From the Department of Biochemistry and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064
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ABSTRACT |
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The first 90 amino acids of the The mitochondrial ATP synthase is a multimeric enzyme composed of
the water-soluble F1 and the membrane-bound F0
complexes. F1-ATPase is composed of five unique subunits in
the stoichiometry of The crystal structures of bovine (15), rat liver (16), and
Bacillus PS3 (17) F1-ATPases indicate that the
Yeast Strains and Media--
A list of the parent strains used
in this study is shown in Table I. The yeast S. cerevisiae
strain BY101 was made by making a null mutation in the ATP1
gene in the host strain W303-1A. The strain BY6A is a meiotic progeny
of the diploid formed from mating BY101 with DMY301.
The yeast media are standard recipes as described (20): YPD, 1% yeast
extract, 2% peptone, and 2% glucose; YPG, 1% yeast extract, 2%
peptone, and 3% glycerol; and YPAD, 1% yeast extract, 2% peptone, 20 mg/liter adenine sulfate, and 2% glucose. Synthetic minimal medium
contained 2% glucose (SD) and was supplemented with adenine,
histidine, arginine, methionine, tyrosine, lysine, leucine, isoleucine,
and tryptophan or uracil at 20 mg/liter.
Gene Disruptions--
The genes encoding the Construction of the Chimeric Genes--
The chimeric subunits
containing the
The DNA encoding the PS3 Phenotypic Testing--
The function of the chimeric subunits
was tested by determining the growth phenotypes of cells containing
each of the chimeric constructs. The plasmids containing the chimeric
constructs were introduced into the yeast strains BY101
(atp1 Gain-of-Function Mutant Isolation--
To obtain strains able to
grow on YPG medium, the colonies obtained after transformation of
DMY111 with the PCR product and the single-cut pRS344ATP2SB/Kan were
replica-plated onto YPG medium and incubated at 30 °C. Colonies that
grew were selected and retested on YPG medium to confirm the phenotype.
Plasmid DNA was isolated from the yeast, transformed into E. coli XL1-Blue, and sequenced as described above. The plasmid was
also retransformed into DMY111 and tested for its ability to rescue the
YPG Biochemical Studies--
N-terminal sequence analysis of the
purified The goal of this study was to determine the role of the The primary sequence comparison of the - and
-subunits of mitochondrial F1-ATPase are folded
into
-barrel domains and were postulated to be important for
stabilizing the enzyme (Abrahams, J. P., Leslie, A. G.,
Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric
enzymes, replacing the domains from the yeast Saccharomyces
cerevisiae enzyme with the corresponding domains from the enzyme
of the thermophilic bacterium Bacillus PS3. The enzymes
containing the chimeric
-,
-, or
- and
-subunits were not
functional. However, gain-of-function mutations were obtained from the
strain containing the enzyme with the chimeric PS3/yeast
-subunit.
The gain-of-function mutations were all in codons encoding the
-barrel domain of the
-subunit, and the residues appear to map
out a region of subunit-subunit interactions. Gain-of-function
mutations were also obtained that provided functional expression of the
chimeric PS3/yeast
- and
-subunits together. Biochemical analysis
of this active chimeric enzyme indicated that it was not significantly
more thermostable or labile than the wild type. The results of this
study indicate that the
-barrel domains form critical contacts
(distinct from those between the
- and
-subunits) that are
important for the assembly of the ATP synthase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
. The
/
- interfaces form six nucleotide-binding sites, three of which
are catalytic and three noncatalytic. F1 is bound to
F0 by one or two stalks that probably comprise the rotor
and the stator of the enzyme (1-9). Possible subunits that provide the
stalks include the oligomycin sensitivity-conferring protein (OSCP)1 and subunits a and b
of F0. There are also various other peptides in the yeast
and mammalian enzymes whose specific role is not certain, including
subunits d-i, and F6 (10-14). Thus, many subunits must
associate to form a complete and functional ATP synthase with
interactions spanning from F0 to F1.
