From the Departments of Anesthesiology and Genetics,
University Hospitals and the § Departments of
Pharmacology and Medicine, Department of Veterans Affairs Medical
Center, Case Western Reserve University, Cleveland, Ohio 44106
Received for publication, December 8, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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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 that
gas-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-1
nematodes compared with those from wild type. These data indicate that
gas-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.
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 (1, 2). Our
laboratory exploits a very simple animal model, the nematode
Caenorhabditis 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 (3, 4).
At least seven genes interact to control the response of C. elegans to volatile anesthetics (3, 4). Mutations in one gene,
gas-1 (for general
anesthetic-sensitive)1
cause 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 (5). 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 (6). 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 (7, 8).
The 49-kDa(IP) proteins are common to both the very complicated
eukaryotic Complex I (41 different subunits) and to the much simpler
Paracoccus 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 (9-10). A knockout mutant of the "49-kDa(IP) gene" in
Neurospora completely lacked NADH-dehydrogenase
activity, because the "matrix arm" of the enzyme complex failed to
assemble (11). All of the Complex I mutants in Neurospora
were 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 (10, 12). 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 (13). Because the
relative importance of these two genes in mitochondrial function is not
known, we studied their expression and the effects of the gas-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 (14) 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 from
gas-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.
Nomenclature--
The conventions for C. elegans
nomenclature have been followed throughout (15). 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 in
gas-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.
Nematode Strains--
The wild type C. elegans, N2,
as well as the mutants mnDp1; unc-3(e151) and
unc-7(e5) were obtained from the Caenorhabditis Genetics Center in Minneapolis, MN. gas-1(fc21) was isolated
in a screen for immobile worms in 3.5% enflurane (4) after mutagenesis of N2 with ethylmethanesulfonate (N2 is immobilized by 6.5%
enflurane). Standard techniques were used for growing and maintaining
cultures of C. elegans and for constructing double mutants,
e.g. unc-7 gas-1 (16, 17).
Aldicarb/Levamisole Assay--
Aldicarb and levamisole were
added to agar plates on which an Escherichia coli lawn had
been grown as in previous studies (14, 18). 24 h after addition of
the drugs, nematodes were placed on the agar plates. Nematodes were
scored through a dissecting microscope for contracted immobility 2 h after exposure to the drugs, during which a steady-state response had
been reached. They were scored as immobile if no locomotion was seen
for 10 s. EC50 values were determined as described
previously (3, 4). At least 50 animals were scored at each
concentration of drug and the EC50 values were calculated
from the responses to five different drug concentrations, done in
duplicate, bracketing the EC50 values.
Microinjection--
Transgenic nematodes such as
unc-7 gas-1 [gas-1+;
rol-6(su1006)] have been created by microinjection as
previously described (6, 19, 20). The dominant mutation
rol-6(su1006) causes nematodes to roll about their
longitudinal axis. A plasmid containing rol-6(su1006) (gift
of Michael Krauss) was coinjected with the test gene to provide an
easily visible phenotype for selection of successful transformants.
gas-1 Promoter Reporter Assay--
The genomic DNA sequence
immediately upstream of the gas-1 coding sequence was
amplified by PCR using the cosmid K09A9 as a template. The primers
introduced an artificial ApaI site on one end of the
resulting 818-bp PCR fragment and a BamHI site on the other.
This sequence was cloned directionally into the ApaI-BamHI-linearized vector pEGFP1
(CLONTECH) upstream of a promoterless gene for
"enhanced green fluorescent protein" (EGFP) (Fig. 1A). The resulting plasmid pPrKE together with a
rol-6-containing plasmid were microinjected into wild type
N2 hermaphrodites. Transformed offspring were selected by picking
rolling F1 larvae. Stable transformants were picked in the F2
generation. In these transformants, transcriptional activity of the
gas-1 promoter was reported by green fluorescence, which was
observed by epifluorescence microscopy (Zeiss Axioscope, Chroma
long-pass filter set 41018). The filter set allows one to distinguish
yellowish gut granule autofluorescence from EGFP's green fluorescence.
