From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095
Received for publication, February 1, 2001, and in revised form, March 7, 2001
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ABSTRACT |
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Ubiquinone (coenzyme Q or Q) is an essential
component of the mitochondrial respiratory chain in eukaryotic cells.
There are eight complementation groups of Q-deficient
Saccharomyces cerevisiae mutants designated
coq1-coq8. Here we report that COQ8 is
ABC1 (for Activity of
bc1 complex), which was originally
isolated as a multicopy suppressor of a cytochrome b
mRNA translation defect (Bousquet, I., Dujardin, G., and Slonimski,
P. P. (1991) EMBO J. 10, 2023-2031). Previous studies
of abc1 mutants suggested that the mitochondrial
respiratory complexes were thermosensitive and function inefficiently.
Although initial characterization of the abc1 mutants
revealed characteristics of Q-deficient mutants, levels of Q were
reported to be similar to wild type. The suggested function of Abc1p
was that it acts as a chaperone-like protein essential for the proper
conformation and functioning of the bc1 and its
neighboring complexes (Brasseur, G., Tron, P., Dujardin, G., Slonimski,
P. P. (1997) Eur. J. Biochem. 246, 103-111).
Studies presented here indicate that abc1/coq8 null
mutants are defective in Q biosynthesis and accumulate
3-hexaprenyl-4-hydroxybenzoic acid as the predominant
intermediate. As observed in other yeast coq mutants,
supplementation of growth media with Q6 rescues the abc1/coq8 null mutants for growth on nonfermentable carbon
sources. Such supplementation also partially restores
succinate-cytochrome c reductase activity in the
abc1/coq8 null mutants. Abc1/Coq8p localizes to the
mitochondria, and is proteolytically processed upon import. The
findings presented here indicate that the previously reported
thermosensitivity of the respiratory complexes of abc1/coq8 mutants results from the lack of Q and a general deficiency in respiration, rather than a specific phenotype due to dysfunction of the
Abc1 polypeptide. These results indicate that ABC1/COQ8 is
essential for Q-biosynthesis and that the critical defect of abc1/coq8 mutants is a lack of Q.
Ubiquinone (coenzyme Q or
Q)1 is a prenylated
benzoquinone found in both prokaryotic and eukaryotic membranes (1). In
prokaryotes Q is found in the plasma membrane and functions in
respiratory electron transport as well as in disulfide bond formation
of periplasmic membrane proteins (2, 3). In eukaryotes Q is a an
integral and essential component in mitochondrial electron and proton
transport in the respiratory complexes I, II, and III (4). In addition to its function in respiratory electron transport, Q forms a critical link between a wide variety of other Q-linked dehydrogenases, including
those acting on fatty acids, glycerol 3-phosphate, dihydroorotate, choline, sarcosine, and dimethylglycine (5). Recently, Q has been
identified as an essential component of the H+ pumping
function of UCP1, an uncoupling protein involved in thermogenesis (6).
UCP1 depends on the availability of oxidized Q (QH2 is inactive), suggesting that the redox state of Q is important in regulating uncoupling activity. It is intriguing that another mechanism
for regulating mitochondrial membrane potential, the permeability
transition, is inhibited by Q and either inhibited or stimulated by
various Q derivatives (7, 8). The permeability transition results in
the opening of an unidentified, proteinaceous channel, releasing matrix
contents with a molecular mass of up to 1500 Da. The opening of this
channel reflects mitochondrial dysfunction, and is one of several
pathways leading to programmed cell death (9).
Q is found in membranes throughout eukaryotic cells (10). In the plasma
membrane, Q is involved in an enzyme-mediated trans-plasma membrane electron transport system that can reduce extracellular compounds such as the ascorbyl radical (11-13). By virtue of its ability to redox cycle, Q also has the capacity to function as a lipid
soluble antioxidant, either directly as a chain-terminating antioxidant
or indirectly by reducing the Studies of Q-biosynthesis in both prokaryotes and eukaryotes have
enabled the elucidation of a putative Q-biosynthetic pathway (1, 15,
16). Tzagoloff and Dieckmann (17) described eight complementation
groups of Q-deficient Saccharomyces cerevisiae mutants,
coq1-coq8. The COQ1 gene product catalyzes
the formation of the polyprenyldiphosphate precursor tail of Q (18).
The COQ2 gene product catalyzes the attachment of the
polyprenyldiphosphate tail to 4-hydroxybenzoate, leading to the
formation of the early Q-biosynthetic intermediate
3-hexaprenyl-4-hydroxybenzoic acid (HHB) (19). From HHB, a series of
ring modifications produce Q and require the COQ3,
COQ4, COQ5, COQ6, COQ7, and
COQ8 genes (20). Yeast coq3-coq8 mutants lack Q
and accumulate HHB as the predominant intermediate (21). Coq3p is
located in the mitochondrial matrix, peripherally associated with the
inner membrane and catalyzes both O-methylation steps in Q
biosynthesis (22). Coq5p functions as a
C-methyltransferase in the mitochondria (23, 24). Coq6p is
likely to function as a flavin-dependent monooxygenase,
adding one or more of the ring oxygens (20). Coq7p is tightly
associated with the mitochondrial inner membrane, and is required for
the last monooxygenase step in Q biosynthesis (25, 26). Coq4p is
located in the mitochondrial matrix peripherally associated with the
inner membrane (27). Although the precise functions of Coq4p and Coq7p
in Q biosynthesis are not known, genetic evidence suggests that many of
the Coq polypeptides may function in a multisubunit complex converting
HHB to Q (27, 28).
