From the Department of Biochemistry and Molecular Biology, P. O. Box 13D, Monash University, Clayton Campus, Victoria 3800, Australia
Received for publication, May 9, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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We have investigated the question of the presence
of a cap structure located at the top of the F1
F1F0-ATP synthase uses energy produced
from the electrochemical gradient to produce ATP from ADP and
Pi. The enzyme complex is described as having two major
structural domains, F1 and F0. The globular
F1 catalytic sector contains three Despite the considerable advances in determining much of the structure
of F1 (2-4), there are many questions that remain unanswered concerning the structure/function relationships of F1F0-ATP synthase components. One important
question relates to the structure of F1 in the vicinity of
the dimple at the very top of F1. A "cap" at the top of
F1 has been visualized in electron microscopic images of
ATP synthase isolated from Escherichia coli (5, 6) and
bovine mitochondria (7). This cap structure is not seen in models of
ATP synthase derived from x-ray crystallographic data of
F1. The identity of the proteins that contribute to the cap
structure is not clear but may represent, in part, the N-terminal 20-30 amino acids of subunits It is not clear whether the cap represents a solid structure closing
off the surface area at the top of F1 as represented by the
mouth of the dimple seen in F1 crystal structures. Under the experimental conditions used by Böttcher and colleagues (6) the cap of the E. coli ATP synthase
(EF1F0) was shown to cover much of the area
represented by the dimple. Thus, upon binding of the nucleotide
analogue AMP-PNP to EF1F0 a cap structure could be detected concomitant with significant shrinkage in the diameter of
F1 (6). It was suggested that some of the differences
observed on binding AMP-PNP were a result of a significant
rearrangement in the N-terminal domains of the In this study we have investigated the question of the presence of a
flexible cap structure in the yeast mitochondrial
F1F0-ATP synthase (mtATPase) complex. Although
direct evidence for a cap structure has yet to be reported for yeast
mtATPase, the high degree of conservation documented between eukaryotic
mtATPase complexes would favor the existence of a cap. Thus we have
sought to determine whether the putative cap precludes the assembly
into mtATPase complexes of subunit Our strategy has been to express in yeast cells subunit Materials--
Dodecyl Construction of Yeast Expression Vectors--
A DNA cassette
encoding a yeast enhanced variant of GFP, referred to here as YEGFP3L,
was retrieved by PCR using the primer pair YGFP3UP/YGFP3DO
(YGFP3UP, 5'-GGATCCATCGCCACCATGTCTAAAGGTGAAGAATTATTCACTGG-3'; YGFP3DO,
5'-CAGCTGTTATTTGTACAATTCATCCATACCATGG-3') with the vector pYGFP3
(15) as template. The PCR product was cloned into the BamHI/NotI site of the multicopy yeast expression
vector pAS1NB to produce pAS1NB::YEGFP3L from which the DNA
cassette encoding wild-type GFP had been removed. pASN1B is a
derivative of pAS1N (16) in which a BamHI restriction site
has been removed from the PGK promoter region. This vector
allows the expression of GFP not fused to a partner protein.
The DNA cassettes encompassing segments of the yeast ATP3
gene for subunit Construction of Yeast
Strains--
pRS306::ATP3-YEGFP3L27 was linearized by
digestion at the unique XbaI site 363 bp upstream of the
initiation codon for subunit
A PCR product was generated with the primer pair ATP3POUP/ATP3TDO using
pRS306::ATP3-YEGFP3L4 as a template and Dynazyme EXT thermostable polymerase (Finnzymes, Espoo, Finland). The product was digested with XbaI and used to transform YRD15 cells.
Transformants (ura+) and then ATP3 gene
replacement events were selected as described above. Strain MP
Expression vector pAS1NB::YEGFP3L was introduced into strain
YRD15 to produce the strain MPGFPc. Cells of this strain express GFP
not fused to another protein, and therefore GFP is localized to the cytoplasm.