This distinction is lost in pictures taken with a "colorblind"
confocal microscope.
Mutant Rescue with a Fluorescent Fusion Protein--
In this
study a translational fusion construct was made consisting of: 1) the
818-bp gas-1 promoter region and the first 123 bp of the
gas-1 coding sequence covering the translational start through 9 codons past a predicted cleavage site for a mitochondrial transit peptidase; 2) the complete coding sequence of EGFP omitting the
stop codon; and 3) the genomic sequence of the gas-1 locus, starting with the first codon after the predicted cleavage site and
extending 451 bp past the stop codon (Fig. 1A). These three subfragments were generated by PCR using primers having 15-mer overhangs with the sequence of the respective neighboring fragment. PCR
with a mix of fragments 1, 2, and 3 as template and the forward primer
to fragment 1 and the reverse primer to fragment 3 assembled the entire
gene. The complete gene was cloned into vector pBluescript II
K+ (Stratagene) and named pKEK. Correct assembly
of pKEK was confirmed by sequencing the construct.
pKEK was microinjected into mnDp1; unc-7 gas-1
yielding unc-7 gas-1[pKEK, rol-6] offspring, which were
tested for sensitivity to halothane. For a detailed description of the
rationale behind using the nematode strain, mnDp1; unc-7
gas-1 to demonstrate mutant rescue of gas-1 (see Ref.
6). The localization of the GAS-1::EGFP internal fusion
protein in animals that were mobile in 2% halothane was determined by
epifluorescence microscopy.
Preparation of Mitochondria--
All preparations were done at
4 °C. Clean worms were suspended in MSM-E (220 mM
mannitol, 70 mM sucrose, 5 mM MOPS, 2 mM EDTA, pH to 7.4 with KOH). A polytron (Brinkman
Instruments) was used for initial rupture: 20 s at 14,000 rpm. 5 mg/(g of worms) proteinase type XXVII (Sigma, St. Louis, MO) was added
to the homogenate and stirred for 10 min. Immediately afterward the
slurry was homogenized in a glass Potter/Elvehjem tissue grinder with a
Teflon pestle. After adding 1 volume of MSM-E containing 0.4% BSA, the
homogenate was centrifuged (300 × g, 10 min, 4 °C).
The supernatant was filtered through three layers of gauze and
recentrifuged (7000 × g, 10 min, 4 °C). The
mitochondrial pellet was resuspended in MSM-E and washed twice by
centrifugation (7000 × g, 10 min, 4 °C). The final
mitochondria pellet was resuspended in 100 µl of MSM-E. Total protein
was determined by the Lowry assay with BSA as a standard (21).
Oxidative Phosphorylation--
Polarigraphic measurement of
oxidative phosphorylation was performed as previously described (22).
Briefly, oxygen uptake was followed with a Clark type electrode (Yellow
Springs Instruments, YSI) connected to a chart recorder via a YSI
Oxygen monitor. 500 µg of mitochondria was injected into 500 µl of
air saturated incubation media (100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM potassium
phosphate, 1 mg/ml defatted BSA) kept at 30 °C. The following
substances were added sequentially: 50 nmol of ADP allowing the
mitochondria to exhaust internal substrates; either 10 mM
malate, 10 mM glutamate, or 20 mM succinate
providing a defined substrate in saturating concentration, then 50-100
nmol of ADP to determine state 3 and state 4 respiration rates; 100 nmol of 2,4-dinitrophenol to uncouple the mitochondria; and 5 mM ascorbate with 0.5 M TMPD as an electron donor for cytochrome c and to quickly determine the point of
0% oxygen saturation for each run. Oxygen uptake rates, respiratory control ratio, and ADP/O were calculated according to previous studies
(23, 24).