- and
-subunits of the ATPase are divided into three domains. The
top domain is composed of a 90-amino acid
-barrel structure that
forms a crown on top of F1. This crown is 50 Å from the
nucleotide-binding site and does not appear to be critical in the
formation of the active site. The role of the
-barrel domains is not
certain, but they were postulated to be important for stabilizing
F1-ATPase (6). This hypothesis is tested here by making
chimeric enzymes that replace the
-barrel domains from yeast
Saccharomyces cerevisiae ATPase with those from the
thermophilic bacterium Bacillus PS3 enzyme. It was predicted
that if the
-barrel structures were critical in stabilizing the
enzyme, then the chimeric enzyme would be more thermostable than the
wild-type yeast enzyme. No support for this hypothesis was obtained,
but instead the results indicate that the crown made by the
-barrel
domains forms critical interactions necessary for the assembly of the
ATP synthase. These interactions appear to map on the face of the
-barrel crown at the noncatalytic interface between the
- and
-subunits.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit
(ATP1)and
-subunit (ATP2) of the ATPase were
disrupted following a single-step procedure using linear plasmid DNA
(21). The plasmid for the null mutation in ATP2 was
described earlier (18). The knockout plasmid for the ATP1
gene was made by digesting ATP1 with BglII and
replacing the 210-base pair fragment with the 1767-base pair
BamHI fragment containing the HIS3 gene. The
resulting ATP1/HIS3 construct was released from
the plasmid after digestion with BamHI and EcoRI and then used to transform (22) yeast W303-1B.
-barrel domain from PS3 and the remaining portion
from S. cerevisiae were made by taking advantage of the
ability of yeast to perform gap repair (23). A schematic diagram for
the production of the chimeric genes for the genes encoding the
-subunit (ATP1) and
-subunit (ATP2) is shown in Fig. 1. The method requires that
the gene of interest (the
- and
-subunits) is cloned into a yeast
centromere vector, in this case, pRS316 and pRS314 (19), respectively.
The method also requires the presence of a unique restriction enzyme
site in the region that is to be exchanged. There was not a unique restriction site available in the DNA encoding the
-barrel domains of the
- or
-subunits. Thus, a unique restriction site was added to the appropriate region by inserting the gene encoding either the
chloramphenicol (Cm) or kanamycin (Kan) resistance into the region of
the portion of the gene encoding the
-barrel domain of the
- and
-subunits, resulting in plasmids pY16ATP1/Cm and pRS344ATP2SB/Kan,
respectively. The Cm and Kan genes were obtained by PCR using the
primers shown in Table I. The Kan primers contained a KpnI
site at their 5'-end to simplify the ligation of the gene into the
KpnI site of ATP2. The Cm gene was blunt
end-ligated into the BseRI site of ATP1 after the
BseRI site was made blunt with the Klenow fragment of DNA
polymerase. The ligation mixture was transformed into Escherichia
coli XL1-Blue, and the transformants were selected on LB medium
containing either Cm or Kan. Selected clones were checked by
restriction digestion to ensure the proper construct as well as to
determine the orientation of the Cm or Kan resistance gene. These
constructs provided unique NcoI and NruI sites in
the plasmids that were used to make the chimeric constructs.
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Fig. 1.
Method used to make the chimeric
constructs. Homologous recombination effected in yeast between a
PCR product and the target gene was used to make chimeric constructs.
The yeast vectors containing the genes encoding the -subunit
(ATP1) and the
-subunit (ATP2) are shown in
A and B, respectively. A unique NcoI
site was added in the ATP1 gene by inserting the Cm
resistance gene into the BseRI site, whereas a unique
NruI site was introduced into the ATP2 gene by
inserting the Kan resistance gene into the KpnI site. The
PS3 replacement sequence was amplified by PCR using primers (Table I)
that primed DNA synthesis at the beginning of the region that encoded
the PS3
- or
-subunit
-barrel domain with cloned DNA
containing the gene encoding the
- or
-subunit. The 5'-tail on
the PCR primer contained the sequence of the desired target for
homologous recombination. Upon transformation of yeast with the PCR
product and the linearized plasmid, recombination closed the gap and
allowed replication of the plasmid. The transformants were identified
by complementation of the auxotrophic markers
ura3-52 (for pY16ATP1/Cm) and trp1
(for pRS344ATP2SB/Kan). CDS, coding sequence.