Previous characterization of a coq8-1 mutant (strain C130)
showed it to be respiratory defective, Q-deficient (29), and to
accumulate HHB as the predominant intermediate (21). This study reports
the isolation of the COQ8 gene and its identification as the
previously described ABC1 gene. ABC1 was
originally isolated as a multicopy suppressor of a cytochrome
b mRNA translation defect (30). Studies of
abc1 mutants have suggested that Abc1p acts as a
chaperone-like protein essential for function of the
bc1 complex (31). The data presented here show
that Abc1/Coq8p is required for Q biosynthesis. The implications of our
findings on the functional role of ABC1/COQ8 and its gene
product are discussed.
Strains and Growth Media--
The strains used in this study are
listed in Table I. Growth media for yeast
were prepared as described (34) and included YPD (1% yeast extract,
2% peptone, 2% dextrose), YPE (1% yeast extract, 2% peptone, 2%
ethanol), YPGal (1% yeast extract, 2% peptone, 2% galactose), YPG
(1% yeast extract, 2% peptone, 3% glycerol), SDC (0.18% yeast
nitrogen base without amino acids, 2% dextrose, 0.14%
NaH2PO4, 0.5%
(NH4)2SO4, complete amino acid supplement), SD-Leu (SDC minus leucine), and SD-Ura (SDC minus uracil).
The complete supplement was modified so that the final concentration of
each component was as described (23). Semisynthetic lactate media was
prepared as described (35). Media for sporulation and tetrad analysis
were prepared according to Adams et al. (34). All components
of growth media were purchased from Difco, Fisher, or Sigma. 2% Agar
was added for solid media.
In Vivo Labeling of Q Intermediates, Lipid Extraction, and
Analysis by HPLC--
Yeast strains CH130-A1 (coq8-1) and
FY Cloning of the COQ8 Gene--
CH130-E1 (coq8-1) yeast
were grown to early-log phase in YPD medium and transformed as
described (38) with a YEp24-based plasmid library of S. cerevisiae genomic DNA (39) containing the URA3 gene as
a selectable marker. A total of 44,000 transformants were obtained by
selection on SD-Ura plates and, after a 3-day incubation at 30 °C,
were replica plated onto YPG plates to test for respiratory growth.
Thirty-eight colonies were complemented for respiratory deficiency as
indicated by growth on YPG plate media. Eleven of the putative
Q-prototrophic transformants were further purified and tested for
cosegregation of uracil prototrophy and respiration competency
following vegetative growth in rich media. All 11 showed co-segregation
of the Ura Disruption of the COQ8 Gene--
Two separate deletion alleles
of ABC1 were prepared. In both cases, a 1.1-kb
AflII/SunI fragment of p3HN4 was replaced (after preparation for blunt end ligation) with either a 1.1-kb
HIS3 or 0.8-kb TRP1 fragment, derived from YDp-H
or YDp-W, respectively (41). The resulting plasmids, pHQ8 Rescue of coq Mutants with Exogenous Q6--
Yeast
cells were grown overnight in 5 ml of YPD to saturation. Overnight
cultures were then diluted into 30 ml of YPE, in 125-ml Erlenmeyer
flasks, to a density of ~0.25 A600 nm, with or
without 15 µM Q6 supplementation.
Q6 supplementation increased the absorbance of the culture
by ~0.01 A600 nm. These cultures were
subsequently incubated at 30 °C with shaking (200 rpm). Growth was
monitored by A600 nm measurements over a period
of 8 days. Samples were taken after 8 days of growth and plated on YPD
plates to confirm that cultures were not contaminated.
Determination of Succinate-cytochrome c Reductase
Activity--
Yeast cells were grown overnight in 5 ml of YPGal
containing 0.1% dextrose. Overnight cultures were diluted into 100 ml
of YPGal (0.1% dextrose) to a density of ~0.004 A600
nm. Cell cultures were grown and harvested at ~4
A600 nm. Yeast crude mitochondrial fractions
were prepared as described (35) and aliquots were stored frozen at
Thermosensitivity Assays--
Crude mitochondria from various
yeast strains of the CEN.PK2-1C background were prepared and stored as
described above. Cytochrome c oxidase activities were
assayed at 30 °C in 40 mM sodium phosphate buffer, pH
7.4, containing 25 µM reduced cytochrome c
(43, 44). Horse heart cytochrome c was reduced in 40 mM sodium phosphate buffer, pH 7.4, with
L-ascorbate, and the ascorbate was subsequently removed by
dialysis. The reaction was initiated by adding 25 µg of mitochondrial
protein and the reaction rate was monitored by following the oxidation
of cytochrome c at 550 Mitochondrial Import Assay--
An in vitro
transcription template plasmid was constructed by inserting the
ABC1/COQ8 open reading frame into the pBluescript SK(+)
vector downstream of the T7 promoter. The ABC1/COQ8 open reading frame was polymerase chain reaction-amplified from pTM with 5'
primer pHAC8-1
(5'-ACGCACGCGTCGACTTATGGTTACAAATATGGTGAA-3') and 3'
primer p2H8R (5'-CGGAATTCTTAAACTTTATAGGCAAAAATC-3') with Vent DNA
polymerase. The polymerase chain reaction product was gel-purified and kinase-treated and inserted into the EcoRV
site of pBluscript SK(+) vector. The resulting clone, pBCOQ8, was
linearized by XbaI and served as template for in
vitro transcription (Promega Ribomax Large Scale RNA Production
System). The resulting mRNA was then translated using Promega Flexi
Rabbit Reticulocyte Lysate System in the presence of
[35S]methionine (1000 Ci/mmol, at final concentration of
0.75 µM). Both the mRNA and the synthesized
polypeptides were stored at Plasmid Construction of HA-tagged ABC1/COQ8--
Two yeast
expression plasmids, one single copy and one multiple copy, were
constructed to express the Abc1/Coq8 polypeptide containing a
carboxyl-terminal peptide (MYPYDVPDYASLDGPMST) corresponding to the
carboxyl terminus of the influenza hemagglutinin (HA) viral protein, an
epitope for the 12CA5 monoclonal antibody (47). Construction began by
directional cloning using SalI and NotI sites in
the plasmid pADCL (48). The ABC1/COQ8 open
reading frame region without the stop codon was polymerase chain
reaction-amplified using 5' primer pHAC8-1 (described above) and 3'
primer pHAC8-2 (5'-ATAAGAATGCGGCCGCAGAACTTTATAGGCAAAAATCT-3'). The
polymerase chain reaction product was directionally cloned into the
multiple copy pADCL vector (SalI and NotI at 5'
and 3', respectively), to generate pHA8-1, a multiple copy vector
containing the ABC1-HA construct. pRSHA8-1, a single copy version of
the ABC1-HA construct, was generated by removing the 3.6-kb
transcriptional cassette from pHA8-1 by BamHI partial
digestion and insertion into the BamHI site of the vector
pRS316 (40). Yeast abc1/coq8 mutants were transformed with
pHA8-1, pRSHA8-1, or pTM (42). Transformants were selected for the
presence of either the LEU2 gene (pHA8-1) or the
URA3 gene (pRSHA8-1) on SD-Leu or SD-Ura plate media.
