From the Departments of Surgery and Biochemistry and Molecular
Biology, Wayne State University School of Medicine,
Detroit, Michigan 48201
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INTRODUCTION |
The ATP synthase of mitochondrial, chloroplast, and bacterial
membranes is a key enzyme involved in energy production (1-3). The
enzyme is composed of a catalytic moiety F1, attached
peripherally to an integral membrane component F0. The
subunit composition of F0 varies among species, while
F1 has been highly conserved in evolution. In most
organisms studied F1 contains five different subunits in
the stoichiometric ratio:
3
3

(1-3). X-ray diffraction data for the F1 have been
obtained with the mitochondrial enzyme from both bovine (4) and rat
liver (5, 6). The
and
subunits alternate in a hexagonal array
(4, 5) surrounding the amino and carboxyl termini of the
subunit
(4). The
and
subunits are not visible in the current crystal
structures. These subunits likely reside at the base of the enzyme
since they are required for binding F1 to F0
(7, 8).
In Saccharomyces cerevisiae the F1 subunits are
encoded by nuclear genes (9-13) and, with the exception of
(11),
are synthesized as precursors containing an amino-terminal
mitochondrial targeting sequence that is cleaved during import (14).
The reactions of sorting the F1 subunits into mitochondria
and the subsequent folding reactions within the organelle are mediated
by heat-shock proteins (Hsps)1 that serve as
"molecular chaperones" in the cell (15). With respect to the
biogenesis of mitochondrial proteins (such as the F1), the
combined activities of the cytoplasmic and mitochondrial Hsp70-Hsp40
chaperone pairs are proposed to mediate the translocation of unfolded
proteins into mitochondria; folding the polypeptide chain in the matrix
is then facilitated by the Hsp60 and Hsp10 proteins (for review, see
Refs. 15 and 16). In the specific case of F1 biogenesis,
the final steps in the enzyme formation require two proteins, Atp11p
and Atp12p, neither of which has significant sequence homology with
other proteins in the data banks (17-19). Yeast mutants that are
deficient for either Atp11p or Atp12p accumulate the F1
and
subunits in large protein aggregates instead of forming the
enzyme oligomer (17). In contrast to Hsp60-deficient strains, which
show aggregation of both the mature and precursor forms of the
F1
subunit (20), only the mature form of the
F1 subunits is observed in atp11 and
atp12 mutants (17). For this reason, Atp11p and Atp12p are
suggested to act at a point in the F1 assembly pathway that
is downstream from the Hsp60 step.
In considering the type of action elicited by Atp11p and Atp12p during
F1 assembly, it is informative to compare the phenotypes of
atp11 and atp12 strains with those of yeast that
have null mutations in the individual F1 structural subunit
genes. For instance, yeast that are deficient for the
subunit
harbor the
subunits as aggregated proteins; likewise, the
subunit aggregates in the
subunit null strains (17). As mentioned
above, aggregation of the F1
and
subunits is the
signature phenotype of atp11 and atp12 strains.
In contrast, the
and
subunits remain soluble in mitochondria of
(13),
(12), or
(11) null mutants, despite the fact that the
absence of any of these subunits blocks F1 assembly.
Moreover, in
subunit-deficient yeast, the
and
subunits
sediment in linear sucrose gradients to the position where 
dimers would be expected (13). Thus, it appears that aggregation of the
F1
and
subunits prevails only under conditions in
which 
dimerization is not possible, such as in the
or
subunit null strains, or in yeast that lack a protein (i.e. Atp11p or Atp12p) whose function may be to mediate
/
oligomerization. The proposal that Atp11p and Atp12p serve as
chaperones during F1 biogenesis is supported by the fact
that Atp11p and Atp12p are present in mitochondria at levels that are
several orders of magnitude lower than the amount of F1
and
subunit protein (21)2
which is in accord with the fact that there is only a small pool of
unassembled F1 proteins in the steady state (22).
The present paper reports on features of the Atp12p protein that are
important for its action as an F1 assembly factor. The primary translation product of the ATP12 gene is a 37-kDa
precursor protein, which is cleaved to generate the mature polypeptide
(33 kDa) following import into mitochondria (18). In previous work Atp12p was shown to be a soluble protein of the mitochondrial matrix
that sediments in linear sucrose gradients as an oligomer of 70-80 kDa
(18). The current work presents evidence that the Atp12p oligomer
observed in mitochondria is likely heterogeneous in content and
indicates a region in the Atp12p primary structure that is important
for intermolecular associations of the protein. In addition, the
functional domain of Atp12p was identified and shown to include an
acidic amino acid that is required for optimal activity of the
protein.
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MATERIALS AND METHODS |
Strains and Growth Media--
The genotypes and sources of the
mutant and wild type yeast strains used in the present study are listed
in Table I. Chemically induced mutations
were obtained as described (24). Yeast was grown in the following
media: YPD (2% glucose, 2% peptone, 1% yeast extract), YPGal (2%
galactose, 2% peptone, 1% yeast extract), YEPG (3% glycerol, 2%
ethanol, 2% peptone, 1% yeast extract), WO (2% glucose, 0.67% yeast
nitrogen base without amino acids (Difco)). Amino acids and other
growth requirements were added at a final concentration of 20 µg/ml.
The solid media contained 2% agar in addition to the components
described above. Escherichia coli RR1 (proA
leuB lacY galK xyl-5
mtl-1 ara-14 rpsL supE
hsdS 
) was the host bacterial strain for
the recombinant plasmids.
DNA Sequencing--
The oligonucleotide primers used for
sequencing and for constructing atp12 deletion/truncation
mutants are listed in Table II. Genomic
DNA was purified from yeast and served as the template for PCR
amplification of the atp12 gene using the primers 1 and 9. The 1048-bp PCR product was digested with BamHI and
SstI and ligated at these restriction sites in the
yeast/E. coli shuttle vector, YEp352 (25). The
atp12 gene carried in the plasmids was sequenced by the
dideoxy method (26) with Sequenase (U. S. Biochemical Corp.). Both
strands of two separate PCR products were sequenced for each
atp12 mutant.
