas-1 [gas-1 have been by microinjection as (6Kayser E.-B. Morgan P.G. Sedensky M.M. Anesthesiology. 1999; 90: 545-554Crossref PubMed Scopus (135) Google Scholar, D. J. PubMed Scopus Google Scholar, C. A. Caenorhabditis of an Scholar). The to their A of was with the gene to an for of The of the gas-1 was by the as a The an site on one of the and a site on the This was cloned into of a gene for green The with were into wild type were by were in the In these activity of promoter was reported by green was by The one to from green This is in with a In this a was the gas-1 promoter and the first of the through a site for a mitochondrial the of the and the of the gas-1 with the first the site and the These were by with the of the with a of and as and the to and the to the gene. The gene was cloned into II and of was by sequencing the was into mnDp1; were tested for sensitivity to For a of the the nematode mnDp1; gas-1 to mutant of gas-1 E.-B. Morgan P.G. Sedensky M.M. Anesthesiology. 1999; 90: 545-554Crossref PubMed Scopus (135) Google Scholar). The of the protein in animals that were in was by All were worms were in to with A was used for of type was added to the and for the was in a with a of the was The was through of and The mitochondrial was in and by The mitochondria was in of protein was by the with as a J. PubMed Google Scholar). of oxidative phosphorylation was as J. PubMed Scopus Google Scholar). oxygen was followed with a type to a via a of mitochondria was into of The following were added of ADP the mitochondria to either or succinate a in of ADP to and of to the and with as an electron for and to the point of oxygen for rates, control and were to studies B. J. Google Scholar, Scopus Google Scholar). of mitochondria were with and rates were for the following enzyme NADH-ferricyanide and succinate The first was for and were as by J. P. J. B. J. 1998; PubMed Scopus Google Scholar). mitochondria were for electron as by J. P. J. B. J. 1998; PubMed Scopus Google Scholar). as an sensitivity to aldicarb was decreased in sensitivity to was from that of is a aldicarb acetylcholine These are used in C. elegans to from effects of Our results indicate that least of the effect of gas-1 results from a presynaptic effect M. Alfonso A. Johnson C.D. Rand J.B. 1995; PubMed Google different from that for N2, of aldicarb and to of were from the response of to different of in the number of is in are were compared different from that for N2, in a of aldicarb and to of were from the response of to different of in the number of is in are were compared Previously, we were to the hypersensitivity of gas-1(fc21) to by a from the into an gas-1(fc21) animal (6Kayser E.-B. Morgan P.G. Sedensky M.M. Anesthesiology. 1999; 90: 545-554Crossref PubMed Scopus (135) Google Scholar). Thus, the not only the wild type but also a functional the in this gas-1 promoter is the of the gas-1 was cloned in of the of a green fluorescence protein, type transgenic for this show green fluorescence in that normally the The fluorescence was in the and widely in the wall and of the system were also with of the it to was expressed in the and expressed the of the promoter in these All of expressed the was seen as as GAS-1 is into a was that expressed an GAS-1 protein the control of the gas-1 We that to the mutant the protein the All mitochondrial proteins encoded in the an to be into the this is to be the protein In the of an by and G. 1990; PubMed Scopus Google Scholar) a site amino acid residue number Thus, the to be of this the other the into the of the of GAS-1 function. this the was to the first amino the site with these The was to the with a site and to have the of the GAS-1 of the gas-1 [gas-1 subcellular green fluorescence the seen with the promoter In body wall these are in to the This suggests mitochondrial of the protein. This pattern was seen in all of as as A with the to the of GAS-1 a pattern but not the anesthetic not The C. elegans sequencing (13The C. elegans Sequencing Consortium.Science. 