Colonies obtained on the respective selective plate media were
subsequently replica plated to YPG plate media. The Abc1-HA epitope
fusion protein retained activity as assayed by the ability of either the single or multicopy plasmid construct to rescue
abc1/coq8 null mutant yeast strains for growth on
media containing a nonfermentable carbon source (YPG plates, data not
shown). Yeast strain CH130-A1 harboring pHA8-1, pRSHA8-1, or pTM was
used for subcellular localization by Western analysis.
Cell Lysis and Fractionation--
Cell cultures (1 liter)
were grown in semisynthetic lactate media to saturation density. Cells
were lysed and fractionated as described (35). Briefly, spheroplasts
were prepared and lysed by Dounce homogenization with a tight fitting
pestle. Total cell lysates were separated from nuclei and unlysed cells
at 1,500 × g for 5 min. Crude mitochondria were
separated from the cytosolic fraction by centrifugation of cell lysates
at 12,000 × g for 10 min. Purified mitochondria were
prepared using a linear Nycodenz gradient as described (35).
Western Analysis--
Fractions were assayed for protein
concentration by the bicinchoninic acid assay. Equal amounts of protein
from the mitochondrial fractions of cells containing the plasmids pTM,
pRSHA8-1, or pHA8-1 were analyzed by electrophoresis on 12% Tris
glycine gels and subsequently transferred to Hybond ECL nitrocellulose.
Western analysis and membrane stripping were performed as described by Amersham Pharmacia Biotech. An exception to the stated protocol was the
use of Western washing buffer: 10 mM Tris, pH 8.0, 154 mM NaCl, 0.1% Triton X-100. The primary antibodies
recognizing the HA epitope or F1 The coq8-1 Mutant Lacks Q and Accumulates a Polar Intermediate That
Co-elutes with HHB--
The coq8-1 mutant strain
C130 (G75 complementation group) fails to respire or grow on
nonfermentable carbon sources (17). The Q-deficient phenotype in the
coq mutants is defined by a profound stimulation of both
NADH- and succinate-cytochrome c reductase activities by the
addition of decyl-Q, Q2, or Q6 to in
vitro assays (29, 49). To study the biochemical defect responsible
for Q-deficiency, D27310-B/A1, a wild-type strain, or strain CH130-A1 (coq8-1) were grown in SDC media in the presence of
[U-14C]4-hydroxybenzoic acid, the aromatic ring precursor
of Q. Lipid extracts were prepared, fractionated by normal phase HPLC,
and monitored for 14C radioactivity (Fig.
1). In lipid extracts of wild-type yeast, the two predominant peaks of radioactivity correspond to Q6
and to the polar Q-biosynthetic intermediate HHB (Fig. 1A;
Ref. 37). Lipid extracts of CH130-A1 (coq8-1) lack
Q6 and contain one large peak of radioactivity in fraction
29 that co-eluted with HHB. HHB accumulates in cultures of CH130-A1
grown in the presence of
[carboxyl-14C]4-hydroxybenozic acid (data not
shown). HHB is present at high levels in wild-type yeast strains grown
in media containing 2% glucose (SDC) and harvested at log phase (37),
and in each of the coq3, coq4, coq5,
coq6, and coq7 mutant strains (Refs. 21 and 25,
and data not shown).
The Yeast coq8-1 Mutant Is Complemented by the ABC1
Gene--
To characterize further the nature of the defect in the
coq8-1 mutant, the COQ8 gene was cloned by
transformation of CH130-E1 (coq8-1) with a yeast genomic DNA
library as described under "Experimental Procedures." Yeast plasmid
DNA was recovered from each of 11 independent clones and found to
contain overlapping segments of genomic DNA. One clone, pTM, contained
an 8-kb insert and transformation of CH130-E1 with pTM restored growth
on YPG media. Partial sequence determination of the insert DNA in pTM
showed the insert to contain the previously characterized
ABC1 gene (for Activity of
bc1 complex) (Ref. 30;
GenBankTM accession number X59027). A subclone containing
the ABC1 gene on a 4.0-kb NheI/HpaI
fragment (Fig. 2) complemented the
glycerol growth deficiency of CH130-E1. The ABC1 gene
encodes a 501-amino acid polypeptide that is required for respiration
and was originally isolated as a multicopy suppressor of a cytochrome
b mRNA translation defect (30). Characterization of an
abc1 null mutant showed the phenotype to be reminescent of
Q-deficient mutants, yet a quinone deficiency was ruled out at that
time (31).
