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INTRODUCTION |
As mitochondria comprise the site of most ATP production in animal
cells by a process known as oxidative phosphorylation, there has been
intense interest in understanding its mechanism (1-3). This remarkably
complex process requires that four major events take place: electron
transport, generation of a proton gradient, transport of Pi
and ADP, and finally coupling the proton gradient to ATP synthesis, a
process catalyzed by the ATP synthase complex
(F0F1).1
The latter two events are closely synchronized as each ATP molecule that is made on the F1 unit of the ATP synthase inside the
mitochondria exits this organelle as new Pi, and ADP
molecules enter simultaneously on separate transporters, referred to
here as PIC and ANC, respectively. As the ATP synthase has long been
known to be associated with inner membrane regions or extensions called
"cristae" (4, 5), it is here that PIC and ANC are also most likely localized.
A major impediment to fully understanding the terminal events of
mitochondrial oxidative phosphorylation is the absence of atomic
resolution structures for the complete ATP synthase, PIC, and ANC. In
this regard, it seems likely that within the mitochondria, as for other
complicated biological systems, supercomplexes exist. One or more may
involve the electron transport chain complexes and another an ATP
synthase-PIC-ANC complex. Significantly, biochemical evidence
for respiratory chain supercomplexes in both mitochondria and bacteria
has been obtained recently (6, 7), as has highly suggestive evidence
for an association of the ATP synthase, PIC, and ANC (8, 9).
Considering recent structural achievements in obtaining high resolution
data on the 70 S ribosome-RNA complex from two different laboratories
(10, 11), it is not unrealistic to assume that similar achievements are
likely to be forthcoming for other "supercomplexes" including those
located in the mitochondrial inner membrane (6, 7). Here, however, the
problem is compounded as the first barrier that must be overcome is not
that of obtaining two- or three-dimensional crystals. Rather it is to
identify an appropriate detergent that will maintain the complex or
supercomplex of interest intact, active, and in soluble dispersed form
(12). This difficulty likely contributes substantially to the fact that of the 19,551 structures currently reported in the Research
Collaboratory for Structural Biology data base, less than 30 are
membrane proteins. Also, in examining several reports where remarkable
success has been achieved (13-16), it is clear that no single
detergent is appropriate for all membrane proteins. Rather, an
exhaustive search must be conducted to identify the appropriate
detergent(s) for each (12).
With the above thoughts in mind, the objectives of the work reported
here were to obtain a highly enriched cristae-like vesicular fraction
containing the ATP synthase in association with PIC and ANC and to
identify detergents most appropriate for solubilizing this membrane
associated supercomplex in an active dispersed form so that future
structural studies could be conducted.
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EXPERIMENTAL PROCEDURES |
Materials
Rats (Harlan Sprague-DawleyCD, white
males) were obtained from Charles River Breeding Laboratories, reagents
for electron microscopy from Pella, digitonin from Calbiochem, and
Lubrol WX from Grand Island Biologicals. Other detergents were from
Anatrace. Linbro 96-well microtiter plates for detergent screening were
from ICN, and the multiwell plate reader was from Labsystems and Flow
Laboratories. Oligomycin was from Sigma, polyvinylidene
difluoride membranes from Millipore, Western blot reagents from
Amersham Biosciences, and Coomassie dye from Pierce. An antibody
to ANC was from Santa Cruz Biotechnology, whereas antibodies to the rat
ATP synthase
-subunit (Walker A region),
-subunit, and PIC
(residues 302-312) were from our own stocks. Antibodies to other ATP
synthase subunits were from Drs. Y. Hatefi and Akemi Matsuno Yagi of
the Scripps Research Institute. The hyperChem version 7 program was
obtained from Hypercube, Inc.
Methods
ATPase and Respiration Assays--
ATPase activity (±0.6 µg
of oligomycin/mg of protein) and respiration (oxygen consumption) were
monitored as described previously (17, 18) in the presence,
respectively, of 3.0 mM ATP and 7.8 mM succinate.
SDS-PAGE, Western Analysis, and N-terminal Sequence
Analysis--
SDS-PAGE was carried out by the method of Laemmli (19),
and Western analysis and N-terminal sequence analysis were carried out
exactly as described previously (20).