Electron Transport Chain (ETC) Assays--
Frozen ( Electron Microscopy--
Freshly prepared mitochondria were
prepared for transmission electron microscopy as described by
Hoppel et al. (25).
Aldicarb/Levamisole Assay--
Using contracted immobility as an
end point, sensitivity to aldicarb was decreased in gas-1
animals, whereas sensitivity to levamisole was unchanged from that of
N2 (Table I). Levamisole is a cholinergic
agonist, whereas aldicarb inhibits acetylcholine degradation. These
agents are used in C. elegans to distinguish pre- from
post-synaptic effects of mutations. Our results indicate that at least
part of the effect of gas-1 results from a presynaptic effect (18).
Promoter Reporter Assay--
Previously, we were able to rescue
the hypersensitivity of gas-1(fc21) to halothane by
introducing a 4.6-kb SfuI restriction fragment from the
cosmid K09A9 into an unc-7 gas-1(fc21) animal (6). Thus, the
fragment contained not only the wild type coding sequence for
gas-1 but also a functional promoter. To visualize the
tissues in which this gas-1 promoter is active, the 818-bp sequence immediately upstream of the gas-1 start codon
(gas-1 promoter) was cloned in front of the promoterless
coding sequence of a green fluorescence protein, EGFP. Wild type
nematodes made transgenic for this reporter construct show green
fluorescence in tissues that normally express the gas-1 gene.
The strongest fluorescence was regularly observed in the pharyngeal
muscles and widely distributed in the tail. Body wall muscles, vulva
muscles, and parts of the nervous system were also labeled, although
with more variability (Fig. 1).
Autofluorescence of the gut granules made it impossible to determine
whether EGFP was expressed in the intestinal cells. Gonads and eggs
never expressed the reporter, suggesting silence of the microinjected
gas-1 promoter in these tissues. All developmental stages of
larvae expressed the construct, which was seen as early as L1.
Rescue with a Fluorescent Fusion Protein--
To assess whether
GAS-1 is indeed directed into mitochondria, a plasmid, pKEK,
was constructed that expressed an EGFP-labeled GAS-1 protein under the
control of the gas-1 promoter. We reasoned that to rescue
the mutant phenotype, the GAS-1::EGFP fusion protein must
reach the mitochondria. All mitochondrial proteins encoded in the
nucleus require an N-terminal transit peptide to be routed into the
mitochondrion; this peptide is believed to be cleaved off once the
protein reaches its destination. In the case of GAS-1, an algorithm
described by Gavel and von Heijne (26) predicted such a cleavage site
after amino acid residue number 32. Thus, the EGFP tag had to be
inserted downstream of this site. On the other hand, inserting the tag
into the sequence of the mature part of GAS-1 might disrupt GAS-1's
function. To solve this potential dilemma, the fusion in
pKEK was designed to repeat the first 9 amino acids beyond
the cleavage site with EGFP sandwiched between these repeats (Fig.
2A). The rationale was to
provide the transit peptidase with a familiar peptide sequence around
its cleavage site and to have the full sequence of the mature GAS-1
downstream of the tag.
EGFP-containing transformants, unc-7 gas-1
[gas-1+-egfp; rol-6], showed subcellular
punctate green fluorescence rather than the fluorescence-filled cells
seen with the promoter reporter transformants (Fig. 2B). In
body wall muscle these fluorescing dots are neatly arranged in lines
parallel to the myofibrils. This suggests mitochondrial localization of
the fusion protein. This pattern was seen in all stages of development,
as early as L1. A simpler construct with the EGFP appended to the C
terminus of GAS-1 yielded a comparable staining pattern but did not
rescue the anesthetic phenotype (data not shown).
T26A5.3--
The C. elegans sequencing consortium (13)
identified a potential isogene to gas-1. Because no
mutations of this gene are known, it is designated by its associate
cosmid name: T26A5.3. The predicted gene product would be a protein
that is 96% identical to GAS-1 and is 65% identical to the bovine
homologue (transit peptides excluded). Worms transgenic for T26A5.3
(unc-7 gas-1 [T26A5.3; rol-6], originating from
mnDp1; unc-7 gas-1 microinjected with the cosmid T26A5),
were immobile in 2% halothane in all (12 out of 12) transformed lines.