Yeast Plasmid Constructions--
The plasmids used in this study
are described in Table III. Atp12p is
numbered from 1 to 325, where residue 1 is the initiator methionine in
the primary translation product (18). In cases where sequences were
removed from the 3
end of the ATP12 gene, the plasmids and
the encoded products are named according to where the mutant protein is
truncated. This is indicated by the single-letter code and number (18)
of the last retained amino acid. For mutant proteins that have
deletions from the amino terminus, the plasmids and the encoded
products are designated by the
symbol, followed by the span of
ATP12 codons removed. The number of amino acids that are
deleted from the carboxyl or the mature amino terminus of the mutant
Atp12p proteins is indicated in Table III. In naming the plasmids
coding for mutant Atp12p proteins harboring missense mutations, the
single-letter code is used to indicate the amino acid substitution at
the designated position in the Atp12p primary structure (18). The
details of the plasmid constructions are provided in Footnote
3. All of the plasmids whose construction utilized PCR (p657/E289D, p657/E289A, pG57/E289Q, pG57/ST21) were sequenced to verify that there were no artificial mutations introduced in the ATP12 coding region due to errors made by the
Taq polymerase.
Preparation of Yeast Mitochondria--
Yeast were grown
aerobically in liquid YPGal at the temperature indicated in the
experiment to early stationary phase. The method of Faye et
al. (28) was used to prepare mitochondria with the exception that
Zymolyase, instead of Glusulase, was used to digest the cell wall.
Phenylmethylsulfonyl fluoride (PMSF) was added to 10 µg/ml final
concentration during the cell-breaking step to minimize
proteolysis.
Solubilization of Atp12p from Mitochondria--
Two different
methods, both of which give comparable results, were used to prepare
mitochondrial extracts containing solubilized Atp12p. For some
experiments, mitochondria were suspended at 10 mg/ml in 10 mM Tris-HCl, pH 8.0, and sodium deoxycholate was added to 1 mg/ml to permeabilize the membranes. Following a 15-min incubation at
0 °C, the suspension was centrifuged for 30 min at 4 °C at 50,000 rpm in a Beckman 70Ti rotor. Alternatively, mitochondria were suspended
at 7-8 mg/ml in 0.4 ml of the buffer specified in the experiment and
exposed to four 10-s bursts, with cooling in between, of sonic
irradiation at 40% output (Branson sonifier, model 450). The sonicated
samples were then centrifuged as described above. The disruption of
mitochondria (by means of detergent or sonic irradiation) was done in
the presence of PMSF (0.5 mM), leupeptin (1 µg/ml), and
pepstatin (1 µg/ml) to minimize proteolysis of the solubilized
proteins.
Purification of Histag-Atp12p from E. coli Expression
Systems--
Two different plasmids were employed for the
overproduction of Histag-Atp12p in bacteria. In one case, PCR was used
to create the bacterial plasmid, pHISATP12, which encodes the mature
form of Atp12p (without the mitochondrial leader sequence (18)) that carries a (6x)histidine sequence at the amino terminus. For this construction, a yeast plasmid coding for wild type Atp12p (pG57/ST4, Table III) served as the template for PCR with the primers 9 and 10 (Table II) to produce a 910-bp DNA fragment. The PCR product was
digested with SmaI and SstI and ligated to
EheI,SstI-cut pPROEXTM HTa to produce
pHISATP12. E. coli cells carrying pHISATP12 were grown in a
0.5-liter LB/ampicillin culture at 30 °C to mid-log phase. Following
a 4-h induction with 0.5 mM isopropylthiogalactoside, the
cells were harvested, sonically irradiated in buffer L (50 mM Tris-HCl, pH 8.5, 10 mM 2-mercaptoethanol, 1 mM PMSF), and centrifuged at 6,000 rpm for 10 min in a
Sorvall SA600 rotor at 4 °C. Recombinant Histag-Atp12p partitions in
the insoluble fraction at this step. The inclusion bodies were
dissolved in 8 M urea, 20 mM Tris-HCl, pH 8.0, 1 mM PMSF, 10 mM 2-mercaptoethanol, after which
the urea-solubilized solution was clarified by centrifugation (6,000 rpm, 10 min), rapidly diluted 20-fold in buffer (20 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol), and loaded on a
DEAE column (bed volume = 6 ml). The column was washed with 20 mM Tris-HCl, pH 8.0, and eluted with a linear 0-2
M NaCl gradient in 20 mM Tris-HCl, pH 8.0. The
fractions containing Histag-Atp12p were pooled and brought to 80%
saturation with (NH4)2SO4. The
protein precipitate was dissolved in 10 ml of buffer L and loaded on a Ni-NTA column (bed volume = 2 ml). The affinity column was washed sequentially with 20 ml of buffer A (20 mM Tris-HCl, pH
8.5, 100 mM KCl, 10 mM 2-mercaptoethanol, 10%
glycerol, 20 mM imidazole), 15 ml buffer of B (20 mM Tris-HCl, pH 8.5, 500 mM KCl, 10 mM 2-mercaptoethanol, 10% glycerol), and 15 ml of buffer
A. Buffer C (20 mM Tris-HCl, pH 8.5, 100 mM
KCl, 10 mM 2-mercaptoethanol, 10% glycerol, 100 mM imidazole) was then used to selectively elute
Histag-Atp12p, which yielded the protein 95% pure (see lane
1, Fig. 1). The second expression
system employed the bacterial vector, pMAL-C2 (New England Biolabs), to
direct the synthesis of a chimeric protein in which maltose binding
protein (MBP) is fused immediately proximal to the (6x)histidine
sequence in Histag-Atp12p. The plasmid pMBPHISATP12 was created by
subcloning a 1-kb SmaI-HindIII fragment encoding the sequence for Histag-Atp12p from the plasmid pG57/ST21 (Table III)
into the pMAL-C2 vector that was prepared as an
XmnI,HindIII-cut DNA. E. coli cells
carrying the plasmid pMBPHISATP12 were grown in 2 liters of
LB/ampicillin at 37 °C to the exponential phase of growth, after
which expression from the plasmid was induced for 3 h using 1 mM isopropylthiogalactoside. Next, the cells were harvested
and suspended in 50 ml of lysis buffer (10 mM
Na2HPO4, pH 7.0, 30 mM NaCl, 0.25%
Tween 20, 10 mM 2-mercaptoethanol, 10 mM EDTA,
10 mM EGTA). Following three cycles of freezing and
thawing, the cell suspension was sonically irradiated, brought to 0.5 M NaCl, and centrifuged at 9,000 rpm in a Sorvall SA600
rotor. The crude extract (containing the recombinant fusion protein)
was diluted five times with column buffer (10 mM
Na2HPO4, pH 7.0, 500 mM NaCl,
0.25% Tween 20, 10 mM 2-mercaptoethanol, 1 mM
EGTA, 1 mM azide) and loaded at a flow rate of 1 ml/min on
an Amylose column (bed volume = 3 ml) that was pre-equilibrated
with the same column buffer. The column was washed with 9 ml of fully
supplemented column buffer and then with 15 ml of column buffer omitted
for Tween 20, and the MBP-Histag-Atp12p fusion protein was finally eluted with 15 ml of column buffer (detergent-free) that contained 10 mM maltose in 95% pure form. The Coomassie-stained gel in
Fig. 2 shows a sample of the purified
MBP-Histag-Atp12p protein (lane 2) from the pooled column
fractions.