Subcellular Fractionation--
Mitochondria were prepared from
spheroplasts (21) and stored in aliquots at Assays of ATP Hydrolysis Activity--
Assays of mitochondrial
lysates were performed as described previously (22). ATPase activity of
mtATPase separated on clear native gels was determined in
situ by incubating the gel slices in a solution of 5 mM ATP, pH 8.6, in 50 mM glycine/NaOH, pH 8.6, 0.05% lead acetate, 1 mM magnesium acetate with gentle
agitation at room temperature (23). A white precipitate in the gel was indicative of ATPase activity. Images of gel sections made for detection of ATPase activity were recorded against a black background using a flat bed document scanner.
Electrophoresis--
Clear native gel electrophoresis was
performed essentially as described previously (24). 200 µg of
mitochondrial protein was pelleted for 10 min at 100,000 × g. Each pellet was solubilized in 20 µl of extraction
buffer (50 mM NaCl, 2 mM 6-aminocaproic acid, 1 mM EDTA, 50 mM imidazole-HCl, pH 7.0, 5 mM phenylmethylsulfonyl fluoride, and 3% (w/v)
dodecyl Western Blotting--
Proteins were transferred to
polyvinylidene difluoride membranes (Gelman Laboratory, Pall
Corporation) after SDS-PAGE by standard procedures. Membranes were
probed with rabbit polyclonal antisera against subunit Modeling of Fusion Proteins--
The x-ray crystal structures of
bovine mitochondrial F1-ATPase (Ref. 2; Protein Data Bank
identifier 1BMF) and GFP (Ref. 28; Protein Data Bank identifier 1EMB)
were obtained from the Protein Data Bank (www.rcsb.org; Ref. 29). Using
the tools available within the program Quanta (Accelrys Inc., San
Diego, CA), and to simplify the modeling procedure, a "template"
structure was created containing subunit
To build the model for the zero length linker, the GFP structure was
positioned "end on" with its N terminus of GFP proximal to the C
terminus of subunit In YRD15 yeast cells lacking endogenous subunit 3
3 hexamer of the yeast mitochondrial
F1F0-ATP synthase complex. Specifically, we
sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the
3
3 hexamer or alternatively whether the
cap is more flexible permitting access to the central shaft space under
certain conditions. Thus, we sought to establish whether subunit
,
an essential component of the F1 central stalk housed
within the central shaft space and whose N and C termini would both lie
beneath a putative cap, could be fused at its C terminus to green
fluorescent protein (GFP) without loss of enzyme function. The GFP
moiety serves to report on the integrity and location of fusion
proteins containing different length polypeptide linkers between GFP
and subunit
, as well as being a potential occluding structure in
itself. Functional incorporation of subunit
-GFP fusions into ATP
synthase of yeast cells lacking native subunit
was demonstrated by
the ability of intact complexes to hydrolyze ATP and retain sensitivity
to oligomycin. Our conclusion is that the putative cap structure cannot
be an inflexible structure, but must be of a more flexible nature
consistent with the accommodation of subunit
-GFP fusions within
functional ATP synthase complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and three
subunits arranged alternately in a hexamer thereby forming a central shaft space that houses part of the
subunit. Each
subunit contains a catalytic site for the synthesis of ATP. These sites are
repetitively and sequentially driven through defined conformational states by the rotation of the
subunit, which is in turn linked to
the translocation of protons through the membrane bound F0 sector (1). Models of the F1 sector derived from x-ray
crystallographic data indicate the presence of an apparent "dimple"
in the top of the F1 sector ~15 Å deep that is
contiguous with the central shaft space housing the C- and N-terminal
portions of subunit
(2).
and
that were disordered and therefore not represented in models derived from the x-ray crystal data, or alternatively, F0 proteins lost upon
crystallization of the complex. Using an immuno-electron microscopy
approach, bacterial subunit
, in particular its C-terminal portion,
has been localized to the dimple region (8). In earlier studies, proteolysis experiments had indicated that the eukaryotic homologue of
subunit
, OSCP,1 binds to
the N-terminal end of the
subunit (9). Subsequent studies have
positioned OSCP in association with F1, but sometimes at
the top and sometimes at the side (10). Recent evidence obtained using
a protein chemistry approach for ATP synthase isolated from rat
mitochondria indicates that specific regions at the top of F1 are shielded by F0 components (11). It was
suggested that this shielding represents an extension of the stator
stalk and that the cap, composed in part by F6 (whose
functional homologue in yeast is subunit h (12)) and possibly subunit
d, completely covers the N termini of all
and
subunits.
and
subunits.