Yeast abc1 Null Mutants Lack Q and Accumulate HHB--
To further
investigate the relationship of ABC1 to the
coq8-1 mutation, a one-step gene replacement procedure (50)
was used to obtain the abc1 null mutant strains W303
To investigate whether the abc1 null mutation impairs the
biosynthesis of Q, the yeast abc1 deletion strain FY
Q-deficient yeast mutants are characteristically hypersensitive to
treatment with polyunsaturated fatty acids. This phenotype is
attributed to the absence of Q and its role in protecting cells from
oxidative stress due to autoxidation of the polyunsaturated fatty acids
(21, 51). When stressed with the polyunsaturated linolenic acid, the
abc1/coq8 null mutants also displayed this hypersensitive
phenotype (data not shown), consistent with a deficiency in Q and its
role as an antioxidant.
Rescue of the abc1 Null Mutant by Q6
Supplementation--
The Q-deficient strains coq3
The effect of the addition of Q6 on the restoration of
succinate cytochrome c reductase activity (complex II + III)
was also investigated. As shown in Fig.
4, addition of Q6 to the
growth media partially restored succinate-cytochrome c
reductase activity in both coq3 Thermosensitivy of abc1 Mutants--
The findings presented
above indicated that abc1 mutants are Q-deficient.
Characterization of abc1 mutants by Brasseur et al. (31) indicated that the activities of the various
mitochondrial respiratory complexes, including cytochrome c
oxidase, were thermosensitive when compared with wild-type. Considering
the defect is in Q availability, these findings are intriguing, as they
imply that Q itself may play a role in the proper assembly and/or
stability of these complexes. To investigate this possibility,
thermosensitivity assays were performed with mitochondria prepared from
late log phase (A600 nm = 4.0) cultures of
wild-type (CEN.PK2-1C) and the abc1 mutant. For these
studies, the thermosensitivity of complex IV (cytochrome c
oxidase) activity was determined as described under "Experimental Procedures." Our results, obtained with mitochondria isolated from
late log phase cultures, indicated that complex IV activities were
similarly thermosensitive in both wild-type and the abc1 mutant strain (Fig. 5A).
Similar thermosensitivity assays were then performed with mitochondria
isolated from stationary phase cultures of CEN.PK2-1C wild-type, or
from the respiratory-deficient mutants abc1,
coq3, cor1, or atp2. Cytochrome
c oxidase activity present in wild-type mitochondria was
less thermosensitive when compared with mitochondria isolated from
Q-deficient mutants (abc1 and coq3) or from a
respiratory deficient control cor1 mutant as well as an
atp2 null (ATP2 encodes the Localization of Abc1p to the Mitochondria--
Previous studies
have suggested that the Abc1 polypeptide is located in the mitochondria
(30, 31). In vitro mitochondrial import assays were
performed to determine whether the putative mitochondrial leader
sequence of Abc1p functions to direct mitochondrial import. The Abc1p
35S-labeled in vitro translation products
(Fig. 6, lane 1) generated an
~56-kDa precursor and several smaller products. Upon incubation with
mitochondria, a new polypeptide corresponding to the mature form of
Abc1p (53 kDa) accumulated (lane 2). The mature form was protected from treatment with proteinase K (lane 3), and the
addition of Triton X-100 rendered the mature product susceptible to
proteinase K treatment (lane 4). The processing of Abc1p was
dependent on the membrane potential, as treatment with valinomycin
inhibited the generation of mature Abc1p and its import (lanes
5 and 6).
A carboxyl-terminal fusion of the Abc1 coding region with the
hemagglutinin viral protein epitope (HA) was constructed in order to
determine the subcellular localization of Abc1p. Both single copy and
multicopy constructs expressing the Abc1-HA polypeptide were able to
rescue W303 The findings presented in this paper demonstrate that
COQ8 encodes a mitochondrial protein necessary for Q
biosynthesis and is allelic to the previously described ABC1
gene. ABC1 was initially isolated as a multicopy suppressor
of a cytochrome b mRNA translation defect due to the
cbs2-223 mutation (30). Cbs2p is a translational activator
of the long untranslated leader in the mRNA of COB, a
mitochondrial gene encoding cytochrome b (55). Yeast strains with either cbs2 null or cbs2-223 mutations fail
to respire due to a defect in translation of cytochrome b.
The ABC1 gene in multicopy cannot function in place of
CBS2 because suppression is specific for the
cbs2-223 mutation. Initial characterization of
abc1 mutants revealed characteristics similar to Q-deficient
mutants (30). However, in the assays performed by Brasseur et
al. (31) no Q deficiency was detected in abc1 mutant
strains. Further analysis of abc1 mutants revealed that
various complexes of the mitochondrial respiratory chain, including
complexes II, III, and IV, were thermosensitive when compared with
complexes from wild-type cells. Based on this information, Abc1p was
hypothesized to function as a chaperone-like protein essential for the
proper conformation and efficient functioning of cytochrome
b and complex III (31).
The results presented here show that Q is not detectable in either
coq8-1 or abc1 Previous work by Brasseur et al. (31) showed the various
complexes of the respiratory chain were more thermosensitive in mitochondria isolated from stationary phase cultures of abc1
mutant strains compared with wild-type strains. In light of the Q
biosynthetic defect, this result suggested that the proper assembly
and/or stability of these complexes may depend on Q availability.
However, the findings presented here indicate that the
thermosensitivity of the respiratory complexes in the abc1
mutant strain is a general characteristic of respiration deficiency
rather than a specific function of either the ABC1 gene or Q
itself. When mitochondria were isolated from log phase cells, the
cytochrome c oxidase was similarly thermosensitive in both
the wild-type and the abc1 mutant strain. However, in
stationary phase cultures, an enhanced thermosensitivity of cytochrome
c oxidase was observed in each of the respiratory deficient
mutants when compared with wild-type mitochondria. In general,
respiratory deficient mutants have defects in activating genes
repressed by glucose (26, 57). Glucose repression is a global
regulatory system in S. cerevisiae that affects expression of genes involved in gluconeogenesis, alternative sugar metabolism, and
respiration (58). Hence, the observed thermosensitivity of cytochrome
c oxidase observed in mitochondria isolated from the
respiratory deficient cells may result from the defects in the glucose
derepression regulatory system.