1998; 282: 2012-2018Crossref PubMed Scopus (3634) Google Scholar) identified a to Because no of this gene are known, it is by The gene be a protein that is to GAS-1 and is to the bovine homologue transgenic for gas-1 gas-1 with the were in in all of In worms transgenic for a gene of the control of the gas-1 promoter were to anesthetic behavior not Thus, the control of the gas-1 the protein can GAS-1. that is a from a of wild type worms was with to either or the In both of the size were and indicating that is not of the with was for the were However, failed gas-1 were used on the with This of the with the from either gene. and is a promoter was to the one for We that the promoter of is of the but no the gene of this was of the this was into a wild type no fluorescence could be detected in the of not We that is expressed very or in the as the or phosphorylation of the of gas-1 on the transport of the chain. In electron as measured by oxygen and of ATP are by the across the mitochondrial from wild type can or succinate Biochem. Scholar). measured as oxygen is electron or and for the and are in Thus, the the of the and are by mitochondrial produce in electrons into the chain via Complex I. Complex I is by of these oxygen not In gas-1 with either or as the is in the wild type by and A and the was decreased in the gas-1 mutant and the other in mitochondria is enhanced relative to that of is by Complex II Complex I C. Biophys. PubMed Scopus Google atom different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from different from gas-1, different from different from gas-1, different from different from gas-1, different from gas-1, different from different from gas-1, different from different from gas-1, different from different from different from gas-1, different from different from gas-1, different from different from gas-1, different from gas-1, rates of isolated mitochondria are as the number of is in parentheses, mitochondria were with The in the of ADP by the one in the of ADP is the control is the number of ADP molecules converted to ATP per oxygen atom Both and are of the and ATP were compared of different from different from gas-1, in a rates of isolated mitochondria are as the number of is in parentheses, mitochondria were with The in the of ADP by the one in the of ADP is the control is the number of ADP molecules converted to ATP per oxygen atom Both and are of the and ATP were compared of We also to show that of gas-1 function could the in oxidative phosphorylation seen ingas-1(fc21). we measured metabolism in transgenic animals carrying the This is as an that it is not into the not all of a wild type and not all animals that as wild type the wild type gene in all We to for the by animals that strongly the i.e. that strongly green to the These animals be but are of the they of the or that are these in we compared mitochondria from these of to gas-1 as for Complex I-dependent metabolism were in mitochondria from the carrying the in Complex II metabolism was in mitochondria from gas-1 animals in the those from the the in the was to relative to that of control transport different from different from different from are as followed by the number of in All are is a first I, NADH-ferricyanide were compared different from in a are as followed by the number of in All are is a first I, NADH-ferricyanide were compared electron transport through of the chain. the results of the of these of Complex I reductase in and the electron transport from the NADH through Complex I, and Complex to the reductase in and electron transport through Complex I alone from NADH to is used of the because of in NADH-ferricyanide reductase in and is an activity of the flavoprotein of Complex I. from NADH are to the they could through the of Complex I. In the mutant gas-1, all Complex I-dependent are decreased and Thus, the mutant subunit electron transport through of Complex I function. The other electron transport not the of Complex I. The of reductase (Complex and (Complex are gas-1 and Thus, the mutation not the complexes of the chain. The of reductase in and reductase in were also and In to the results for oxidative of the electron transport Complex II were increased in the dehydrogenase is an activity of the two of Complex The activity electron through Complex II and Complex We were to the of the increase in Complex II-dependent oxidative phosphorylation seen in were by electron All by their However, the not of mitochondria but also including of or of as well as a number of the same of they were isolated from or gas-1 Thus, gas-1 not the of the the of in from both indicating that the seen in the mutant are not by a of or The of in the mitochondrial the as to the to the oxygen measured in the oxidative phosphorylation with the alone a of oxygen could not be by of of the used or by ADP or from N2, on the other were to all of the indicating that the of oxygen is mitochondrial in The is in for measured in the of In these we show that gas-1 was expressed and in consistent with and the mutation gas-1(fc21) mitochondrial function. We gene expression with function with