When a three-dimensional reconstruction of
EF1F0 was viewed from the top looking down the axis of the central stalk a triangular shaped cap appeared to cover the
central shaft space. A flexible cap that shifts its position throughout
the catalytic synthesis of ATP on F1 would be consistent
with the idea that the conformationally flexible N termini of the
and
subunits contribute to its structure. The appearance of the cap
would alter depending on the state in which the complex was
"captured," sometimes completely covering the top of the
F1 and sometimes not.
, an essential component of the central stalk, fused to a reporter protein, green fluorescent protein
(GFP). The N and C termini of subunit
would both lie beneath the
cap within the central shaft space formed by the F1
3
3 hexamer, with the C-terminal glycine
residue about 15 Å below the position of the N-terminal end of
subunits
and
(2).
fused at
its C terminus to GFP via a polypeptide linker. Correctly folded GFP
forms a very rigid and stable 11-stranded
-barrel structure 24 Å in
diameter and 48 Å in length threaded by an
-helix running up the
axis of the barrel (13) that is remarkably stable to the action of a
range of denaturing agents and proteases (14). The fluorescence
properties of GFP have been utilized widely as a convenient tag to
report the presence of a protein in various subcellular locations. We
have used these properties of GFP to report on the integrity and
location of fusion proteins containing different length polypeptide
linkers between GFP and mtATPase subunit
. Functional incorporation
of subunit
-GFP fusions into mtATPase would suggest that the
putative cap structure in vivo is not at any time a
"solid" inflexible structure that completely occludes the dimple in
the top of the F1, but must be of a more flexible nature
consistent with the incorporation of subunit
-GFP fusions within
functional ATP synthase complexes. In such complexes it is presumed
that that the polypeptide linker connecting subunit
with GFP would
not be obstructed in exiting the central shaft space at the top of
F1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-maltoside and complete protease
inhibitors were purchased from Roche Molecular Biochemicals (Sydney,
Australia). Vistra ECF substrate was purchased from Amersham
Biosciences (Sydney, Australia).
were recovered by PCR from YRD15 genomic DNA (17).
The first, ATP3PO, encompasses 729 bp of sequence upstream of the
initiation codon flanked by HindIII and nested
BamHI/BglII/NotI restriction sites at
the 5' and 3' ends, respectively. The second, ATP3T, encompasses a
transcription terminator cassette representing the terminator region of
the ATP3 gene flanked at the 5' and 3' ends by restriction
sites for NotI and SacII, respectively. The oligonucleotide pairs ATP3POUP/ATP3PODO and ATP3TUP/ATP3TDO,
respectively, were used for these amplifications (ATP3POUP,
5'-TGAAAGCTTGAATGTACAGGTTATGACAACC-3'; ATP3PODO,
5'-AACGATGCGGCCGCAGATCTGAGGATCCCAAAGAGGAAGCACCAG-3' and ATP3TDO, 5'-ATCGTTGCGGCCGCGCATTGCCTCTTTATTTGACG-3';
ATP3TDO, 5'-TACTCCGCGGTCTGGGCATACGCTTGGTAAAAAACCAAATC-3'; in
each case the underline indicates the position of relevant restriction
sites). The ATP3PO and ATP3T DNA cassettes were cloned sequentially
into the yeast expression vector pRS306 (18) as
HindIII/NotI and NotI/SacII
fragments, respectively, to produce the expression vector denoted
pRS306::ATP3. A BglII/NotI DNA fragment
encoding YEGFP3L was excised from pAS1NB::YEGFP3L and then
cloned into the BglII/NotI site of
pRS306::ATP3 to produce a vector
(pRS306::ATP3-YEGFP3L27) encoding subunit
fused to YEGFP3
with a polypeptide linker of 27 amino acids (
-27-GFP; Fig. 1). A
vector (pRS306::ATP3-YEGFP3L4) encoding subunit
fused to
YEGFP3 with a polypeptide linker of 4 amino acids (
-4-GFP) was
derived from pRS306::ATP3-YEGFP3L27 by removal of a 69-bp
fragment flanked by BamHI sites (Fig. 1).