Cristae-like Inner Membrane Vesicles--
Each preparation
commenced by preparing from four rats an inner mitochondrial membrane
fraction (IMF) (18), after which a previously described procedure (21)
was modified to prepare the cristae-like membranes. Specifically, the
IMF was suspended first in 16 ml of Buffer A (300 mM
potassium Pi, 4 mM ATP, 10% ethylene
glycol, 5 mM EDTA, and 0.5 mM dithiothreitol,
pH 7.9), frozen in dry ice and acetone, and stored overnight at
20 °C. The IMF was then slowly thawed, made up to 65 ml with
Buffer B (300 mM potassium Pi, 50 mM EDTA, pH 7.9), and washed for 15 min by stirring in an
ice-cold 100-ml beaker. Centrifugation was then carried out for 30 min
in a Ti 70.1 rotor at 50,000 rpm in a Beckman LE 80K ultracentrifuge.
The combined pellets in the multiple tubes were suspended in 65 ml of
Buffer C (300 mM potassium Pi, 50 mM EDTA, 1 mM ATP, pH 7.9) for 15 min and
centrifuged again for 1 h at 50,000 rpm. The combined pellets were
now suspended in 32 ml of Buffer C and centrifuged for 10 min at 6,000 rpm in a SS-24 rotor in a Sorvall RC-2B centrifuge. After saving the
supernatants, the combined pellets were suspended in 16 ml of the same
buffer and centrifuged as before in the Sorvall SS-24 rotor. The
supernatants were saved again, and once more the pellets were suspended
in 16 ml of the same buffer and centrifuged. Then pellets from the two
previous steps were discarded, while the supernatants were combined
and, after 15 min, subjected to centrifugation for 30 min at 50,000 rpm
in the Beckman ultracentrifuge as described above. The pellets were
suspended in 32 ml of Buffer C and centrifuged for 20 min at 10,000 rpm
in the SS-24 rotor in a Sorvall RC-2B centrifuge. After saving the
supernatants, the tubes were tapped gently to dislodge the membrane
pellets from underlying glycogen pellets. Then the membrane pellets
were rinsed out of the tubes, suspended in 16 ml of Buffer C, and
centrifuged in the SS-24 rotor as before. The supernatants were saved,
suspended in 16 ml of Buffer C, and centrifuged as before at 10,000 rpm. The pellets were discarded, while the saved supernatants were
pooled. Ethylene glycol was then added to the pooled supernatants to
give a final concentration of 10%, and after 30 min this fraction was
diluted to 65 ml with Buffer A and centrifuged for 15 min at 20,000 rpm in the Beckman ultracentrifuge. The supernatants were saved and the
pellets discarded. The combined supernatants were then subjected to
centrifugation for 45 min at 50,000 rpm in the same centrifuge. Now,
the supernatants were discarded and the pellets suspended in 16 ml of
Buffer A and centrifuged for 15 min at 20,000 rpm. The resultant
supernatants were centrifuged then for 1 h at 50,000 rpm. Finally,
the supernatants were discarded and the pellets suspended in 32 ml of
Buffer A and centrifuged again for 1 h at 50,000 rpm. The final
pellets comprising the cristae-like inner membrane vesicles were
suspended to 10 mg/ml in Buffer A, frozen in dry ice and acetone, and
stored at
80 °C until use.
Electron Microscopy, Transmission (TEM), and Scanning
(SEM)--
For TEM the cristae-like membranes (20 µg/ml) were
adsorbed onto glow-discharged, carbon-coated parlodion grids, rinsed in distilled water, negatively stained with 1% uranyl acetate + 0.04% tylose, dried, and then viewed and photographed using a Phillips CM 120 transmission electron microscope at 80 kV. After scanning the
negatives, tif images were created with a Zeiss Scanner (SCAI) utilizing Phodis software version 2.1 and then processed with Adobe
Photoshop version 7.0. For SEM the same samples were adsorbed onto glow
discharged silica chips (Pella 16008), fixed for 5 min at 25 °C in
1% glutaraldehyde + PBS, pH 7.4, rinsed twice with PBS and 0.1 M sodium cacodylate, postfixed for 5 min in 2% osmium tetraoxide + 0.1 M sodium cacodylate, and after washing
twice with distilled water, stained enbloc in 2% uranyl acetate. After complete dehydration using 100% ethanol and a critical point dryer (Baltec CPD 30), samples were sputter-coated with 2-nm particles of chromium (Denton, DV 502A) under high vacuum and viewed with a LEO
1530 FIE scanning electron microscope operating at 1 kV. Tif images
were stored and processed with Adobe Photoshop version 6.0.