The results presented here argue against the model that the Abc1
polypeptide acts as a chaperone essential for the assembly or stability
of the respiratory complexes (30, 31). However, the multicopy
ABC1 suppression of the cytochrome b mRNA
translation defect in cbs2-223 mutants remains an intriguing
observation. Could higher levels of Q, hypothetically resulting from
the ABC1 gene present at multicopy, mediate the suppression
of the cbs2-223 mutation? In this model the function of the
mutant Cbs2p as a translational activator of COB mRNA
would be rescued, albeit inefficiently, by an increased amount of Q
acting as a cofactor. Another scenario is that increased levels of Q
might be expected to stabilize a very low amount of cytochrome
b polypeptide produced in the cbs2-223 mutant.
Although cytochrome b in the cbs2-223 mutant is
undetectable in whole cell absorption spectra (59), it has been
hypothesized that low levels might be translated (30). One prediction
of these models of Q as cofactor (either with Cbs2 in translational activation or in stabilizing cytochrome b) is that
supplementation of media with Q6 would rescue the
respiratory defect in mutants harboring the cbs2-223 allele.
Alternatively, the suppression of the cbs2-223 allele could
be mediated by a function of Abc1p distinct from its role in Q
biosynthesis. However, since exogenous Q6 restores growth
of the abc1 null mutant on media containing a nonfermentable
carbon source, such a secondary function of Abc1p would not be
essential for translation of COB mRNA or assembly of the
bc1 complex.
Unlike Cbs2, a protein that appears to be specific to S. cerevisiae (60), proteins with amino acid sequence similarity to ABC1 have been identified in many species of eukaryotes,
including human, mouse, and Caenorhabditis elegans (61). The
ABC1 homologs from Schizosaccharomyces pombe (62)
and Arabidopsis thaliana (61) show functional conservation
to ABC1 in S. cerevisiae, since both were
isolated by complementation of the S. cerevisiae abc1
mutant. These studies strongly suggest Q biosynthesis in these
eukaryotes is highly conserved, while the suppression of the
cbs2-223 mutation is not. The respiratory chain complexes in
the S. pombe abc1Sp::LEU2 mutant were
severely affected since cytochrome aa3 was
undetectable, cytochrome c oxidase activity was decreased
7-fold, and the mutant cells showed very slow growth on glucose
containing media (62). Thus Q deficiency in S. pombe produced drastic effects on respiratory complexes and growth. These
respiratory defects were rescued by transformation with either the
S. pombe or S. cerevisiae ABC1 gene. In S. cerevisiae abc1 mutants, the ABC1 homolog of A. thaliana only partially restored the activity of
succinate-cytochrome c reductase and NADH-oxygen oxidoreductase (complexes I-IV), because levels of activity were only
24 and 13% of wild type (61). It seems likely that this observed
inefficient rescue is due to incomplete restoration of Q biosynthesis.
A similar phenomenon of partial restoration of Q biosynthesis was
observed in the coq3 S. cerevisiae mutant rescued with a
human COQ3 homolog (63). In this case, Q levels in the coq3 yeast mutant harboring the human COQ3
cDNA on a multicopy plasmid were only 20% of wild type, and Q was
undetectable when the human COQ3 cDNA was present on a
single copy plasmid.
The specific function of Abc1p in the biosynthesis of Q has yet to be
defined. Like many mutants in the Q-biosynthetic pathway, abc1 mutants accumulate HHB as the predominant intermediate
(21). It has been suggested that the accumulation of this single
intermediate may result from the role of these polypeptides in a
multisubunit protein complex involved in the biosynthesis of Q (28).
Yeast abc1 mutants have greatly reduced levels of Coq3p and
its corresponding O-methyltransferase activity (28). These
findings suggest that Abc1p may be an essential component of this
complex, required for one or more of the various monooxygenase and/or
methylation steps in the modification of the quinone ring. However, it
is unlikely that Abc1p is itself catalyzing this step as analysis of
the encoded amino acid sequence does not reveal any similarities to any
known hydroxylases or methyltransferases. Interestingly, both
prokaryotic and eukaryotic ABC1 homologs possess conserved kinase motifs belonging to a class of eukaryotic serine/threonine protein kinases (64). One prokaryotic homolog of ABC1 is
E. coli ubiB, a gene required for the first monooxygenase
step in Q-biosynthesis; the conversion of 2-octaprenylphenol to
2-octaprenyl-6-hydroxyphenol. E. coli ubiB mutants lack Q
and accumulate 2-octaprenylphenol as the predominant intermediate
(65-67). Based on the conserved kinase motifs in UbiB, it has been
suggested that UbiB plays an indirect role in the first monooxygenase
step of Q biosynthesis, perhaps activating proteins necessary for
monooxygenase activity via phosphorylation (67). The analogous
hydroxylation step in Q biosynthesis in yeast catalyzes the
hydroxylation of HHB to 3,4-hydroxy-5-hexaprenylbenzoic acid. Based on
the homology between Abc1p and UbiB, and their involvement in Q
biosynthesis, it is tempting to speculate that Abc1p may share a
similar function in yeast Q biosynthesis. Work is ongoing to
investigate this possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopheroxyl radical (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Genotypes and sources of S. cerevisiae strains
ABC1 (abc1
::TRP1) were grown in
1 liter of SDC media supplemented with 0.65 µCi of [U-14C]p-hydroxybenzoic acid (365 Ci/mol),
synthesized from L-[U-14C]tyrosine by alkali
heat fusion (36). After 3 days of incubation at 30 °C with shaking
(200 rpm), cultures were harvested (A600 nm = 10), and lipids were extracted as described (25). Approximately 10-12% of the 14C-radiolabel added to the cultures was
recovered in the lipid extracts. Extracts were concentrated and
aliquots containing 104 cpm were analyzed by normal phase
HPLC employing a cyanopropyl column (Zorbax, 5 µm, 4.6 mm × 250 mm, MacMod Analytical, Chadds Ford, PA) as described by Poon et
al. (37). The column was equilibrated with a mobile phase composed
of 98% solvent A (hexane) and 2% solvent B (isopropyl
alcohol/hexane/water/methylene chloride, 52:41:5:2) at a flow rate of 1 ml/min. Ten minutes after sample injection, the percentage of solvent B
was increased linearly from 2 to 27% in 25 min (35 min from the start)
and then from 27 to 100% in 20 min (55 min after the start). In the
next 5 min, percentage of solvent B was decreased linearly from 100 to
2% and remained at 2% for 30 min to equilibrate the column before next sample injection. The radioactivity of 1-ml fractions were measured by scintillation counting in 10 ml of BIOsafe nonaqueous scintillation mixture (Research Products International) with a Beckman
scintillation counter (model LS-3133P).