and used to transform YRD15 cells by
the method of Schiestl and Gietz (19). Transformants were
selected by plating onto solid minimal medium supplemented with
histidine and leucine. Loss of plasmid sequences leaving only sequences
encoding a
-GFP fusion at the chromosomal ATP3 gene locus
was induced as follows. Individual ura+ transformant
colonies were isolated, cultured in non-selective glucose-containing
medium, and then subjected to positive selection for the loss of the
URA3 marker (ura
) by plating onto medium
containing 5'-fluoro-orotic acid (20). One isolate was designated
strain MP
-27 and expresses
-27-GFP.
-4
expresses
-4-GFP. A PCR product encoding YEGFP3 linked, without any
intervening amino acids, to the C terminus of subunit
(
-0-GFP),
the yeast ADH1 terminator, and the Kluyveromyces lactis URA3-selectable marker and bearing regions of homology sufficient for recombination with the ATP3 gene was prepared
using a template to be described elsewhere. The PCR product was used to
transform YRD15 cells. Following selection of ura+
transformants and then ATP3 gene replacement events (as
described above), one isolate was designated strain MP
-0 (expresses
-0-GFP).
70 °C until required.
For the isolation of cytoplasmically expressed YEGFP3, MPGFPc cells
were cultured in a glucose-containing medium. Cells were harvested,
resuspended in 50 mM Tris/HCl, pH 7.5, 1 mM
EDTA containing Complete Inhibitors (Roche Molecular
Biochemicals) and broken open by vortexing with glass beads at
4 °C for 1 min. Soluble proteins were retrieved by centrifugation
(20,000 × g for 20 min) and stored in aliquots at
20 °C.
-maltoside), incubated on ice for 20 min, and centrifuged
100,000 × g for 10 min. Supernatants (20 µl) were
loaded into wells of 4-16% gradient acrylamide gels (13 × 10 × 0.075 cm). Gels were run for 1 h at 100 V, then 2-3 h
at 500 V with current limited to 5 mA/gel. Gels, while still between
the glass plates, were then imaged for GFP fluorescence using a Wallac
multi-wavelength imager in "edge-illumination mode" equipped with
filters for excitation (480 ± 20 nm) and emission (535 ± 20 nm). After imaging, gels were separated from gel plates and sliced into
sections for further individual analysis. Protein was stained with
Coomassie Brilliant Blue G-250 (24). Proteins extracted from cells (25)
were subjected to SDS-PAGE on 4-20% gradient acrylamide gels
according to standard procedures (26) and using a Bio-Rad mini-gel apparatus.
(diluted
1:1000). Secondary antibodies were alkaline phosphatase-conjugated
anti-rabbit. Signals were generated using chemifluorescent Vistra
substrate and incubating for 10 mins at room temperature.
Chemifluorescence was detected using a Amersham Biosciences
Storm PhosporImager (27). Image data were analyzed using ImageQuant
software (Amersham Biosciences). Integrated volumes for each of
the relevant bands were determined and expressed as a percent of the
relevant intact fusion protein after background correction.
plus all residues within a
40-Å radius of its C terminus. Thus, the template contained the dimple and surrounding regions located at the top of F1.
. Manual positioning of the GFP structure was
carried out so that steric clashes were minimized (using the contacts
package within Quanta to highlight close contacts) in the merging of
the two termini such that the GFP moiety became a C-terminal extension
of subunit
. The entire F1-derived portion of the model
was then constrained (i.e. so that only the GFP portion of
the model was allowed to move) and the model subjected to CHARMm minimization. Further minimization was performed using dihedral constraints applied to residues in the N-terminal region of GFP. Minimization was performed to convergence, and upon completion of the
modeling procedure all residues in the GFP molecule were in allowed
conformations. We predict that in order for the GFP molecule to
correctly link to subunit
, the N-terminal helix must partially
unwind. To create the full model of F1 containing subunit
fused to GFP, the coordinates of the fusion were copied back into
1BMF (since the F1-derived portion of the template was
constrained during minimization the subunit
chain of 1BMF could be
simply replaced with that of the template). Models containing longer
linkers were created similarly, the linkers being added to the C
terminus of subunit
using the "edit protein" facility available
within Quanta. Linkers were modeled as extended regions so as to create
the maximum possible distance between the C terminus of subunit
and
the GFP moiety.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, we expressed
individually the fusions
-27-GFP,
-4-GFP, and
-0-GFP (Fig. 1) and tested their ability to act as a
functional replacement for the native subunit. Yeast cells lacking
expression of subunit
are unable to grow on non-fermentable
substrates because of the absence of a functional ATP synthase (30).