-barrel domain sequence and the junction
point of the PS3/yeast sequence was amplified by PCR. PCR was performed
with forward and reverse primers that contained ~30 bases at the
5'-end that corresponded to the desired site of recombination. The last
15 bases served to prime the PS3 sequence and corresponded to the
border encoding the
-barrel domain from PS3 (Table
I). PCRs were performed using plasmids
containing the genes for the
- and
-subunits of the ATPase from
PS3, pUC118
, and pUC118
(kindly provided by Dr. Masasuke
Yoshida). The PCR products consisted of the
-barrel region of either
the
- or
-subunit of PS3 flanked by 30 base pairs of DNA, which
correspond to the desired sites of recombination in either the yeast
ATP1 (encodes the
-subunit) or ATP2 (encodes
the
-subunit) gene. The PCR products were used to transform yeast
strain BY101 (atp1
) or DMY111 (atp2
) with
the NcoI- or NruI-cut plasmid DNA, respectively.
(Note that this method does not require that the yeast strain contain a
null mutation in the target gene. Use of these strains, however, allowed us to directly test the ability of the chimeric constructs to
complement the respective null mutation.) Upon transformation in yeast,
homologous recombination occurs between the PCR product and the
corresponding linear plasmid, thereby replacing the DNA encoding the
yeast
-barrel domain with the PS3 DNA and circularizing the plasmid
DNA. Since only the circular plasmid DNA is replicated in yeast, nearly
all the resulting transformants result from the correct integration
event. The transformed yeast cells were selected on minimal medium
lacking the appropriate auxotrophic requirement. Colonies were taken
and grown in liquid minimal medium; the plasmid DNA was isolated from
the yeast; and the DNA was transformed into E. coli
XL1-Blue. The plasmid DNA was isolated from the E. coli, and
the DNA was sequenced by cycle sequencing (Amersham Pharmacia Biotech)
using 33P-dideoxynucleotides and dITP in place of dGTP. The
entire region that was amplified by PCR and the DNA that encodes the
upstream and downstream borders of the ligation sites were sequenced to ensure that accurate homologous recombination had occurred and to
ensure the absence of other mutations generated by PCR.
Strains and oligonucleotides used in this study
), DMY111 (atp2
), and BY6A
(atp1
,atp2
); selected on minimal glucose
medium; and tested on YPG medium at 18, 30, and 37 °C. Growth on
medium containing glycerol as the sole carbon source indicates a
functional ATP synthase.
phenotype of DMY111. For the isolation of strains
14.1-14.20, BY6A [cATP1][cATP2-4]
(the brackets indicate nonchromosomal plasmids) was grown on SD-Trp-Ura
medium (3 ml); the cells were washed once with sterile water; and
~107 cells were plated onto YPG medium and incubated at
30 °C. The plasmids were isolated from each of the 20 strains,
amplified in E. coli, and retransformed into BY6A. The
plasmids did not provide BY6A the ability to grow on YPG medium,
indicating that the YPG+ phenotype was not due to a
mutation on either cATP1 or
cATP2-4.
- and
-subunits of the ATPase was performed as described
(24). Mitochondria were isolated from yeast after growth on minimal
medium containing the auxotrophic requirements (25). The extraction of
the mitochondria with chloroform, Western blot analysis, and ATPase
measurements were done as described (25). Protein concentration was
determined by the BCA method (26) for samples that contained membrane
proteins and by a modified Bradford method for soluble proteins (26). All biochemical experiments were repeated at least twice starting from
the growth of the cells, and all assays were done in at least duplicates for each preparation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel
domain in the structure and function of F1-ATPase. The
initial hypothesis was that the
-barrel domains are important for
stabilizing F1-ATPase (15). To test this hypothesis,
chimeric subunits were made in which the
-barrel domains from the
- and
-subunits of PS3 replaced the
-barrel domains of the
corresponding subunits from yeast.