In contrast, worms transgenic for a chimeric gene consisting of T26A5.3
coding sequence under the control of the gas-1 promoter were
rescued to normal anesthetic behavior (data not shown). Thus, when
under the control of the gas-1 promoter, the T26A5.3 protein
can functionally replace GAS-1.
To rule out that T26A5.3 is a non-transcribed pseudogene, RNA from a
mixed stage culture of wild type worms was amplified using RT-PCR with
primer pairs designed to either specifically amplify the
gas-1 mRNA or the presumed T26A5.3 mRNA. In both
cases products of the expected size were obtained (1633 and 1423 bp, respectively), indicating that T26A5.3 is transcribed (data not shown).
Re-amplification of the products with nested primers was successful if
primers specifically designed for the respective cDNA were
employed. However, amplification failed when gas-1 primers were used on the PCR product obtained with T26A5.3 primers (and vice
versa). This ruled out cross-reactivity of the primers with the
mRNA from either gene.
To visualize where and when T26A5.3 is expressed, a promoter reporter
construct was made analogous to the one for gas-1. We assumed that the promoter of T26A5.3 is situated upstream of the predicted start codon but no farther away than the next predicted gene
upstream of T26A5.3; this entire 1.5-kb fragment was placed upstream of
the EGFP code. When this construct was introduced into a wild type
nematode, no fluorescence above background could be detected in the
transformants at any stage of development (data not shown). We conclude
that T26A5.3 is naturally expressed at very low levels or in tissues
where the assay cannot detect it, such as the germline or gut.
Oxidative Phosphorylation--
Oxidative phosphorylation allows
assessment of the impact of gas-1 on the proton transport
capacity of the whole respiratory chain. In intact mitochondria,
electron transport, as measured by oxygen uptake (respiration), and
generation of ATP (phosphorylation) are tightly coupled by the proton
gradient across the inner mitochondrial membrane.
Mitochondria from wild type nematodes can metabolize malate, glutamate,
pyruvate, or succinate (27). Oxidative phosphorylation, measured as
oxygen uptake, is maximal when electron donor substrates (malate,
glutamate, pyruvate, or succinate) and substrates for the
F0F1-ATPase (ADP and inorganic phosphate) are
present in saturating concentrations (state 3 respiration). Thus, the
state 3 rate represents the maximum respiratory capacity of the
mitochondrion. Malate, glutamate, and pyruvate are oxidized by
mitochondrial enzymes, which produce NADH, which in turn feeds
electrons into the respiratory chain via Complex I. When Complex I is
blocked by rotenone, none of these substrates promotes oxygen uptake
(data not shown). In gas-1 state 3 respiration with either
glutamate, malate, or pyruvate/malate as the substrate is lower than in
the wild type by 66%, 64%, and 62%, respectively (Fig.
3A and Table
II). Similarly, the ADP/O ratio was
decreased in the gas-1 mutant (Fig. 3B and
Table II). On the other hand, in mitochondria from
gas-1animals, succinate-dependent state 3 respiration is enhanced relative to that of N2. Succinate is directly
oxidized by Complex II rather than Complex I (28).