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Fig. 1.
SDS-polyacrylamide gel analysis of
Histag-Atp12p purified from E. coli. The figure shows
a Coomassie-stained 12% SDS-polyacrylamide gel that was used to
analyze recombinant Histag-Atp12p, which was purified from E. coli according to the procedure described under "Materials and
Methods." Lane 1 shows 2 µg of the Histag-Atp12p that
was eluted from the Ni-NTA column. The migration of molecular mass
standards is shown in lane 2. The size of the marker
proteins is indicated in kilodaltons in the right-hand
margin.
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Fig. 2.
SDS-polyacrylamide gel analysis of the
MBP-Histag-Atp12p fusion protein purified from E. coli. The figure shows a Coomassie-stained 12%
SDS-polyacrylamide gel that was used to analyze the recombinant MBP-Histag-Atp12p fusion protein that was purified from E. coli according to the procedure described under "Materials and
Methods." The migration of molecular mass standards is shown in
lane 1; the size of the marker proteins is indicated in
kilodaltons in the left-hand margin. Lane 2 shows
2 µg of the MBP-Histag-Atp12p fusion protein that was eluted from the
Amylose column.
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Affinity Precipitation of Histag-Atp12p from Mitochondrial
Extracts--
Mitochondria were suspended in 50 mM
Tris-HCl, pH 8.5, 10 mM 2-mercaptoethanol, 1 mM
PMSF, and soluble extracts were prepared by sonic irradiation (see
above). A 50-µl slurry of Ni-NTA resin was pre-equilibrated with
buffer M (10 mM Tris-HCl, pH 8.0, 5 mM
imidazole, 140 mM NaCl, 1% Triton X-100, 1 mM
PMSF) and added to 0.14 ml of sonic supernatant containing solubilized
mitochondrial proteins. The suspension was mixed end-over-end for 30 min at room temperature and then centrifuged for 3 min in a
microcentrifuge at room temperature. The supernatant was collected, and
the Ni-NTA beads were washed five times for 5 min with 0.14 ml of
buffer M, and finally suspended in SDS-gel loading buffer in
preparation for Western analysis.
Sedimentation Analysis of Atp12p--
Mitochondria were
suspended in 20 mM Tris-HCl, pH 8.0, and soluble extracts
were prepared by sonic irradiation (see above) at 4 °C. Molecular
weight markers (hemoglobin and myokinase, or hemoglobin and lipoamide
dehydrogenase) were added to the solubilized protein samples, and the
mixtures were centrifuged through 7-20% linear sucrose gradients
under the conditions of sedimentation described (18). Previously
described methods (18, 21) were used to define the positions of
myokinase, hemoglobin, and lipoamide dehydrogenase peaks in the
gradients. The gradient fractions were assayed by Western analysis for
Atp12p.
Chemical Cross-linking--
For cross-linking with
amine-reactive bifunctional reagents, purified recombinant
Histag-Atp12p was incubated at 0.4 mg/ml in 20 mM
Na2HPO4, pH 7.5, 150 mM NaCl with
either 5 mM disulfosuccinimidyl tartarate, 5 mM
ethylene glycolbis(sulfosuccinimidylsuccinate), or 5 mM dithiobis(sulfosuccinimidylpropionate) at 22 °C
for 30 min. The modification reactions were quenched by the addition of
Tris-HCl, pH 8, to 50 mM concentration and denatured in
SDS-gel loading buffer in preparation for electrophoresis. For
experiments that employed glutaraldehyde, the purified Histag-Atp12p
protein was incubated in 10 mM Tris-HCl, pH 7.5, at 0.4 mg/ml with 0.05, 0.1, or 0.5% glutaraldehyde at 22 °C for 10 min,
at which time SDS-gel loading buffer was added, and the samples were
loaded on SDS-polyacrylamide gels. In control experiments, 0.5 mg/ml hemoglobin was incubated under the same conditions with 0.5%
glutaraldehyde.