Thus, strains MP
-27, MP
-4, and MP
-0 were assessed for growth
in liquid SaccE medium containing ethanol as carbon source (31). All
three strains grew on ethanol-containing medium, enabling comparison of
their growth rates at 28 °C with that of the parent strain YRD15
(Table I). There was no significant
difference2
(p > 0.05) in generation time for yeast MP
-27 (10.9 h) and the control strain YRD15 (10.4 h). The generation times for
MP
-4 (14.7 h) and MP
-0 (23.9 h), however, were significantly
longer than that for the control strain (p > 0.05).
Collectively these results indicate that functional mtATPase complexes
are assembled in each of the strains MP
-27, MP
-4, and MP
-0 at
levels sufficient to support growth on a non-fermentable substrate.
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Fig. 1.
The structure of the linker joining
subunit and GFP in fusion proteins. The
position of the BglII and BamHI sites, within the
polylinker into which the DNA encoding the subunit
precursor was
cloned, is underlined. The length of the polypeptide linker
for each fusion protein is defined by the number of amino acids between
the C-terminal amino acid of subunit
(G in shaded
box at left) and the initiating methionine of GFP
(M in open box at right). An
arrow indicates for each of the fusion proteins,
-27-GFP,
-4-GFP, and
-0-GFP, the position at which the C-terminal amino
acid of subunit
was fused to the polypeptide linker. The number
below each arrow indicates the length of the linker in amino
acids.
Generation times of strains and oligomycin-sensitive ATPase activities
of isolated mitochondria
Individual cells of strains MP-27, MP
-4, and MP
-0 were
observed by fluorescence microscopy. For cells of each strain
fluorescence due to GFP was distributed in a filamentous manner
characteristic of a mitochondrial location (data not shown). This
result indicated that GFP had been correctly targeted to the
mitochondrion and that the correct folding of the protein and
maturation of the chromophore had occurred.
The integrity of the -GFP fusion proteins was further investigated
as follows. Proteins were extracted from cells of yeast strains
MP
-27, MP
-4, MP
-0, and YRD15 and subjected to SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride membrane. Polypeptides of Mr ~63,800 (Fig.
2, lane 2) and
Mr ~61,600 (Fig. 2, lanes 3 and
4) were detected when a blot was probed with polyclonal antibodies against subunit
. The polypeptides migrated with mobility corresponding to a size slightly larger than that predicted for the
corresponding fusion proteins (Mr 59,997, 57,891, and 57,495 respectively, for
-27-GFP,
-4-GFP, and
-0-GFP). Native subunit
(not fused to GFP) from YRD15
mitochondria (Fig. 2, lane 1) was also found to migrate more
slowly, Mr 32,400, than its predicted size
(Mr 30,661). Probing an equivalent blot with
antibodies against GFP confirmed that each of these polypeptides
contained GFP (data not shown). An additional band
(Mr ~38,000) not present in YRD15 and
representing only minor amounts (1.9%, MP-27; 0.47%, MP-4; and 2.8%,
MP-0) of intact fusion protein was observed when blots were probed with
antisera against subunit
(Fig. 2). If it is assumed this
polypeptide was assembled into functional mtATPase complexes, the
amounts of this polypeptide relative to the intact fusion protein
cannot explain the levels of oligomycin sensitive ATPase assayed in
isolated MP
-27, MP
-4, and MP
-0 mitochondria (see Table I and
below). Thus, compared with YRD15 reductions in ATPase activity of
greater than 97% would be expected instead of the 40% observed for
MP-0 (Table I). These findings confirm the identity of the fusion
proteins and indicate that only complete fusion proteins are present
within the vast majority of ATP synthase complexes. In other
experiments in which a hexahistidine tag was added to the C terminus of
the fluorescent protein moiety, it was possible using