-barrel domains of the
-
and
-subunits from yeast and PS3 is shown in Fig.
2A. The yeast and PS3
- and
-subunits are 45 and 57% identical in this region as compared with
an overall identity of 59 and 71%, respectively. Thus, this region is
less well conserved than the other portions of the molecule.
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Fig. 2.
A, homology of the -barrel domains in
the
- and
-subunits of PS3 and yeast F1-ATPases. The
numbering system is based on the sequence of the mature subunit. The
vertical lines and double dots indicate identical
and similar residues, respectively. The mutant number and mutations
correspond to the following: cATP2-4,
T2K;
G4, cATP2-5,
P24L;
cATP2-6,
A63V; cATP2-7,
E49D; cATP2-8,
L30Q;
cATP2-10,
L54H;
cATP2-11,
T58V;
cATP2-12,
L54H. B, primary sequence
of the chimeric PS3/yeast
- and
-subunits. The site of maturation
was determined by direct N-terminal sequence analysis of the purified
yeast subunits. This site of processing of the chimeric proteins is
assumed to occur at the same site as the yeast subunits. The amino
acids in lowercase letters correspond to the sequence
derived from PS3, whereas the remaining sequence is derived from yeast.
Only the first 128 residues are shown.
The primary sequences of the - and
-subunits of the chimeric
constructs are shown in Fig. 2B. The genes encoding the
chimeric PS3/yeast
- and
-subunits will henceforth be referred to
as cATP1 and cATP2, respectively, whereas the
wild-type yeast genes will be indicated as ATP1 and
ATP2, respectively. The numbering system used in Fig.
2B assigns position 1 to the first residue of the mature
protein. For yeast S. cerevisiae, the first residue shown
here is different from those reported earlier (27, 28). However, these
sites were determined here by direct N-terminal amino acids analysis of
the purified peptides, whereas the prior published determinations were
obtained either by homology (27) or by an indirect method (28). The
amino acids in lowercase letters correspond to the sequence
derived from PS3, whereas the remaining sequence is that from yeast.
For the
-subunit (Atp1p), the resulting chimera (cAtp1p) had the
same number of residues as the yeast
-subunit. However, the sequence
QHKARNE (cf. Fig. 2A) is unique to the
-subunit of PS3 and thus to cAtp2p. These 7 residues (residues
39-45) make the chimera 7 amino acids longer (485 residues) than the
wild-type (478 residues) yeast
-subunit (Atp2p). This difference
results in the slower migration of cAtp2p as compared with yeast Atp2p
on SDS-polyacrylamide gel (cf. Fig. 5).
The chimeric constructs were tested individually and together to see if
they were able to complement the corresponding null mutations. The
tests were performed in strains DMY111 (atp2) and BY6A
(atp1
,atp2
). These strains had null
mutations in the gene encoding the
-subunit (ATP2) or the
-subunit (ATP1) and
-subunit of the ATPase and thus
are unable to grow on medium containing glycerol as the sole carbon
source (e.g. YPG). In addition, the ATP1 and
ATP2 genes (both the wild type and PS3/yeast chimeras) were
carried on yeast vectors that also contained the URA3 and
TRP1 genes, respectively, which allowed selection of cells
containing the plasmids.
The functional complementation of cATP1 and
cATP2, as assessed by the cells' ability to grow on YPG
medium, is shown in Fig. 3. The wild-type
yeast ATP1 and ATP2 genes complement the
respective mutations as seen in positions 1 and 5. However,
cATP2 did not complement the mutations either alone
(position 3) or together with cATP1 (position 7). Thus,
despite forming a clear distinct domain structure separate from the
rest of the - or
-subunits, the
-barrel domains must have some
species-specific structural requirements beyond just the interactions
between themselves.