the effects of and and mitochondrial function with of oxidative phosphorylation and electron transport chain is an of the acetylcholine of the neuromuscular junction mutation the of to is seen as to in the worm. Because gas-1 worms normally to the mutation not with acetylcholine Mutations or the of into the are seen as hypersensitivity or to aldicarb in the worm. gas-1 worms are to that the mutation decreases the of by the Thus, the effect of mutation is We also expression of gas-1 via microinjection of a promoter carrying green protein. genes normally in C. elegans and are during a C. A. Caenorhabditis of an Scholar). to the they are to gene J. J. C. A. 1999; PubMed Scopus Google Scholar). Thus, fluorescence from transgenic in C. elegans, genes are in the their are normally J. Google Scholar). Thus, the of fluorescence from the gas-1 the of fluorescence not that the is in a The pattern of that gas-1 is expressed in the and neurons of body and the subcellular of the the mutant is consistent with mitochondrial of the GAS-1 protein. Our results indicate that the second also is expressed in a GFP promoter M. G. 1994; PubMed Scopus Google we were to a or in this gene was expressed we were to an of Our is thatgas-1 is the gene for expression of the subunit in C. elegans. The oxidative phosphorylation the of mitochondria by following oxygen of for both electron transport and phosphorylation of ATP from ADP and Because both are by the established by complexes I, and oxygen is a of ATP In this we a in Complex I activity in the gas-1 mitochondria compared with those of Thus, oxidative phosphorylation and both indicate that GAS-1 is to the function of Complex I. The seen in the that the seen gas-1 and are the result of in the GAS-1 protein. We these data to indicate that GAS-1 is the functional 49-kDa(IP) subunit of Complex I in C. elegans. of Complex I function was two other were The of activity, and the increase of Complex II NADH-ferricyanide reductase is an activity of the flavoprotein of Complex I and is used as a of NADH dehydrogenase is known to function from the other the iron protein and the protein of bovine Complex I Rev. Biochem. PubMed Google Scholar). wild type GAS-1 protein, is a subunit of be for the flavoprotein activity per The fact that with activity of the suggests the mutant subunit of the Complex I or has an effect on the The has been for a knockout mutation of the 49-kDa(IP) homologue in U. C. Nehls U. A. T. Weiss H. Biophys. 1994; PubMed Scopus Google Scholar). Previously, we a of to those in this as a in NADH dehydrogenase B. U. J. PubMed Scopus Google Scholar). However, results show that a mutation in functional in Complex I the activity of NADH The fact that a subunit of activity of the of activity as the of The apparent increase of Complex II activity, seen as rates for oxidative suggests a of Complex However, a enzymatic could not be identified in the studies that with the in Complex II-dependent oxidative An increase in Complex II activity with in Complex I has been in with J. 1996; PubMed Scopus Google Scholar). It be to the mutant worms increase the of succinate for the of M. M. S. D. Nature. 1998; PubMed Scopus Google Scholar) studied a mutation in a subunit of Complex and that succinate dehydrogenase activity was decreased in animals. that animals are to hyperoxia and free and that life span is in both In S. J. J.C. S. J. 1999; 18: PubMed Scopus Google Scholar) that in gene, a mitochondrial protein and also have decreased Complex II-dependent to animals. is not to volatile anesthetics gas-1 are to these P.G. Sedensky M.M. Anesthesiology. 1994; 81: 888-898Crossref PubMed Scopus (57) Google P. S., Ishii, N., Kayser, E.-B., Morgan, P. G. and Sedensky, M. M. (2000) Mech. Ageing Dev., in press.Google Scholar). G. Morgan and M. M. Sedensky, rates of and anesthetic sensitivity are not in a simple There are a number of mitochondrial function anesthetic It is that the anesthetics Complex I-dependent metabolism a for is in gas-1 animals. In of this have indicated that Complex I is the most sensitive component of mitochondrial function to the effects of volatile anesthetics (7Cohen P.J. Anesthesiology. 1973; 39: 153-164Crossref PubMed Scopus (121) Google R.A. Munroe J. Farmer B. Kim K.C. Jenkins P. Arch. Biochem. Biophys. 1971; 142: 435-444Crossref PubMed Scopus (68) Google Scholar). We are in the of this by the effects of anesthetics on oxidative phosphorylation in We and for their was by We also and for we the of the of of for their in these
Kayser et al. (Mon,) studied this question.