nickel-nitrilotriacetic acid chromatography, to recover assembled
mtATPase complexes that contain only intact
-fusion proteins. Under
conditions where mtATPase complexes from YRD15 mitochondria did not
bind to nickel-nitrilotriacetic acid resin, complexes containing
-4-GFP or
-0-GFP were recovered from lysates of mitochondria that
when assayed for ATPase were found to be oligomycin sensitive (data not
shown). Thus, it can be concluded that
-27-GFP,
-4-GFP, and
-0-GFP are able to functionally replace native subunit
in ATP
synthase complexes. This conclusion is consistent with the results of
other studies from our laboratory that show different subunits of the
yeast mtATPase complex can be functionally replaced by the cognate GFP
fusion protein (16, 32,
33).3
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The integrity and function of isolated ATP synthase complexes
containing -GFP fusion proteins were next investigated. Lysates of
mitochondria isolated from cells of yeast strains MP
-27, MP
-4, MP
-0, and YRD15 were subjected to clear native gel electrophoresis. This gradient gel electrophoresis technique (23) is capable of
separating proteins and protein complexes over a wide size range. After
electrophoresis, gels were sliced longitudinally into several sections.
Each section was subjected to one of the following: staining for
protein, fluorescence imaging, or an in situ assay for
ATPase activity (Fig. 3). When stained
for protein (Fig. 3A), a band having mobility corresponding
to that of assembled monomer ATP synthase, purified by the method of
Rott and Nelson (34), was observed for each of the mitochondrial
lysates isolated from YRD15, MP
-27, MP
-4, and MP
-0 cells.
Since proteins were extracted from mitochondria using the detergent
dodecyl
-maltoside, the species of ATP synthase observed in this
study corresponds, as expected, to the monomeric form. The Coomassie
Blue staining and fluorescence profiles for monomeric mtATPase
complexes from MP
-0 cells shown in Fig. 3 (A and
B) were of reduced intensity and presumably result from a
decreased extractability of complexes. However, similar amounts of each
of the
-GFP fusion proteins were present in whole cell lysates (Fig.
2). ATP synthase complexes in a dimer form can be isolated if digitonin
is used to extract the ATP synthase (35). In a separate experiment
fluorescent bands of lower mobility corresponding to ATP synthase
dimers were observed on clear native gels when samples of MP
-27,
MP
-4, and MP
-0 mitochondria were extracted using digitonin (data
not shown).
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One gel slice was imaged for fluorescence due to GFP (excitation
480 ± 20 nm, emission 535 ± 20 nm). A single fluorescent species with mobility similar to that observed for mtATPase
stained with Coomassie Blue (Fig. 3A, lanes 1-4)
was observed for mitochondria lysates isolated from the three strains
expressing a subunit -GFP fusion protein (Fig. 3B,
lanes 2-4). As expected, fluorescence was not detectable
for the mitochondrial lysate isolated from YRD15 cells (Fig.
3B, lane 1). A cytosolic extract prepared from cells of strain MPGFPc (Fig. 3B, lane 5) was used
to indicate the mobility of GFP not fused to another protein.
Collectively, these results indicate that mtATPase complexes isolated
from MP
-27, MP
-4, and MP
-0 cells are stably assembled and
contain fluorescent GFP. In other experiments regions of clear native
gels corresponding to mtATPase complexes extracted from mitochondrial
isolates of MP
-27, MP
-4, and MP
-0 cells were excised and
subjected to a second dimension of reducing SDS-PAGE. Proteins were
then transferred to polyvinylidene difluoride membrane and probed with
antibodies against subunit
. Polypeptides with mobilities
corresponding to each of the fusion proteins were detected (data not
shown). These findings provide additional confirmation that complete
fusion proteins are present in assembled and functional mtATPase complexes.