We also wished to show that restoration of gas-1 function
could correct the defects in oxidative phosphorylation seen in
gas-1(fc21). Therefore, we measured metabolism in transgenic
animals carrying the translational fusion, pKEK, which
rescues gas-1(fc21). This fusion is carried as an
extrachromosomal array, that is, it is not incorporated into the
genome. Thus not all offspring of a rescued parent remain wild type and
not all animals that appear as wild type express the wild type gene in
all cells. We tried to enrich for the presence of
gas-1+ by picking animals that strongly express
the EGFP fusion, i.e. that strongly glow green under
fluorescence, to seed the culture flasks. These animals may be genetic
mosaics but are of the genotype unc-7 gas-1(pKEK rol-6);
they throw offspring of the identical genotype (albeit mosaic) or
offspring that are purely unc-7 gas-1. With these
limitations in mind, we compared mitochondria from these cultures of
mixed genotype to unc-7 gas-1 as controls (Table III). Values for Complex
I-dependent metabolism were significantly higher in
mitochondria from the line carrying the pKEK transgene than
in controls. Complex II metabolism was significantly higher in
mitochondria from unc-7 gas-1 animals than in the those from the pKEK strain. Also the ADP/O ratio in the rescued line
was restored to normal relative to that of control animals.
Electron Transport Chain (ETC) Assays--
ETC assays measure
electron transport through individual components of the respiratory
chain. Table III summarizes the results of the ETC assays. Three of
these provide measures of Complex I activity: 1) Rotenone-sensitive
NADH-cytochrome-c reductase (I-III in Fig.
4 and Table III) determines the electron
transport from the donor NADH through Complex I, ubiquinone (Q), and
then Complex III to the acceptor cytochrome c. 2)
Rotenone-sensitive NADH-decylubiquinone reductase (I in Fig.
4 and Table III) measures electron transport through Complex I alone
from NADH to decylubiquinone. Decylubiquinone is used instead of the
natural e
In the mutant gas-1, all three Complex
I-dependent activities are decreased (Fig. 4 and Table
III). Thus, the mutant subunit significantly impedes electron transport
through each measurable step of Complex I function. The other electron
transport steps examined do not require the participation of Complex I. The activities of antimycin A-sensitive
decylubiquinone-cytochrome-c reductase (Complex III), and
cytochrome-c oxidase (Complex IV) are indistinguishable between gas-1 and N2. Thus, the mutation does not affect the
downstream complexes of the respiratory chain. The activities of
succinate:cytochrome-c reductase (II-III in
Table III) and TTFA-sensitive succinate:DQ reductase (II in
Table III) were also unchanged between N2 and gas-1. In
contrast to the results for oxidative phosphorylation, none of the
electron transport activities involving Complex II (II-III,
II, SDH) were significantly increased in the
mutant. Succinate dehydrogenase (SDH) is an activity of the proximal
two subunits of Complex II. The II-III activity measures electron flow
through Complex II and Complex III. We were unable to identify the
specific cause of the increase in Complex II-dependent
oxidative phosphorylation seen in gas-1(fc21).
Electron Microscopy--
Mitochondrial preparations were examined
by electron microscopy. All preparations contained intact mitochondria,
identifiable by their cristae. However, the preparations did not
consist exclusively of mitochondria but also contained debris,
including shreds of cuticle or broken mitochondria, various vesicles of
undetermined origin, as well as a small number of bacteria.
Mitochondria looked the same regardless of whether they were isolated
from N2 or gas-1 (Fig. 5).
Thus, gas-1 does not visibly alter the morphology of the
organelle. Furthermore, the amount of contamination appears equal in
preparations from both sources, indicating that the lower specific
activities seen in the mutant are not caused by a higher fraction of
non-mitochondrial or non-nematode proteins.
The presence of E. coli in the mitochondrial preparations
raises the question as to the bacterial contribution to the oxygen consumption measured in the oxidative phosphorylation assay. Controls with the food bacteria alone had a constant rate of oxygen uptake, which could not be modulated by addition of any of the substrates used
or by ADP or 2,4-dinitrophenol. Mitochondria preps from N2, on the
other hand, were responsive to all of the above, indicating that the
majority of oxygen uptake is mitochondrial in origin. The bacterial
contamination is probably responsible, in part, for respiratory rate
measured in the absence of substrates.