Yeast Two-hybrid Screen--
The yeast two-hybrid screen
(29) employed yeast vectors (pACT2 and pAS2-1) and a host strain
CG-1945 (ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3, 112 gal4-542 gal80-538 cyhr2
LYS2::GAL1UAS-GAL1TATA-HIS3
URA3::GAL417-mer(x3)-CYC1TATA-lacZ) that were supplied in the MATCHMAKER Two-hybrid System 2 purchased from CLONTECH. The construction of
ATP12 plasmids with pACT2 and pAS2-1 (pG57/ST22, pG57/ST23,
Table III) is described in Footnote 3. Expression from the
GAL417-mer(x3)-CYC1TATA-lacZ reporter gene
was determined using 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside as a chromogenic substrate for
-galactosidase in the colony-lift assay described in the
CLONTECH manual.
Assays--
Protein concentrations were estimated by the
method of Lowry et al. (30). ATPase activity was measured by
the colorimetric determination of inorganic phosphate as described
previously (27).
Miscellaneous Procedures--
Standard techniques were used for
restriction endonuclease analysis of DNA, purification and ligation of
DNA fragments, and transformations of and recovery of plasmid DNA from
E. coli (31). Yeast transformations employed the LiAc
procedure (32). The method of Laemmli (33) was used for
SDS-polyacrylamide gel electrophoresis. Western blotting followed the
procedure of Schmidt et al. (34). Antibodies against the
full-length, mature Atp12p protein were prepared as described (18) and
used at a dilution of 1:100.
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RESULTS |
Characterization of the atp12 Mutants Obtained by Chemical
Mutagenesis--
Thirteen independent yeast isolates, with mutations
in the ATP12 gene, were obtained by chemical mutagenesis
with nitrosoguanidine and ethyl methanesulfonate (24). These strains
are respiratory-deficient due to a defect in the F1-ATPase
assembly pathway and fail to utilize non-fermentable carbon sources for
growth (17). The biochemical properties of some of the mutant
atp12 strains were reported previously (18). In the current
work, the mutant genes were cloned and sequenced from each of the
atp12 strains to determine the type of genetic lesions that
alter Atp12p function (see under "Materials and Methods"). This
analysis disclosed a single missense allele (carried in strains E822
and E823) and nine mutant alleles (eight nonsense and one frameshift)
that cause early termination of the Atp12p protein (Table
IV). The genetic lesions in the latter strains should lead to the synthesis of Atp12p with deletions of 29, 48, 76, 86, 172, 187, 223, 271, or 274 amino acid residues from the
carboxyl terminus (Table IV). The positions of the chemically induced
mutations are indicated in the Atp12p sequence shown in Fig.
3, using arrows that are flagged with the
name of the mutant strain (highlighted in the black
boxes). Western analysis (data not shown, see also Ref. 18) shows
the presence of the mutant form of Atp12p in mitochondrial samples
prepared from the atp12 strains, C264, E822, E695, N242, and
P366. Such proteins are deleted for up to 86 amino acids from the
carboxyl terminus of Atp12p. Mutant Atp12p proteins that are predicted
to have 172 or more amino acids removed from the carboxyl terminus were
not detected in Western blots of total mitochondrial protein.

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Fig. 3.
Description of Atp12p mutants. The
protein sequence for the "mature" form of Atp12p is shown in
single-letter code. The names of the atp12
strains, which were obtained by chemical mutagenesis, are
highlighted in the black boxes; the
arrow indicates the amino acid residue that is mutated in
each of the strains. The mutation in strain E695 shifts the frame at
Asp-267. Strains E822 and E823 produce Atp12p with a Glu-289 Lys
missense mutation. In the remaining atp12 strains, the
denoted amino acid is mutated to a stop codon. The white
boxes bear the names of the plasmid-borne Atp12p deletion mutants.
Arrows are used to indicate the first Atp12p amino acid in
the 1-81, 1-124, 1-180, and 1-224 Atp12p proteins, and
the last retained amino acid in the V306, V283, and P239 Atp12p
proteins. Since the construction of the plasmids coding for the
1-81, 1-124, 1-180 Atp12p proteins resulted in the addition
of sequence between the mitochondrial leader peptide (of Atp11p) and
the mature Atp12p polypeptide fragments, any or all of these proteins
might have amino acids coded for in the linker sequence preceding the
Atp12p amino acid that is indicated in the figure (see Footnote
3).
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Respiratory Properties of Yeast That Produce Genetically Engineered
Forms of Atp12p with Deletions of Sequences from the Carboxyl or the
Amino Terminus--
The atp12 nonsense alleles (see above)
encode truncated forms of Atp12p, which are inactive when produced in
single copy from the chromosome. To investigate the possibility that
carboxyl sequences could be dispensed with if the level of truncated
Atp12p is raised in the cell, a series of multi-copy plasmids were
constructed with atp12 genes that have deletions of
sequences from the 3
end of the gene (Table III). These plasmids
direct the synthesis of the mutant proteins, Atp12(P239)p,
Atp12(V283)p, and Atp12(V306)p, which are missing 86, 42, and 19 amino acids, respectively, from the carboxyl terminus (Table III). The
position of the last retained amino acid in these mutant proteins is
indicated with an arrow in the Atp12p sequence shown in Fig.
3.
The properties of the plasmid-borne Atp12p mutant proteins were
evaluated in the genetic background of a respiratory-deficient yeast
strain that harbors a disruption at the ATP12 locus
(aW303
ATP12 (18)) (Table V). None of
the mutant proteins that have sequences deleted from the carboxyl
terminus conferred to the host strain the ability to grow within
48 h using ethanol-glycerol (EG), a non-fermentable carbon source
(Table V). Low levels of respiratory activity were observed only for
the strain that produces Atp12(V306)p, which eventually grows on EG
plates after 3-4 days at 30 °C. Western analysis established that
the plasmid-produced Atp12(V306)p, Atp12(V283)p, and Atp12(P239)p
proteins were present in mitochondria isolated from the respective
yeast transformants at 30-80% of the wild type level (Fig.