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A further understanding of the secondary requirements for function was
obtained by isolating gain-of-function mutations in cATP2.
Cells were isolated that were able to grow on YPG medium from DMY111
(atp2) containing the chimeric
-subunit gene. One
example of these cells is shown in Fig. 3 (position 4, cATP2-4). The plasmids in these cells were
isolated and amplified in E. coli, and the sequence of
cATP2 was determined. In all but one case
(cATP2-4), a single missense mutation in a codon
in the
-barrel domain of the
-subunit was identified as
summarized in Fig. 2A. The clone
(cATP2-4) had a deletion of the codon for G4 in
addition to the missense mutation, T2K. Note that all of the mutations
identified changed residues that are conserved between PS3 and yeast,
rather than mutating nonconserved residues. Thus, single-base mutations
in the
-barrel domain were able to provide a functional ATP synthase
containing the chimeric PS3/yeast
-subunit.
The -barrel domain structures of the PS3
- and
-subunits are
nearly identical to those of the bovine subunits (15, 17). The main
difference is the presence of the 7 additional residues in the PS3
-subunit, which extends the second
-sheet, forming a finger-like
projection from the domain. Otherwise, the overall conformation and the
relative positions of the individual residues are comparable between
the bovine and PS3 proteins. Inspection of the crystal structure of PS3
F1-ATPase indicates that all of the gain-of-function
mutations face the interface of the
- and
-subunits that forms
the noncatalytic nucleotide-binding site (Fig.
4). None of the gain-of-function
mutations lie at the interface between the
- and
-subunits, where
residues from the
- and
-subunits make contact. Instead, the
residues (especially
T2,
P24, and
E49) appear to map out a
region on the surface of the
-barrel domain located on the front
face on the surface of the interface that forms the noncatalytic
nucleotide-binding site.
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The identification of the gain-of-function mutation in cATP2
led to the question of whether this mutation would be sufficient to
allow the functional expression of the chimeric enzyme when paired with
cATP1. To test this, the cATP2 genes containing
the gain-of-function mutations (Fig. 2A) were cotransformed
with cATP1 into a yeast strain (BY6A) devoid of the yeast
ATP1 and ATP2 genes. These cells remained unable
to grow on YPG medium, which indicates that these single missense
mutations were not sufficient to allow the functional expression of
both cATP1 and cATP2, as is seen in Fig. 3
(position 8). In this example, the gain-of-function -subunit chimera
(cATP2-4) was coexpressed with cATP1
in BY6A, and together, they were unable to allow growth on glycerol
medium. Thus, either cAtp1p must lack species-specific requirements
present in Atp1p, or the gain-of-function mutations in cATP2
must not be compatible with cAtp1p.
However, gain-of-function mutations were obtained in BY6A
(atp1,atp2
) containing cATP1 and
cATP2-4. The growth phenotype of one such strain,
14.1, is shown in Fig. 3 (position 9). For >20 separate isolates
(14.1-14.20), the gain-of-function mutation did not lie in either
cATP1 or cATP2-4. This was determined
by isolating the plasmids containing cATP1 and
cATP2-4, followed by retransformation of BY6A
with the plasmids. In all cases, the cells were unable to grow on YPG
medium, indicating that the gain-of-function mutation did not lie in
either cATP1 or cATP2-4. Furthermore,
retransformation of the gain-of-function strain, e.g. 14.1, which had been cured of the plasmids with cATP1 and
cATP2-4, with cATP1 and
cATP2-4 resulted in strains that were able to
grow on YPG medium. Thus, the gain-of-function mutation(s) allowing the
functional expression of cATP1 with
cATP2-4 were not in either cATP1 or
cATP2-4, but must be in either the chromosomal or mtDNA.