In these studies, we show that 1) gas-1 was expressed
and functioned in tissues consistent with its phenotype; and 2) the mutation gas-1(fc21) significantly disrupted mitochondrial
function. We evaluated gene expression with EGFP constructs, cellular
function with the pharmacological effects of levamisole and aldicarb,
and mitochondrial function with measurements of oxidative
phosphorylation and electron transport chain activity.
Levamisole is an agonist of the acetylcholine receptor of the
neuromuscular junction (NMJ). Any mutation directly impairing the
ability of muscle cells to contract is seen as resistance to levamisole
in the intact worm. Because gas-1 worms react normally to
levamisole, the mutation does not noticeably interfere with muscle
contraction/function. Aldicarb inhibits acetylcholine (ACh) esterase.
Mutations increasing or decreasing the release of ACh into the NMJ are
seen as hypersensitivity or resistance, respectively, to aldicarb in
the intact worm. gas-1 worms are slightly resistant to
aldicarb, suggesting that the mutation decreases the amount of ACh
released by the motorneurons. Thus, the main effect of the
gas-1 mutation is presynaptic.
We also examined cellular expression of gas-1 via
microinjection of a promoter reporter construct carrying green
fluorescent protein. Microinjected genes normally form extrachromosomal
tandem arrays in C. elegans and are lost during mitosis at a
high frequency (20). Furthermore, due to the repetitive nature they are
subject to gene silencing (29). Thus, fluorescence patterns resulting from transgenic constructs commonly differ between individuals. Furthermore, in C. elegans, microinjected genes are silenced
in the germline, even if their chromosomal counterparts are normally active (30). Thus, although the presence of fluorescence indicates transcription from the gas-1 promoter, the absence of
fluorescence does not rule out that the chromosomal gas-1
promoter is active in a tissue. The observed pattern of its reporter
indicates that gas-1 is expressed in the muscles and neurons
of pharynx, body wall, and vulva. Furthermore, the subcellular
distribution of the translational fusion construct, which rescues the
mutant phenotype, is consistent with mitochondrial localization of the
GAS-1 protein.
Our results indicate that the second 49-kDa(IP)-like gene,
T26A5.3, also is expressed in N2. Using a GFP
promoter reporter (31), we were unable to find a time or tissue in
which this gene was expressed even though we were able to obtain an
RT-PCR product of its predicted message. Our interpretation is that
gas-1 is the predominant gene for expression of the
49-kDa(IP)-like subunit in C. elegans.
The oxidative phosphorylation assay determines the respiration capacity
of intact mitochondria by following oxygen uptake under saturating
concentrations of substrates for both electron transport and
phosphorylation (generation of ATP from ADP and inorganic phosphate).
Because both processes are tightly coupled by the proton gradient
established by complexes I, III, and IV, oxygen uptake is a measure of
ATP generation. In this case also, we see a profound decrease in
Complex I activity in the gas-1 mitochondria compared with
those of N2. Thus, oxidative phosphorylation assays and ETC assays both
indicate that GAS-1 is integral to the function of Complex I. The
return toward normal values seen in the rescued line pKEK
confirms that the differences seen between gas-1 and N2 are
the result of alterations in the GAS-1 protein. We interpret these data
to indicate that GAS-1 is indeed the functional 49-kDa(IP) subunit of
Complex I in C. elegans. Although impairment of Complex I
function was expected, two other findings were not: The decrease of NFR
activity, and the increase of Complex II activity.
NADH-ferricyanide reductase (NFR) is an activity of the flavoprotein
(FP) subcomplex of Complex I and is used as a measure of NADH
dehydrogenase activity. NFR is known to function even after perchlorate
treatment, which dissociates FP from the other subcomplexes, the iron
protein (IP) and the hydrophobic protein of bovine Complex I (32).