4 and Table V). Similar results were
obtained when mitochondrial extracts (solubilization with 0.1% sodium
deoxycholate), rather than total mitochondria, were analyzed by Western
blots (data not shown). With respect to the Western analysis it is
important to note that while the antigen used to raise the polyclonal
Atp12p antiserum in previous studies (18) included the full sequence for mature Atp12p, there is no proof that epitopes for the antibody are
distributed along the entire length of the protein. Thus, truncated
forms of Atp12p might, in fact, be missing epitopes or could have an
altered structure that compromises epitope presentation. It is for
these reasons that the levels of the truncated forms Atp12p detected by
Western analysis of mitochondria (Table V) are taken to represent the
minimal amount of the mutant proteins in the organelle. In other
experiments, the mitochondrial samples prepared from the mutant yeast
were assayed for ATPase activity in the absence and presence of
oligomycin (Table V). Since the amount of oligomycin-sensitive ATPase
activity indicates the amount of F1 that is properly
assembled and bound to F0, this analysis provides an
indication of whether or not the resident Atp12p protein is competent
for mediating F1 assembly. Virtually no ATPase activity (<5% wild type level) was detected in the yeast transformants that
overproduce the Atp12(V306)p, Atp12(V283)p, and Atp12(P239)p proteins (Table V), indicating that the removal of sequences from the
carboxyl terminus of Atp12p severely compromises its action in the
F1 assembly pathway.
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Table V
Respiratory phenotype of yeast that produce truncated forms of Atp12p
Yeast were grown at 30 °C unless indicated otherwise.
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Fig. 4.
Western blot of mitochondria prepared from
yeast transformants cultured at 30 °C, which produce truncated forms
of Atp12p from plasmids. Mitochondria were prepared from
aW303 ATP12 transformants that produce the Atp12p proteins indicated
in the figure. The following amounts of total mitochondrial protein
were loaded in each lane of a 12% SDS-polyacrylamide gel: wild type Atp12p, Atp12(V306)p, Atp12(V283)p, 20 µg; Atp12(P239)p, 40 µg; Atp12( 1-81, 1-124, 1-180, 1-224)p, 60 µg. Gel
electrophoresis and Western blotting were done as described under
"Materials and Methods." After probing with antibody against
Atp12p, the blot was reacted with 125I-protein A and
exposed to x-ray film. The migration of molecular mass markers (given
in kilodaltons) is indicated in the left-hand margin of the
figure.
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Deletion mutagenesis was also employed to analyze the functional
significance of amino-terminal sequences of Atp12p using aW303
ATP12
as the yeast host for recombinant plasmids. For this work, multi-copy
plasmids were constructed in which 5
-end-deleted portions of the
ATP12 coding region were fused in-frame with the sequence
for the mitochondrial targeting signal of the Atp11p protein (21) to
encode the mutant proteins, Atp12(
1-81)p, Atp12(
1-124)p, Atp12(
1-180)p, and Atp12(
1-224)p (Table III). Following
cleavage of the Atp11p targeting signal, these mutant Atp12p proteins
are missing 51, 94, 150, or 194 residues from the amino terminus of mature Atp12p. The first amino acid of Atp12p in these proteins is,
respectively, Leu-82, Cys-125, Gln-181, and Ile-225 (see Fig. 3).
Of the four mutant proteins that are deleted for amino-terminal
sequences, two were shown to confer respiratory competence (e.g. growth on EG plates) to aW303
ATP12 within 24-48 h
of incubation at 30 °C (see results for Atp12(
1-81)p and
Atp12(
1-124)p, Table V). Western analysis performed with
mitochondria from these yeast transformants disclosed that only
Atp12(
1-124)p was observed at an appreciable level (18% relative
to the control) in samples of total mitochondrial protein (Fig. 4, see
data for cells grown at 30 °C in Table V) and in soluble extracts of
mitochondria (data not shown). The yeast transformant that produces
this protein was also shown to display 14% the control level of
oligomycin-sensitive ATPase activity in mitochondria isolated from
cells grown in galactose at 30 °C (Table V). Despite the fact that
Atp12(
1-81)p was not detected in Western blots of isolated
mitochondria, yeast carrying the plasmid for this protein showed 78%
the wild type level of oligomycin-sensitive ATPase activity (Table V).
The evidence for high levels of assembled F1 in yeast that
produce Atp12(
1-81)p suggests that the mutant protein is active
in vivo. To explain the absence of Atp12(
1-81)p from
Western blots, we suggest that either epitopes for the antibody are
masked in the mutant protein or that the mutant protein might be
unusually unstable in vitro.
Two of the yeast transformants that produce truncated forms of Atp12p
were found to be temperature-sensitive for respiratory function. At
23 °C, yeast that overproduce either Atp12(
1-124)p or
Atp12(
1-180)p showed wild type growth on EG plates. Moreover, there
was 60% of the control level of oligomycin-sensitive ATPase activity
measured in mitochondria isolated from these transformants, following
their growth at 23 °C in galactose media (Table V). The effect of
temperature is most notable for yeast that synthesize Atp12(
1-180)p, as this strain is completely respiratory-deficient at 30 °C (see Table V). The enhancement or acquisition of
respiratory activity at 23 °C was not accompanied by an increased
amount of Atp12(
1-124)p or Atp12(
1-180)p detected by Western
analysis in samples of isolated mitochondria (see Table V). Again,
altered epitope display or marked instability in vitro offer
potential explanations for the difficulties in detecting forms of
Atp12p deleted for amino-terminal sequences in Western blots.
Respiratory Properties of Yeast That Produce Atp12p with Mutations
at Glu-289--
The identification of a Glu-289
Lys missense
mutation in the Atp12p sequence of two independent atp12
isolates (E822, E823; Table IV) suggests that Glu-289 may be important
for the activity of the protein. For this reason, the effects of
changes at Glu-289 were further analyzed by generating three additional
substitutions; E289D, E289A, and E289Q (see Footnote 3 for method
details). The respiratory phenotypes of yeast that produce Atp12p with
substitutions at Glu-289 are given in Table
VI. Under conditions in which Atp12p is
produced from multi-copy plasmids in the genetic background of the
disruption strain aW303
ATP12, all four mutant proteins were
detected by Western analysis at near wild type levels in isolated
mitochondria (data not shown) and in soluble extracts of
deoxycholate-treated mitochondria (Fig.