and YPG
phenotypes, indicating
these traits were plasmid linked. Yeast plasmid DNA was recovered from
each transformant and each of the 11 clones contained overlapping
segments of genomic DNA inserts as confirmed by restriction enzyme
mapping and Southern analysis (data not shown). One clone, pTM, was
chosen for further study. A 4.0-kb HpaI/NheI
fragment of pTM containing the ABC1 gene was isolated from
pTM, processed for blunt end ligation and inserted into a
SmaI-digested CEN-based vector pRS316 (40) generating p3HN4.
Transformation of CH130-E1 with p3HN4 complemented the glycerol growth deficiency.
and
pWQ5
, were each used to obtain a linear
BamHI/ClaI fragment with the disrupted gene. W303-1A was transformed with 1 µg of a linear fragment from pHQ8
by the procedure of Elble (42). Most of the histidine
prototrophic clones from the transformations were respiration-defective
and were complemented by the
o strain, JM8, but not by
the coq8-1 strain CH130-E1. Nuclear DNAs from 8 independent
transformants were digested with BamHI and ClaI
and separated on 1% agarose. The probe, a 4-kb
HpaI/NheI fragment from pTM, hybridized to three
fragments of 1.6-, 1.4-, and 1.2-kb in the genomic DNA of wild-type
strain W303-1A. The genomic DNA of the mutant strain (W303
ABC1)
contained two fragments of 1.4 and 2.8 kb as expected for the disrupted
allele. Alternatively, linearized pWQ5
was used to transform FY251.
Most of the tryptophan prototrophic clones issued from the
transformations were respiration-defective and were complemented by the
o strain JM8, but not by the coq8-1 strain
CH130-E1. Nuclear DNA from one transformant was digested with
BamHI and ClaI and separated on 1% agarose. The
probe, a 4-kb HpaI/NheI fragment from pTM, hybridized to three fragments of 1.6, 1.4, and 1.2 kb in the genomic DNA of FY251. The genomic DNA of the mutant strain (FY
ABC1)
contained two fragments of 1.4- and 2.5-kb, as expected for the
disrupted allele.
80 °C. Succinate-cytochrome c reductase activities were
assayed at 30 °C in 40 mM sodium phosphate buffer, pH
7.4, containing 20 mM sodium succinate, 500 µM EDTA, 250 µM potassium cyanide, and 50 µM horse heart cytochrome c (43). Briefly,
12.5 µg of mitochondrial protein was incubated in buffer without
cytochrome c for 15 min. The reaction was initiated by
addition of cytochrome c and monitored at 550
540 nm
for 3 min. Specific cytochrome c reductase activities were
determined from the initial linear rate of reaction at 550
540 nm using an extinction coefficient of 18.5 mM
1 cm
1 (43). All assays were
performed in triplicate. For assays in the presence of added
Q6, mitochondria were incubated 15 min in buffer containing
10 µM Q6, 1% EtOH, prior to initiation by
addition of cytochrome c. This concentration of
Q6 was sufficient to saturate the succinate-cytochrome
c reductase activity.
540 nm for 30 s (44).
Cytochrome c oxidase activities were determined from the
initial linear rate of reaction using an extinction coefficient of 18.5 mM
1 cm
1 (43). To determine the
thermosensitivity of cytochrome c oxidase activities,
mitochondria were incubated at 37 °C and over time aliquots were
assayed. All assays were performed in triplicate.
80 °C. The isolation of mitochondria
(from D273-10B) and import reaction conditions were performed based on
Yaffe (45) and described in Hsu et al. (46). Each import
reaction contained 14 µl of radiolabeled in vitro
translated product and mitochondria containing 200 µg of protein.
Valinomycin was added to a final concentration of 10 µg/ml where
appropriate. Following 30 min of incubation at 30 °C, the
mitochondria were reisolated and washed once. Triton X-100 and
proteinase K treatment after the import was performed by adding 1%
Triton X-100 and/or 50 µg/ml proteinase K, final concentration, to
resuspended mitochondria. The proteolytic digest was allowed to proceed
at 0 °C for 20 min and was terminated by the addition of
phenylmethylsulfonyl fluoride at a final concentration of 1 mM. The samples were mixed with sample buffer and heated at
100 °C for 5 min prior to analysis by SDS-polyacrylamide
electrophoresis (12% polyacrylamide) and autoradiography.
-ATPase were used in a
1:10,000 dilution. Horseradish peroxidase-linked secondary antibodies
to rabbit and mouse IgG were used in a 1:1000 dilution.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
Yeast coq8 mutants lack
Q6 and accumulate a polar intermediate that corresponds to
HHB. Lipid extracts were prepared from D27310-B/A1 (wild-type
parent, Panel A), CH130-A1 (coq8-1 allele,
Panel B), or from FY ABC1 (abc1/coq8 deletion,
Panel C) and separated by normal phase HPLC as described
under "Experimental Procedures." 1-ml fractions were collected and
14C radioactivity determined by scintillation counting.