Therefore, wild type GAS-1 protein, which is a subunit of IP, cannot be
directly necessary for the flavoprotein (FP)-dependent NFR
activity per se. The fact that gas-1(fc21)
interferes with activity of the FP suggests the mutant subunit prevents
proper assembly of the entire Complex I or has an allosteric effect on
the FP. The former has been described for a knockout mutation of the
49-kDa(IP) homologue in Neurospora crassa (33). Previously,
we interpreted a profile of ETC changes similar to those in this study
as a deficit in NADH dehydrogenase (34). However, our present results
show that a mutation in another functional unit (IP) in Complex I may
affect the activity of NADH dehydrogenase. The fact that a subunit of
IP affects activity of FP should caution against the interpretation of
NFR activity as directly reflecting the status of FP subunits.
The apparent increase of Complex II activity, seen as higher state 3 rates for oxidative phosphorylation, suggests a compensatory up-regulation of Complex II. However, a specific enzymatic change could
not be identified in the ETC studies that correlated with the change in
Complex II-dependent oxidative phosphorylation. An increase
in Complex II activity associated with defects in Complex I has been
described in patients with Leber's Hereditary Optic Neuropathy (35).
It would be interesting to know whether the mutant worms actually
increase the use of succinate for the regeneration of ATP.
Ishii et al. (36) studied mev-1, a mutation in a
subunit of Complex II, and presented indirect evidence that succinate
dehydrogenase activity was decreased in mev-1 animals.
Hartman et al.(5) showed that mev-1 and
gas-1 animals are hypersensitive to hyperoxia and free
radical damage, and that life span is shortened in both strains. In
contrast, Felkai et al. (37) showed that mutations in the clk-1 gene, which alter a mitochondrial protein and also
have decreased Complex II-dependent metabolism, give rise
to long-lived animals. Additionally, mev-1 is not
hypersensitive to volatile anesthetics whereas gas-1 and
clk-1 are hypersensitive to these drugs (4,
5).2 Therefore, rates of
metabolism, aging, and anesthetic sensitivity are not related in a
simple manner.
There are a number of plausible hypotheses explaining how mitochondrial
function may affect anesthetic sensitivity. It is possible that the
anesthetics acutely decrease Complex I-dependent metabolism
below a threshold necessary for mobility, which is reached more easily
in gas-1 animals. In support of this possibility, previous
reports have indicated that Complex I is the most sensitive component
of mitochondrial function to the effects of volatile anesthetics (7,
8). We are in the process of testing this possibility by measuring the
effects of anesthetics on oxidative phosphorylation in N2 and
gas-1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 °C)
samples of mitochondria preparations were solubilized with cholate, and
zero order rates were determined spectrophotometrically for the
following enzyme activities: Citrate synthase,
rotenone-sensitive NADH-cytochrome-c reductase,
succinate-cytochrome-c reductase, antimycin A-sensitive
decylubiquinol-cytochrome-c reductase, NADH-ferricyanide reductase, rotenone-sensitive NADH-decylubiquinone reductase, succinate-DCPIP reductase, and duroquinone-stimulated TTFA-sensitive succinate DCPIP reductase. The first order rate constant was determined for cytochrome-c oxidase. Assays and calculations were
performed as described by Hoppel et al. (25).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Aldicarb/levamisole doses causing contracted immobility
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Fig. 1.
gas-1 promoter reporter expression in wild
type C. elegans. N2 worms expressing the
enhanced green fluorescence protein (EGFP) under the control of the
gas-1 promoter. A, view of the ventral nerve
cord, the major nerve tract in C. elegans, along with its
associated cell bodies. The inset shows the structure of the
promoter construct. B, a higher magnification of a
neuromuscular junction (NMJ). Rhomboid-shaped muscle cells send
extensions of their membrane to the axons of the nerve cord as they
pass.
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Fig. 2.
GAS-1::EGFP internal fusion protein
expression in C. elegans. The coding sequence for
the fluorescent tag (EGFP) was inserted into the gas-1 gene,
between the sequence for the mitochondrial transit peptide and the main
part of the mature GAS-1 (inset, B). This
construct was expressed in worms carrying the gas-1(fc21)
mutation and rescued the halothane hypersensitivity of fc21
to normal. A, the head of a worm. Fluorescent mitochondria
illuminate the pharynx and are seen extending forward in the snout.