For reasons that will be more evident under "Discussion," the initial hypothesis was that the gain-of-function mutations, which allowed the functional expression of cATP1 and cATP2-4, were in the gene encoding OSCP (ATP5 gene). To test this, ATP5 was amplified by PCR from five different strains that contained cATP1 and cATP2-4 and that were able to grow on YPG medium, and the DNA was directly sequenced. No mutations were identified in the coding region of ATP5, which indicates that the gain-of-function mutations must lie in another gene. Thus, at this point, the identity of the second gain-of-function mutations is unknown.
The ability of the cells to grow on YPG medium does not provide quantitative information on the effectiveness of the ATP synthase to make ATP. This is because only 15% of the wild-type activity of the ATP synthase is necessary for the cell growth on YPG medium (29). Therefore, the functional complementation of the chimeric genes was tested by measuring the oligomycin-sensitive ATPase from isolated mitochondria. The results of this study are shown in Table II. In general, the amount of oligomycin-sensitive ATPase in the mitochondria correlated well with the ability of the cells to grow on YPG medium. The gain-of-function mutations allowed the expression of the oligomycin-sensitive ATPase at nearly the same level as the wild-type enzyme. In a second set of experiments, F1-ATPase was released from the membrane by chloroform, and the efrapeptin-sensitive ATPase activity was measured. The level of the ATPase activity in the chloroform extract correlated well with that in the mitochondria.
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The inactivity of the chimeric enzymes may be due to either the lack of
assembly or an assembled but inactive enzyme. To test between these two
possibilities, Western blot analysis using antibodies against yeast
F1 was performed on the mitochondrial and
chloroform-extracted preparations, as tested in Table II (Fig.
5). Chloroform has been used to
selectively release assembled F1 from the membrane (30) and
thus can be used as a test for assembly of F1-ATPase. This is illustrated in lane 1, where the -subunit is present
in the mitochondria isolated from cells with a null mutation in
ATP2, but not in the chloroform extract of the mitochondrial
membranes from the same cells. The analysis shows that cAtp2p in the
presence of the wild-type
-subunit resulted in the degradation of
wild-type Atp1p and cAtp2p (lane 2). This is unusual since
the
- and
-subunits are normally stable when expressed in yeast
strains with the null mutations ATP1 and ATP2,
respectively (31), as is seen in lane 1 for the
-subunit.
The gain-of-function mutation in cATP2-4 restored
the stable expression of the
- and
-subunits of F1 to
near wild-type levels (lane 3). Note also that the migration of cAtp2-4p (e.g. lane 3) was slightly slower
than that of wild-type Atp2p (e.g. lane 4). This
difference is due to the 7 added residues present in cAtp2p
(cf. Fig. 2A).
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The chimeric PS3/yeast - and
-subunits were not stable when they
were expressed together in yeast (Fig. 5, lane 7). Upon adding the gain-of-function mutation in the chimeric
-subunit (cATP2-4), the
-subunit, but not
cATP1, was present together with a degradation product of
either the
- or
-subunit (lane 8). Finally, strain
14.1, the strain containing cATP2-4 with
cATP1 and an additional undetermined mutation that allowed
the functional expression of the PS3/yeast chimeras, had a stable,
assembled, and functional F1-ATPase (lane 9).
Thus, the gain-of-function mutations stabilized F1-ATPase
assembled from the chimeric PS3/yeast
- and
-subunits.
The stable expression of a functional chimeric ATPase that contained
the -barrel domains for both the
- and
-subunits of the
thermophilic enzyme PS3 allowed the biochemical analysis of the effect
of these replacements on the thermostability of the enzyme. The
thermostability of the membrane-bound enzyme was assessed using
isolated mitochondria, whereas the thermostability of
F1-ATPase was determined using the chloroform extract of
the mitochondrial membrane. Thermodenaturation studies at 55 and
60 °C failed to show any differences between the thermostability of
the wild-type and chimeric enzymes (data not shown). Although the
chimeric enzymes were not any more thermostable than the wild-type
enzyme, it is important to note that the enzymes were not any less
thermostable, which suggests that the conformation of the chimeric
enzymes was not grossly altered.