Q6 and HHB standards eluted in fractions 7-9 and 28-30,
respectively.
View larger version (11K):
[in a new window]
Fig. 2.
Restriction map and strategy of
ABC1 deletion. A map of the 4.0-kb
HpaI-NheI fragment in pTM is presented. The
long black arrow indicates the position of the
ABC1 open reading frame and its direction of transcription.
Also present on this fragment is the amino-terminal two thirds of the
PRP43 gene (YGL120C), with transcription proceeding in a
direction opposite to ABC1. Deletion constructs were
prepared by replacing a 1.1-kb AflII/SunI
fragment (hatched bar) with either the HIS3 or
TRP1 gene as described under "Experimental Procedures."
Restriction sites are abbreviated and correspond to the following DNA
restriction enzymes: A, AflII; B,
BamHI; H, HindIII; Hp,
HpaI; N, NheI; P,
PvuII; S, SunI.
ABC1
and FY
ABC1 (Fig. 2). These yeast strains are either histidine or
tryptophan prototrophic, respectively, and are respiration deficient.
Both were complemented by a
o strain, but not by the
CH130-E1 (coq8-1) strain. These results imply that the
abc1 null and the coq8-1 mutant are allelic. To verify this, W303
ABC1 was mated to CH130-E1(coq8-1), and
the diploid cells were transformed with pTM to maintain the respiratory competency necessary for sporulation. Tetrads from this cross (28 complete sets) were dissected and segregated 2:2 for leucine and
histidine prototrophy. After selection on 5-fluoro-orotic acid
plate media for loss of pTM, all 28 sets of tetrads failed to grow on
YPG and were
+, confirming the allelism between the
ABC1 gene and the original coq8-1 mutation.
ABC1
was grown in the presence of [U-14C]4-hydroxybenzoic acid
and lipid extracts were separated by normal phase HPLC. As shown in
Fig. 1C, no Q was detectable in the abc1 null
mutant strain, and HHB accumulated as the sole predominant intermediate, as observed for the coq8-1 mutant strain (Fig.
1B). Together these data show that ABC1 is the
same as COQ8 and is essential for the biosynthesis of Q. Henceforth the gene will be called ABC1.
and
coq7
can be rescued for growth on ethanol, a
nonfermentable carbon source, by supplementation of liquid media with
Q6 (26). Such rescue by exogenous Q6 provides strong evidence that the sole defect leading to lack of growth is the
availability of Q itself. To test whether the abc1 null mutant can be rescued for growth by the addition of exogenous Q6, wild-type yeast and various respiratory deficient
mutants were incubated in growth media containing ethanol in the
presence or absence of 15 µM Q6. Fig.
3 shows that both coq3
and
abc1
mutants failed to grow on media containing ethanol,
yet growth can be rescued by exogenous Q6. Although both
strains showed slower growth in Q-supplemented media relative to the
wild-type strain, the same final cell density was eventually achieved.
As expected, the addition of exogenous Q6 did not rescue
the respiratory deficient mutant cor1
(an essential
subunit of the bc1 complex) on a nonfermentable carbon source. Since the growth defect of the abc1
mutant
strain can be completely suppressed by the addition of Q6,
the defect is due to Q availability.
View larger version (21K):
[in a new window]
Fig. 3.
Exogenous Q6 rescues the
abc1 and coq3
yeast mutants. Yeast strains CEN.PK2-1C (wild
type:
and
), CEN
ABC1 (abc1
:
and
),
CEN
COQ3 (coq3
:
and
), and CEN
COR1
(cor1
:
and
) were grown in YPE supplemented
without (
,
,
, and
) or with (
,
,
, and
) 15 µM Q6 as described under "Experimental
Procedures." Samples were taken over a period of 8 days and growth
monitored by A600 nm measurements.
and abc1
mutant strains, but failed to restore this activity in the
cor1
mutant. A similar trend was observed when the
Q6 was added to isolated mitochondria prior to the assay of
succinate-cytochrome c reductase activities. The greatest
degree of rescue was observed when Q6 was present during
growth and also included in the subsequent in vitro assay.
The extent of rescue by exogenous Q6 is similar in
magnitude to the observed decyl-Q-mediated restoration of
succinate-cytochrome c reductase activity in isolated mitochondria by Brasseur et al. (31).
View larger version (38K):
[in a new window]
Fig. 4.
Q6 rescues the
succinate-cytochrome c reductase activity in
abc1 and coq3
mutants. Wild-type and mutant cells (CEN.PK2-1C
background) were grown in liquid media containing galactose as carbon
source (YPGal + 0.10% dextrose) in the presence or absence of 15 µM Q6. Mitochondria were isolated and
succinate-cytochrome c reductase activities were measured in
the presence or absence of 10 µM Q6 as
described under "Experimental Procedures." No addition of
Q6, (
); addition of Q6 to isolated
mitochondria (
); addition of Q6 to growth media (
);
and addition of Q6 to both growth media and isolated
mitochondria (
). The level of activity in the CEN.PK2-1C wild type
strain, 522 nmol cytochrome c reduced min
1 mg
of protein
1, has been set at 100%.
subunit of the
ATPase) mutant (Fig. 5B). The data indicate that the
stationary phase thermosensitivity of cytochrome c oxidase
in the abc1 mutant strain is a general characteristic of
respiration deficiency rather than a specific function of Abc1p.
View larger version (19K):
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Fig. 5.
Thermosensitivity of cytochrome
c oxidase activities. Wild-type ( ),
abc1 (
), coq3 (
), atp2 (
),
and cor1 (
) mutants (CEN.PK2-1C background) were grown
in YPGal (0.10% dextrose) and cultures harvested at late log phase
(4.0 A600 nm, Panel A) or from
stationary phase cultures (greater than 10 A600
nm, Panel B). Crude mitochondria were prepared and
thermosensensitivity of cytochrome c oxidase activities were
determined as described under "Experimental Procedures." Briefly,
mitochondria were incubated at 37 °C and samples were assayed for
cytochrome c oxidase activities at various time points.