The ATP hydrolytic activity of mtATPase complexes containing -GFP
fusions was investigated for osmotically lysed mitochondria using a
spectrophotometric assay. ATPase activity was measured in the absence
and presence of oligomycin, an inhibitor of the F0 proton
channel. Such an assay gives an indication of the degree of functional
coupling between the F1 and F0 sectors of the
complex, since ATPase activity is only sensitive to inhibition by
oligomycin if the two sectors are functionally coupled. Inhibition by
oligomycin (Table I) was found to be in the range of 64-83%, within
the range observed for ATPase isolated from wild-type yeast cells. These findings indicate that the fusion of GFP to the C terminus of
subunit
does not compromise the ability of the
subunit to
assemble into active mtATPase complexes.
In a separate experiment the ATP hydrolytic activity of mtATPase
complexes containing -GFP fusions and separated on clear native gel
electrophoresis was confirmed using an in situ gel ATPase
assay. In the presence of lead acetate, free phosphate liberated by
hydrolysis of ATP forms a white precipitate indicating the site of
ATPase action within the gel (23). Enzyme activity (as indicated by the
presence of a white precipitate) was observed for mitochondrial lysates
(Fig. 3C) isolated from the control strain YRD15 and the
three strains expressing a subunit
-GFP fusion at a position in the
gel corresponding to the Coomassie-stained or fluorescent species
detected in the corresponding lanes of A and B.
This result confirms that mtATPase complexes migrating with the
expected mobility of monomers and containing GFP retain ATPase activity.
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DISCUSSION |
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In this study we have shown that fusion proteins in which GFP is
linked to the C terminus of mtATPase subunit with a polypeptide linker of 0, 4, or 27 amino acids are able to assemble into complexes that are functional in vivo. Furthermore, ATP synthase
complexes isolated from these yeast cells expressing each of these
fusion proteins are correctly assembled, fluorescent, and functionally coupled.
There are little structural data available concerning the nature of the
cap structure. The results in this study indicate that any putative cap
structure in yeast mtATPase complexes containing the -GFP fusion
proteins cannot entirely cover the dimple located at the top of
F1. There must be a route passing through, or around, the
cap structure sufficient to accommodate the polypeptide chain linking
the C terminus of subunit
to GFP. Furthermore, such a route
accommodating the linker must be present throughout all states of the
catalytic cycle of the mtATPase. It would be expected that ATP synthase
complexes prevented from undergoing the full range of co-operative
subunit interactions required for multisite catalysis would be severely
compromised in their ability to synthesize ATP. Such complexes would
not be capable of providing sufficient ATP synthetic capacity to
support growth on respiratory substrates such as ethanol. Cells in
which subunit
was replaced by each one of the fusion proteins were
capable of growth on ethanol and exhibited specific activities for
oligomycin-sensitive ATPase similar to that of the control. Unless the
linker of the
-GFP fusion causes some particular displacement of the
cap compatible with continued mtATPase function, it can be concluded
that complexes containing native subunit
would also possess
potential polypeptide "threading" routes through the cap structure.
A significant displacement of the cap might be expected to result in
some uncoupling of the F1 and F0 sectors of the
complex because of an adverse influence on interactions between OSCP
and
/
pairs. OSCP is known to support important structural
interactions between F1 and F0 and also
modulates proton channel function at a distance (36, 37). However,
instability of mtATPase arising from uncoupled
F1-F0 was not apparent according to the results
presented here.
The generation time for yeast expressing -27-GFP is not
significantly different from that of the cells expressing
subunit not fused to GFP. The x-ray crystal structure coordinates for F1 and GFP were used to model the position of GFP in
relation to the top of the F1
3
3 hexamer (Fig.
4). Given the distance of the GFP moiety
in
-27-GFP from the top of F1 (estimated to be some 40 Å), an effect on growth would not be expected. However, expression of
the shorter linker length fusions,
-4-GFP and
-0-GFP, resulted in
longer generation times. Since growth under these experimental
conditions is dependent upon ATP production by mtATPase, increased
generation times can be taken to indicate that the net rate of ATP
production by the complex is compromised when GFP is tethered closer to
the top of the F1 catalytic sector. The model of
F1 incorporating
-4-GFP (Fig. 4) indicates the distances between the loops extending from the end of the
-barrel of GFP and
the proximal loops of the
and
subunits are of the order of 10 Å such that some interference may occur. Much more extensive contacts
between GFP and the top of F1 are predicted for complexes containing
-0-GFP (Fig. 4). The bottom of the
-barrel of GFP in
these complexes may be expected to make significant contacts with the
surface of the
and
subunits at the top of F1 (Fig. 4). If the putative cap structure includes those N-terminal amino acids
of
and
not resolved in the x-ray crystal structure then these
segments must be placed to one side to prevent interference with the
-barrel of GFP. For complexes containing
-0-GFP the N-terminal
domains of the
and
subunits of F1 may be moved apart to accommodate the GFP like a "cork in a bottle."