B, the parallel rows of mitochondria in body wall
muscle.
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Fig. 3.
Oxidative phosphorylation of isolated
mitochondria. A, the state 3 rate is a measure of the
respiratory capacity of mitochondria under maximal phosphorylating
conditions (ATP production). Electrons derived from glutamate and
malate enter the respiratory chain exclusively via complex I while
those from succinate enter via complex II. B, ADP/O is a
measure of the efficiency of ATP production equivalent to the number of
ATP molecules produced per electron pair transferred to oxygen. Note
that both the rate of metabolism of Complex I-dependent
substrates and the efficiency of ATP production are reduced in the
mutant. Values were compared using ANOVA. The asterisk
indicates values different from N2; significance was defined as
p < 0.05.
Oxidative phosphorylation
Electron transport assays
-acceptor, ubiquinone, because of its better
solubility in water. 3) NADH-ferricyanide reductase (NFR in
Fig. 4 and Table III) is an activity of the flavoprotein moiety of
Complex I. Electrons from NADH are diverted to the artificial
e
-acceptor ferricyanide before they could pass through
the rotenone-inhibitable section of Complex I.
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Fig. 4.
Electron transport chain assays for Complex
I. The electron transport activities I-III, I, and NFR (see Table
III) of mitochondria isolated from wild type N2 (solid bars)
and mutant gas-1 (open bars) have been normalized
to citrate synthase (a) and to cytochrome-c
oxidase (b) activity measured in the same samples. Averages
and standard deviations of the normalized data then have been expressed
relative to wild type activity, thus the value for N2 is always 100%.
The abbreviations are defined in the legend of Table III. Values were
compared using Student's t test. The asterisk
indicates values different from N2; significance was defined as
p < 0.05.
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Fig. 5.
Electron micrographs of mitochondrial
preparations. Mitochondrial preparations from wild type N2
(A) and mutant gas-1 nematodes (B).
The scale bar represents 1 µm. No morphologic difference
between wild type and mutant preparations was seen.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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ACKNOWLEDGEMENTS |
---|
We thank Art Zinn, Helen Salz, and Phil Hartman for their helpful discussions. Electron microscopy was done by Medhat Hassan. We also thank Judy Preston, Shawna Boyd, Kalpana Patel, and Hiral Patel for providing invaluable technical assistance. Finally, we thank the members of the Department of Anesthesiology at University Hospitals of Cleveland for their ongoing support in these studies.
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FOOTNOTES |
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¶ Supported by the Veterans Administration Hospital Medical Services and by National Institutes of Health Grant PO1 AG15885.
To whom correspondence should be addressed: Dept. of
Anesthesiology, 2400 Bolwell Bldg., University Hospitals, 11100 Euclid Ave., Cleveland, OH 44106. Tel.: 216-844-7334; Fax: 216-844-3781; E-mail: margaret.sedensky@uhhs.com.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M011066200
* This work was supported in part by National Institutes of Health Grants GM58881 and GM45402 (to M.M.S. and P.G.M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 P. G. Morgan and M. M. Sedensky, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: gas-1, general anesthetic-sensitive gene; Complex I, the first protein complex of the electron transport chain in mitochondria; PCR, polymerase chain reaction; bp, base pair(s); GFP, green fluorescence protein; EGFP, enhanced GFP; MOPS, 4-morpholinepropanesulfonic acid; BSA, bovine serum albumin; ADP/O, number of ADP molecules converted to ATP per oxygen atom respired; ETC, electron transport chain; kb, kilobase(s); SDH, succinate dehydrogenase; NMJ, neuromuscular junction; ACh, acetylcholine; NFR, NADH-ferricyanide reductase; FP, flavoprotein; IP, iron protein; TTFA, thenoyltrifluoroacetone, an inhibitor of electron transport through Complex II.
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