Activities are expressed as a percentage of the initial cytochrome
c oxidase activities before heat shock, defined as 100%. In
Panel A initial activities were 2244 and 1628 nmol of
cytochrome c oxidized min
1 mg of
protein
1, for the CEN.PK2-1C wild type and
abc1 mutant, respectively. In Panel B, initial
activities were 2128 cytochrome c oxidized
min
1 mg of protein
1, for the CEN.PK2-1C
wild type, and 509, 113, 143, and 25 nmol of cytochrome c
oxidized min
1 mg of protein
1, for the
abc1, coq3, cor1, and atp2
mutants, respectively.
View larger version (47K):
[in a new window]
Fig. 6.
In vitro mitochondrial import and
proteolytic processing of the Abc1 polypeptide. In
vitro mitochondrial import assays were performed as described
under "Experimental Procedures." Lane assignment: Lane
1, 10% of the radiolabeled in vitro translation
product used in each import reaction. Lane 2, in
vitro translation product incubated with isolated mitochondria.
Lane 3, same as lane 2 but including a
post-import proteinase K treatment. Lane 4, same as
lane 3 except Triton X-100 was added to disrupt the
mitochondria before proteinase K treatment. Lanes 5 and
6, in vitro translation product was incubated
with mitochondria in the presence of the uncoupler valinomycin (10 µg/ml final concentration). After the incubation, the reisolated
mitochondria were either treated with proteinase K, as in lane
5, or were directly analyzed in lane 6. The samples
were mixed with sample buffer and heated at 100 °C for 5 min prior
to analysis by SDS-polyacrylamide electrophoresis (12% polyacrylamide)
and autoradiography. P and M indicate the
positions of the precursor and mature form of Abc1p, respectively. The
mitochondrial leader sequence of Abc1p is typical of mitochondrial
leader sequences (52) and contains a 3-amino acid consensus sequence
motif (designated by 10,
8, and
5) common to leader sequences of
mitochondrial matrix polypeptides (53). Based on this motif, the
predicted mature amino terminus is designated as +1.
ABC1 for growth on media containing a nonfermentable
carbon source (data not shown). Western analysis of yeast subcellular
fractions indicated that the Abc1-HA fusion protein cofractionates with
the mitochondria (Fig. 7). Although the
expressed Abc1-HA fusion protein was targeted to mitochondria and
rescued the abc1 mutant when present at single copy,
submitochondrial localization studies revealed the majority of
Abc1-HA-tagged polypeptide was found as an insoluble form, resistant to
extraction with Triton X-100 in the presence of high salt. This problem
was also observed upon analysis of other single copy HA-tagged
constructs, including Coq6, Coq7, and
Coq4.2 A similar problem has
been reported in studies of COX3 mRNA-specific translational activator proteins (54). Submitochondrial localization studies will require antisera that recognize the non-tagged Abc1p.
View larger version (20K):
[in a new window]
Fig. 7.
Abc1p localizes to mitochondria.
W303 ABC1 mutant yeast harboring the plasmids pTM (N),
pRSHA8-1 (S), or pHA8-1 (M) were generated as
described. pTM is a multiple copy plasmid containing the wild-type
ABC1 gene. pRSHA8-1 and pHA8-1 are single and multiple copy
plasmids, respectively, harboring the HA-tagged ABC1 gene to
express Abc1p with a carboxyl-terminal epitope tag from the
hemagglutinin viral protein. These yeast strains were grown, lysed,
and fractionated as described under "Experimental Procedures."
50 µg of protein from the total cell lysates (Total
Lysate), cytosolic fractions (Cyto), or mitochondrial
fractions (Mito) were subjected to Western analysis and
visualized by chemiluminescence detection using purified 12CA5
antibodies to the HA-epitope tag (A) and antibodies to the
mitochondrial protein F1
-ATPase (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants. This was determined by
labeling yeast cells with the Q-biosynthetic intermediate
[U-14C]4-hydroxybenzoic acid, subsequent extraction of
lipids, and separation of Q and Q-intermediates by HPLC. This
finding contradicts the previous characterization of abc1
mutants (31), in which a method of detection and measurement of Q
relied on difference spectra of reduced versus oxidized
total lipid extracts (56). It is possible that this latter method may
be prone to interference from other components within the lipid
extracts. Growth of the abc1 null mutant and other
coq null mutants on a nonfermentable carbon source was
rescued by supplementation of media with Q6 (Fig. 3). Such
Q6 supplementation also restored, at least in part, the
activity of succinate cytochrome c reductase. These findings indicate that a defect in Q biosynthesis is responsible for the respiratory deficiency in abc1 mutants.
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ACKNOWLEDGEMENTS |
---|
We thank Wayne Poon for help with the analysis of 14C-labeled yeast lipid extracts and the members of the Clarke laboratory, and Drs. Pamela L. Larsen, Patrice Hamel, and Carlos Santos-Ocaña for helpful comments and suggestions. The yeast coq mutant strains were generously provided by Dr. Alexander Tzagoloff, Columbia University.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM45952 (to C. F. C.).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.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive E., Los Angeles, CA 90095-1569. Tel.: 310-825-0771; Fax:
310-206-5213; E-mail: cathy@mbi.ucla.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100952200
2 T. Jonassen, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: Q, ubiquinone or coenzyme Q; decyl-Q, coenzyme Q with a n-decane tail; HHB, 3-hexaprenyl-4-hydroxybenzoic acid; Q2 and Q6, coenzyme Q with either 2 or 6 isoprene groups in the tail; kb, kilobase(s); HA, hemagglutinin; HPLC, high performance liquid chromatography.
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REFERENCES |
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