5). The mutant protein Atp12(E289K)p
fails to confer respiratory function (growth on EG plates) to the host
strain aW303
ATP12, even when produced from a multi-copy plasmid
(Table VI). In this case only 11% of the wild type level of
oligomycin-sensitive ATPase activity can be detected in isolated
mitochondria. This observation is in accord with the report that the
ability of yeast to utilize non-fermentable substrates for growth
correlates with the presence of at least 15% mitochondrial ATPase
activity (35). Yeast transformants harboring the atp12
mutations E289A or E289Q show normal growth on EG plates but exhibit
60-70% reduction in the level of mitochondrial oligomycin-sensitive
ATPase activity (Table VI). In the case of the mutation E289D, wild
type levels of mitochondrial ATPase activity are observed whether the
mutant protein is produced from a multi-copy plasmid (Table VI) or from
a single copy plasmid (data not shown). Since all four mutant proteins
are detected in mitochondria at near wild type levels, the data suggest
that the reduced activity of Atp12p harboring either the E289K, E289A,
or E289Q mutations is not due to a reduction in the amount of the
mutant proteins but is likely to be a consequence of having a
non-acidic residue at position 289 in the sequence.
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Table VI
Respiratory phenotype of yeast that produce forms of Atp12p with
missense mutations at Glu-289
Yeast were grown at 30 °C.
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Fig. 5.
Western blot of soluble mitochondrial
extracts prepared from yeast transformants that produce plasmid-borne
Atp12p with mutations at Glu-289. Mitochondria were isolated from
aW303 ATP12 transformants that produce the Atp12p proteins indicated
in the figure, and sodium deoxycholate (0.1%) was used to prepare
soluble extracts of mitochondria as described under "Materials and
Methods." Equivalent amounts of total solubilized protein (10 µg)
were loaded in each lane of a 12% SDS-polyacrylamide gel. Gel
electrophoresis and Western analysis were done as described in the
legend to Fig. 4. The migration of molecular mass markers (given in
kilodaltons) is indicated in the left-hand margin of the
figure.
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Sedimentation Analysis of Plasmid-borne Atp12p Variants--
The
molecular mass of mitochondrial Atp12p was estimated from its
sedimentation properties in linear sucrose gradients to be in the range
of 70-80 kDa (18). The molecular mass of the mature protein, as
estimated from its migration in SDS gels relative to the precursor
(18), is 33 kDa. These results suggest that the protein observed in
sucrose gradients is oligomeric. Sedimentation analysis was used to
determine if the ability of Atp12p to form higher ordered oligomers is
lost when sequences are deleted from the amino or carboxyl terminus or
as a result of mutations at Glu-289. Soluble mitochondrial extracts
were prepared from the yeast transformants that overproduce mutant
forms of Atp12p and centrifuged through linear sucrose gradients in the
presence of molecular weight standards. Only the deletion mutants of
Atp12p, whose presence was detected in Western blots of mitochondrial samples (see Table V), were analyzed in these studies, i.e.
Atp12(V306)p, Atp12(V283)p, Atp12(P239)p, and Atp12(
1-124)p.
The sizes predicted for monomers of mutant forms of the protein are
given in Table VII. The three proteins
that are missing carboxyl sequences (Atp12(V306)p, Atp12(V283)p, and
Atp12(P239)p) co-sediment with myokinase (Mr = 21,000) in sucrose gradients (Fig. 6).
This result suggests that these three mutant proteins are present as
monomers in the mitochondrial matrix. On the basis of sequence, the
size of the Atp12(P239)p monomer is comparable to that of
Atp12(
1-124)p (Table VII), yet the sedimentation profiles for these
two proteins are significantly different (Fig. 6). The apparent larger
size noted for the amino-terminal deletion mutant suggests that
Atp12(
1-124)p forms an oligomer in vivo (Table VII).
With respect to the mutant proteins that are substituted at Glu-289,
the sedimentation behavior of all four proteins (Fig. 6) was shown to
be comparable with that of wild type mitochondrial Atp12p (18). These
results indicate that the E289K, E289D, E289A, and E289Q mutations have
no effect on Atp12p oligomerization.

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Fig. 6.
Western blots showing the sedimentation
properties of mutant forms of Atp12p. For sedimentation analysis
of Atp12p variants, mitochondria were isolated from aW303 ATP12
transformants that produce the mutant proteins indicated in the figure
and sonicated as described under "Materials and Methods." The
solubilized mitochondrial protein samples (200 µl) were each mixed
with hemoglobin (Mr = 64,500) and myokinase
(Mr = 21,000), and loaded on 4.8-ml 7-20% sucrose gradients. After centrifuging the samples at 42,000 rpm for
16.3 h in an SW55Ti rotor at 4 °C, 16 fractions were collected from the bottom of the tube. The position of Atp12p protein in the
gradients is indicated in the Western blots shown in the figure. The
numbers above each blot indicate fractions of the gradient; sucrose density increases going from right (high
numbers) to left (low numbers).
Spectrophotometric and enzymatic assays, respectively, were used to
define the peak positions in the gradients for hemoglobin (Hb, filled arrowhead) and myokinase
(MK, open arrowhead) as described (21). Western
analysis followed the procedures described in the legend to Fig.
4.