|
To maintain chemical equivalency of the three /
pairs during
catalysis two models of the stator stalk have been proposed recently by
Pedersen and colleagues (38). In these models it is envisaged that the
top of the stator stalk makes contact with the top of F1
via a cap consisting of the
subunit in E. coli or
F6, OSCP, and possibly subunit d in the complex
isolated from rat liver mitochondria (11, 38). The cap covers the
dimple region in the "swinging" stator model or alternatively dips
into the dimple in the "stepping" model. In both cases the cap is
anticipated to exchange contacts between the three
/
pairs as
either the cap undergoes rotation (swinging model) or the
3
3 hexamer rotates (stepping model). The
position of GFP in the model of a complex containing the fusion protein
-0-GFP suggests that contact between subunits of the stator stalk
and
/
subunit pairs must occur close to the edge of the rim of
the dimple formed by several
sheets at the N termini of
and
and that contact regions cannot extend downwards into the central shaft
space housing subunit
. It is noteworthy that the exact location of
the OSCP (or subunit
) component of the cap remains uncertain and
variously has been positioned at the top of F1 or
alternatively on the outer face of F1 extending from the
nucleotide binding sites toward the top of F1 (7, 39).
Does GFP rotate during catalysis? It is now accepted that that ATP
synthase is a rotatory motor and that subunit together with subunit
(
in mitochondria) and the subunit c (nine in mitochondria) ring
forms the rotor of the motor (40-42). Rotation of the
subunit
appears to be an absolute requirement for the synthesis or hydrolysis
of ATP. However, ATP synthase is capable of synthesizing ATP through
uni-site catalysis where the subunit
is not required to rotate
(43). In this case ATP synthesis occurs at a greatly reduced rate
compared with complexes in which the subunit
is allowed to rotate
without interference. No evidence is yet available concerning rotation
of GFP in the strains investigated here, but rotation of subunit
is
assumed on the basis of restoration of mtATPase function in host cells
lacking native subunit and the results of in situ ATPase
assays for purified mtATPase complexes. However, it is conceivable that
sufficient freedom exists in the polypeptide linker, joining the
C-terminal region of subunit
to GFP, to allow an uncoupling of
rotation between subunit
and the relatively bulky GFP. Evidence
suggesting such a possibility is feasible is provided by the
observation of forced full rotation of subunit
in
EF1F0 complexes when the penultimate residue of subunit
was cross-linked to subunit
(44). Further experiments are now under way in an attempt to obtain evidence for the rotation of
GFP in these complexes. As we have already shown that other subunits of
yeast ATP synthase can be functionally replaced with GFP fusions
proteins (16, 32, 33),3 complexes containing binary
combinations of appropriate fusion proteins may provide the means for
monitoring catalytic activity by ATP synthase in live cells.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Australian Research Council.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.
These two authors contributed equally to this study.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, P. O. Box 13D, Monash University, Clayton Campus, Victoria 3800, Australia. Tel.: 61-3-9905-3782; Fax: 61-3-9905-4699; E-mail: Rodney.Devenish@med.monash.edu.au.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M204556200
2
Significance levels of difference between
generation times were determined using Student's t test,
comparing control strain YRD15 and cells expressing subunit -GFP fusions.
3 M. Prescott, P. Gavin, K. McKee, S. Kashyap, and R. J. Devenish, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
OSCP, oligomycin sensitivity-conferring protein;
AMP-PNP, adenosine 5'-(,
-imino)triphosphate;
mt, mitochondrial;
GFP, green
fluorescent protein.
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