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DISCUSSION |
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This work used a novel method to make fusion protein constructs, although it is based on a prior method used to make new plasmids (23). The method (see Fig. 1 and "Experimental Procedures") takes advantage of the high rate of homologous recombination in yeast and the ability to perform gap repair of plasmids. The method is efficient, quick, and not restricted to any sequence or gene. In addition to the constructs presented in this study, this laboratory has used this method for making numerous other fusion constructs with 100% success. Also, the number of incorrect clones obtained or identified was only ~10%, making this method very efficient. Thus, this method can be used as a general tool for making protein or gene fusion constructs.
The initial hypothesis that was being tested in this study was that the
-barrel domains of the
- and
-subunits function to increase
the stability of F1-ATPase. This hypothesis was originally proposed based on the crystal structure of bovine F1-ATPase
(15), which showed that these domains were far from the catalytic site (50 Å) and clearly formed a domain structure in the form of a crown.
The
- and
-subunits interact with each other along a single face
that forms the catalytic and noncatalytic nucleotide-binding sites. As
such, the
-barrel domain structures of the
- and
-subunits have large faces of interactions, but the
-barrel domains are far
from the nucleotide-binding sites and do not contribute directly to
their formation.
Initially, it was predicted that the chimeric PS3/yeast - and
-subunits would not be able to function without each other. This
prediction was based on a prior study that indicated that the top
domain of the E. coli
-subunit could not be replaced with
the corresponding region from Bacillus megaterium, whereas the catalytic domain could be exchanged (32). Furthermore, the E. coli uncD strain could not be complemented by expression of the
chloroplast
-subunit, although the chloroplast chimera containing the E. coli
-barrel domain was able to restore oxidative
phosphorylation (33). These results suggested that there was a greater
requirement for species-specific residues in the
-barrel domains as
compared with those in the remaining portions of the
-subunit.
The species-specific primary structural requirements in the -barrel
domain of the
-subunit might be due to the interactions with the
corresponding domain of the
-subunit, interactions with another
subunit(s) of the ATP synthase, or a combination of both types of
interactions. However, the structural requirement does not appear to be
due to the interactions with the corresponding
-barrel domain of the
-subunit for two reasons. First, expression of the chimeric
PS3/yeast
- and
-subunits together does not provide a functional
enzyme. This indicates that despite restoring the putative interactions
between the domains, the enzyme is still unable to function. Second, a
number of gain-of-function mutations in the
-barrel domain of cAtp2p
allowed the functional expression of the ATP synthase. These mutations
were all in residues that were conserved between PS3 and yeast,
suggesting that the mutations were not simply restoring specific
interactions that were lost in cAtp2p (Fig. 2A). These
mutations (especially those at T2, P24, and E49) map to the groove that
lies between the
- and
-subunits in the interface that forms the
noncatalytic nucleotide-binding site.
The gain-of-function mutation T2K, G4 may be very telling because it
is at the very end of the N terminus of the
-subunit. The T2 side
chain points directly into the groove formed at the interface of the
- and
-subunits, but does not interact with residues in either
the
- or
-subunit. The simplest explanation for the mechanism of
this gain of function is that the mutation affects the interactions
with another subunit(s) of the ATPase and that this interaction occurs
within this groove. This is also the simplest explanation for the other
gain-of-function mutations, although some of the mutant residues must
act indirectly with the putative subunit as the side chains are not
positioned at the surface of the domain. It is important to remember
that although the gain-of-function mutations are different, they must
all act by the same mechanism, i.e. restoring interactions
that were lost by replacing the
-barrel domain of the yeast subunit
with that of the PS3 subunits. As such, the simplest explanation for
the mechanism of gain of function also seems to be the most likely mechanism.