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Analysis of the Oligomeric State of Atp12p--
Efforts were made
to determine if mitochondrial Atp12p is a homo- or hetero-oligomer. As
part of this work, Histag-forms of Atp12p synthesized in E. coli were used to evaluate the size of Atp12p in the absence of
other mitochondrial proteins. Control experiments had shown that the
presence of the (6x)histidine tag sequence does not alter the
functional properties of Atp12p. Such experiments employed an
atp12 null mutant transformed with a yeast plasmid
(pG57/ST21, Table III) that encodes a protein chimera in which the
Atp11p leader sequence is used to sort the tagged Atp12p protein
(called YHistag-Atp12p for Yeast Histag-Atp12p) to
mitochondria. The (6x)histidine sequence is retained on YHistag-Atp12p
following cleavage of the heterologous leader peptide as indicated by
the fact that YHistag-Atp12p (but not the non-tagged form of the
protein) is selectively precipitated from mitochondrial extracts with
Ni-NTA beads (procedure described under "Materials and Methods,"
data not shown). Notably, the atp12 null mutation is
complemented by the plasmid-borne YHistag-Atp12p as indicated by the
ability of the transformed strain to grow on ethanol-glycerol plates at
the wild type rate and to exhibit wild type levels of
oligomycin-sensitive mitochondrial ATPase activity (data not
shown).
Histag-Atp12p was overproduced in E. coli from the plasmid
pHISATP12 and purified to homogeneity (see Fig. 1). The
sedimentation behavior of Histag-Atp12p (mass = 35.7 kDa) in
7-20% sucrose gradients (Fig. 7) is
significantly different from that of Atp12p that is lacking the last 19 amino acids, which migrates as a monomer (see sedimentation profile for
Atp12(V306)p (mass = 30.9 kDa) in Fig. 6). The size
difference (~5 kDa) between Histag-Atp12p and Atp12(V306)p is too
small to account for the different migration of the two proteins in
this experiment; similar work performed with the native and
biotin-tagged forms of Atp11p indicated comparable sedimentation profiles for the proteins despite a difference of ~9 kDa in their size (21). The sedimentation profile of bacterially produced Histag-Atp12p is much more similar to that of the wild type
mitochondrial protein (18) and to the mitochondrial Atp12p variants
substituted at Glu-289 (Fig. 6), which sediment in sucrose gradients as
oligomers. This analysis suggested that the native form of Atp12p is a
homo-oligomer. However, the results from additional experiments
conflict with this proposal. Studies with purified bacterial
Histag-Atp12p that were designed to visualize a covalently linked
Atp12p dimer gave negative results. SDS gels run with purified
Histag-Atp12p in the absence of 2-mercaptoethanol did not show evidence
of disulfide-linked dimers; such linkages were predicted as possible
since there are three cysteines in the Atp12p polypeptide chain. In
other work, three amine-reactive bifunctional chemical reagents
(disulfosuccinimidyl tartarate, ethylene
glycolbis(sulfosuccinimidylsuccinate),
dithiobis(sulfosuccinimidylpropionate)), and glutaraldehyde, were used
to cross-link purified recombinant Histag-Atp12p as described under
"Materials and Methods"; there was no evidence in SDS gels for
cross-linked Histag-Atp12p dimers generated by any of the chemical
modification reactions (data not shown). Notably, under the same
conditions cross-linking of hemoglobin by glutaraldehyde was observed
suggesting that the experiment was properly designed to identify the
presence of oligomers. Additional evidence against the formation of
Atp12p homo-oligomers was obtained using the yeast two-hybrid screen
(29), in which the two plasmids used in the assay both carried the gene
for Histag-Atp12p. No activation of the lacZ reporter gene
was observed suggesting that Atp12p monomers do not interact under the
conditions of this assay. Although the negative result with the
two-hybrid screen is not final proof against Atp12p
homo-oligomerization, it is in agreement with the results from the
cross-linking experiments that were performed with a battery of
chemical modifiers.

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Fig. 7.
Western blots showing the sedimentation
properties of recombinant forms of Atp12p purified from bacteria.
Histag-Atp12p and the fusion protein MBP-Histag-Atp12p were each
purified from E. coli as described under "Materials and
Methods" (see Figs. 1 and 2). The experiment with purified Histag
Atp12p (loaded as 2 µg in 200 µl of Tris-HCl, pH 7.5) was performed
as described in the legend to Fig. 6. The conditions for the
sedimentation experiment with the MBP-Histag-Atp12p fusion protein
(loaded as 2 µg in 200 µl of Tris-HCl, pH 7.5) were the same with
the following exceptions. First, 10 mM maltose was included
in the buffer to prevent aggregation of the maltose binding protein,
which oligomerizes in the absence of maltose (36). Second, lipoamide
dehydrogenase (100 kDa) was included with hemoglobin as a size standard
in the gradient; lipoamide dehydrogenase activity was assayed as
described (18). Finally, 15 fractions of identical volume were
collected from the bottom of the tube. Western analysis to determine
the position of the recombinant Atp12p proteins in the gradients was done as described in the legend to Fig. 4, and the results are illustrated as in Fig. 6.
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The possibility was considered that the apparent migration of
recombinant Histag-Atp12p as a homodimer in sucrose gradients (Fig. 7)
was due to an artifact imposed by the purification procedure, which
included extraction from inclusion bodies under denaturing conditions
followed by renaturation, and efforts were made to overproduce the
protein in bacteria in soluble form. To this end, a recombinant fusion
protein formed between maltose binding protein (MBP) and Histag-Atp12p
was found to remain soluble following bacterial cell lysis (see under
"Materials and Methods"). Purified MBP-Histag-Atp12p (Fig. 2)
(mass = 77 kDa) sediments in 7-20% sucrose gradients to a
position between the hemoglobin (mass = 66 kDa) and lipoamide
dehydrogenase (mass = 100 kDa) marker proteins (Fig. 7). This
result indicates that the fusion protein migrates as a monomer. Thus,
cumulatively, the results from cross-linking studies, the yeast
two-hybrid screen, and sedimentation analysis of recombinant
MBP-Histag-Atp12p suggest that pure, homogeneous Atp12p does not form
homo-oligomers and that the oligomeric form of Atp12p observed in
mitochondrial samples most likely originates from the interaction of
Atp12p with one or more different gene products.