Other studies have also indicated that the -barrel domain is
important for the assembly of the ATP synthase, and in E. coli, for specific interactions with the E. coli
-subunit. First, mutations in the
-barrel domains of the E. coli
- and
-subunits have been shown to impair the assembly
of the ATP synthase by inhibiting binding of F1 to
F0 (34-36). Second, limited trypsin digestion of bovine
F1 removed 15 and 7 amino acids from the N terminus of the
- and
-subunits, respectively, resulting in the decoupling of ATP
hydrolysis from proton transport (37). Similarly, proteolytic removal
of the first 30 amino acids of the E. coli
-subunit
impaired binding of F1 to F0 (38). Finally,
cross-linking studies have indicated that the E. coli
-subunit interacts with the
- and
-subunits and likely with
the
-barrel domain of the
-subunit (6, 7). Thus, it appears that
a probable reason why cAtp1p and cAtp2p are not functional either alone
or together in yeast is because of structural requirements in the
-barrel domain for interactions with other subunits of the ATP
synthase. Since OSCP is homologous to the E. coli
-subunit, it seemed likely that the interaction with OSCP was
disrupted in the ATPase formed with cAtp1p and cAtp2p.
The isolation of gain-of-function mutations in BY6A
(atp1,atp2
) with the expression of cAtp1p
and cAtp2-4p (e.g. strain 14.1) provided a possible source
for genetic evidence to support the presence of an interaction of OSCP
with the
-barrel domains. The gain-of-function mutations in these
strains were not in either the
- or
-subunits based on a number
of observations. First, after curing the plasmids from the strain,
retransformation of the strain with cATP1 and
cATP2-4 was sufficient to restore cell growth on
YPG medium. Second, purification of the plasmids from the strains that
were able to grow on YPG medium and the subsequent transformation of
the purified plasmids into BY6A did not provide a cell able to grow on
YPG medium. Therefore, the gain-of-function mutation must be in a
gene(s) other than cATP1 or cATP2-4.
The first postulate was that the mutation was in the gene encoding OSCP
(ATP5). However, we sequenced ATP5 from five
different gain-of-function mutants, including 14.1, and were unable to
identify any mutations in the coding region. Although some of the other
isolates may indeed have mutations in ATP5, for at least the
five studied, a mutation in another gene must be responsible for the
gain of function. Possible candidate genes include those that encode
subunit b of F0 or possibly genes encoding other ancillary
proteins, such as subunits d-i.
The initial hypothesis that was tested in this study was that the
-barrel domains are important for stabilizing the ATPase (15). The
prediction that followed from this hypothesis was that an increase in
the thermostability of this domain structure would result in an
increase in the overall thermostability based on the ATPase activity of
the enzyme. However, the chimeric enzyme containing the
-barrel
domains from PS3 showed no significant difference in the
thermostability of the enzyme. Although this negative result did not
disprove the initial hypothesis, the data do not support it either.
With the wealth of data that show the importance of this region in the
assembly of the ATP synthase, it is likely that a major role of this
domain in the enzyme is its interactions with other subunits helping in
forming the ATP synthase. Indeed, as it appears that the subunits that
interact with the domain form the stator of the enzyme (6-9, 37-41),
the easiest way to hold F1 in place during rotation of the
-subunit would be by holding it at the top of the molecule.
Furthermore, subunit-subunit interactions occurring in the groove
formed at the noncatalytic nucleotide interface would be expected to be the least disruptive during the catalytic cycle.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John Keller for critically
reading the manuscript. We especially thank Dr. M. Yoshida for the
plasmids pUC118 and pUC118
.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM44412.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.
Present address: Dept. of Molecular Biology, Princeton University,
Princeton, NJ 08540.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Chicago Medical School, 3333 Greenbay Rd., North Chicago, IL 60064. Tel.: 847-578-8606; Fax: 847-578-3240; E-mail: muellerd{at}mis.finchcms.edu.
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ABBREVIATIONS |
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The abbreviations used are: OSCP, oligomycin sensitivity-conferring protein; Cm, chloramphenicol; Kan, kanamycin; PCR, polymerase chain reaction.
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REFERENCES |
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