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DISCUSSION |
Sequence analysis of 13 independent isolates of yeast harboring
chemically induced mutations in the ATP12 gene identified eight nonsense alleles, one frameshift mutation that leads to premature
termination of the protein, and a Glu-289
Lys substitution in the
Atp12p primary structure (Table IV). The low frequency of missense
mutations (1 in 10 alleles) is not a common feature of the
pet mutant collection (24) from which the atp12
strains were obtained. A plausible reason why more missense mutations are not present among the atp12 strains is because the
genetic screen that was used originally to select the strains (24) was based on the ability of cells to grow on non-fermentable carbons (ethanol-glycerol, EG). Thus, only the most severe mutations, which
prevent Atp12p from assembling F1 in amounts sufficient to
support growth on EG plates, were selected by the screen. Since most
mutations found in the atp12 genes produce deletions, it would appear that Atp12p is relatively resilient to substitutions of
individual residues. However, it was found that at least one position
(residue 289) in the amino acid sequence may be of particular importance. Our study indicates that an acidic residue is required at
this position for optimal Atp12p activity.
Identification of Domains in Atp12p--
A schematic map of wild
type Atp12p is presented in the upper part of Fig.
8. The mitochondrial targeting sequence
(black-shaded region in the wild type Atp12p map) is
estimated to be 30 amino acids long (Met-1 through Leu-30) on the basis
of the relative migrations of the precursor and mature forms of Atp12p
in SDS gels (18). Two domains were mapped in the mature protein using deletion mutants of Atp12p (for reference, Fig. 8 also includes schematic protein maps for the Atp12p deletion mutants and provides a
summary of the characteristics of these proteins). The sequence between
Gln-181 and Val-306 constitutes the functional domain of the protein
(see hatched region in the wild type Atp12p protein map).
The amino-terminal boundary of this domain was assigned on the basis of
the results obtained with the mutant protein, Atp12(
1-180)p (see
Atp12(
1-180)p map in Fig. 8). The overproduction of
Atp12(
1-180)p confers conditional growth properties to the atp12 disruption strain. At the permissive temperature
(23 °C) this mutant protein fosters assembly of a functional
mitochondrial ATPase and thereby enables the host strain to use
non-fermentable carbon sources for growth. No evidence of
F1 assembly was found in yeast that produce Atp12p with a
longer deletion from the amino terminus (see
Atp12(
1-224)p, Fig. 8). The carboxyl-terminal boundary of the functional domain in Atp12p is assigned to Val-306, which is the
last retained amino acid in the mutant protein, Atp12(V306)p (see
Atp12(V306)p map in Fig. 8). Notably, this mutant protein is
significantly impaired for activity, as indicated by the phenotype of
the yeast transformant that produces it from a multi-copy plasmid (Table V). When cultured in media containing the fermentable sugar,
galactose, there was no evidence for functional mitochondrial ATPase
(Table V). However, this strain displays partial respiratory function
(which means there must be some level of assembled ATPase) as indicated
by the slow growth phenotype on EG plates (discussed under
"Results"). Therefore, it is proposed that the minimal sequence necessary for the action of Atp12p in F1 assembly is
contained within the Atp12(V306)p protein.

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Fig. 8.
Protein maps for wild type Atp12p and Atp12p
deletion mutants. A protein map for the wild type Atp12p primary
sequence is shown in the upper part of the figure. The
black-shaded, hatched, and
gray-shaded regions of the map indicate discrete
domains in Atp12p (see text for details). The open
rectangles in the lower part of the figure are maps
that show the region of the mature Atp12p protein, which is retained in
the mutant proteins that are indicated in the figure. The
table in the right-hand portion of the figure
summarizes the salient features of the mutant Atp12p proteins. The
oligomerization state was not determined (n.d.) for the
mutant proteins whose presence was not detected in samples of isolated
mitochondria.
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The results from sedimentation analysis with mitochondrial samples that
harbor variant forms of Atp12p identified a second domain in the
protein, which is important for intermolecular associations. Native
Atp12p is observed to form a higher-ordered oligomer in mitochondria
(18). Deletions made from the amino terminus, and substitutions at
Glu-289 in the sequence, did not show evidence for interfering with the
ability of Atp12p to oligomerize (see data for Atp12(
1-124)p and
the E289K, E289D, E289A, and E289Q proteins in Fig. 6). However,
removing as few as 19 amino acids from the carboxyl terminus yields a
mutant protein (Atp12(V306)p) that sediments like a monomer (Fig. 6).
On this basis the carboxyl-terminal sequence between Asp-307 and
Gln-325 is designated as a discrete domain in Atp12p that is involved
in its oligomerization. The loss of this domain, which reduces the
ability of Atp12(V306)p to form stable oligomers, likely explains the
defective behavior of the protein (see above).
Studies performed to determine if mitochondrial Atp12p is a homo- or
hetero-oligomer provided conflicting results initially. A preparation
of (6x)histidine-tagged Atp12p produced in E. coli was found
to show sedimentation properties similar to the native mitochondrial
protein (Fig. 7), which was interpreted to be representative of
homo-oligomerization. However, such putative homo-oligomers were not
visualized in cross-linking studies performed with the purified
recombinant protein. There was also no evidence for intermolecular associations of Atp12p monomers using the yeast two-hybrid system. The
ambiguity in the data was resolved by examining the sedimentation properties of a chimera (Fig. 2) between maltose binding protein and
Histag-Atp12p. The MBP-Histag-Atp12p fusion protein purified from
bacteria was shown to migrate as a monomer in linear sucrose gradients
(Fig. 7). The results of this analysis, along with the chemical
modification and genetic studies described above, argue against the
formation of homo-oligomers of Atp12p. On this basis we suggest that
the native form of Atp12p observed in mitochondrial samples is a
hetero-oligomer.
We gratefully acknowledge Domenico Gatti for
helpful discussions and for critical evaluation of the manuscript.