Multiwell Detergent Screening Assay--
The screen was
conducted using 96-well microtiter plates equipped with a multiwell
plate reader. In each plate four different detergents at 12 different
concentrations ranging from 0 to 2× CMC (in mM) were
tested at 4 °C. For example, detergent "X" and ~1 mg of
cristae-like membranes in a total volume of 100 µl were placed in
wells in row A at increasing concentrations of detergent. Row B was the
same as row A except the cristae-like membranes were not included.
After 12 h the absorbance in all wells was measured at 405 nm
within 5 s in a multiwell plate reader. Absorbance readings in row
A minus those in row B were used to determine the degree of solubility
of the cristae-like membranes. Finally, a 1-µl aliquot was removed
from each well in row A to assay for ATPase activity ± oligomycin.
Sedimentation Analysis in Sucrose--
The membrane solution to
be sedimented contained the following ingredients in a final volume of
5 ml: 10 mg cristae-like membranes, 0.5% detergent as indicated, 1 mM ATP, 25 mM EDTA, 0.5 mM
dithiothreitol, 5% ethylene glycol, and 50 mM Tricine, pH
7.9. This solution was layered onto a bed of 25 ml 25% sucrose
containing the same components except the detergent concentration was
0.25%. This solution was then subjected to centrifugation for 10 h at 25,000 rpm in a SW 28 rotor at 4 °C in the Beckman LE 80K
ultracentrifuge. Aliquots were then removed from the top and subjected
to ATPase assays, SDS-PAGE, and, where indicated, to Western blot
analysis for PIC and ANC.
Protein Determinations--
For determining membrane protein the
biuret procedure (22) was used. Other protein determinations were made
using either the method of Lowry et al. (23) or the
Coomassie dye binding procedure (Pierce). In all cases the standard was
bovine albumin.
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RESULTS |
Purification of an Oligomycin-sensitive ATPase-enriched Subfraction
of the Mitochondrial Inner Membrane--
The first step employed the
widely used digitonin/Lubrol WX method (18) to obtain a highly purified
mitochondrial inner membrane fraction (Fig.
1A). This fraction exhibits
heterogeneity both in vesicle size (60-400-nm diameter) and in content
of ATP synthase complexes (projecting in part as "lollipop-like"
structures) with some vesicles being saturated and others completely
nude (18). For this reason, in studies reported here, we subjected the
purified inner membrane fraction to an extensive subfractionation approach (see "Methods") in which steps involving lower centrifugal forces were used first to remove larger inner membrane fragments while
retaining in the supernatant the smaller fragments.

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Fig. 1.
A, outline of the procedure used
to prepare ATP synthase-enriched cristae-like membranes. These
membranes were obtained from rat liver mitochondria exactly as
described under "Methods." The ATPase specific activity (see
"Methods") was 14.8 ± 0.32 µmol of ATP hydrolyzed per
min/mg of protein, values 5-6-fold greater than that of the starting
inner membrane fraction. Moreover, this activity was inhibited >90%
by oligomyin. B, characterization of the cristae-like
fraction by SDS-PAGE and N-terminal sequence analysis. SDS-PAGE
(presented) and N-terminal sequence and Western analysis (not
presented) were carried out as described under "Methods." Results
presented in the figure show that 15 subunits types of the ATP synthase
as well as polypeptides corresponding to PIC and ANC are present in the
cristae-like membrane fraction. The only undetected ATP synthase
components are its regulators IF1 (24) and Factor B (25).
C and D, electron microscopy of the cristae-like
membranes. Sample preparation and electron microscopy were carried out
as described under "Methods." TEM of the cristae-like membrane
fraction (C) following negative staining with uranyl acetate
shows vesicles with an average diameter of about 120 nm that are so
densely packed with ATP synthase particles that they give a
para-crystalline-like appearance. SEM (D) gives a view of
the surface of one such negatively stained vesicle showing a more in
depth portrayal of particle distribution (scale bar = 60 nm). Particles are somewhat larger here than in C because
they have been coated with chromium.
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By monitoring ATPase specific activity inhibited by oligomycin, a
potent ATP synthase inhibitor, it became immediately apparent that this
enzyme is greatly enriched in the combined supernatants. When these
were subjected to a final step involving a high centrifugal force, the
resultant membrane fraction (Fig. 1A) was found to have a
very high specific ATPase activity (14.8 ± 0.32 µmol of ATP
hydrolyzed per min/mg of protein), 5-6-fold higher than the mean value
of 2.76 ± 0.61 obtained for the starting inner membrane fraction.
Moreover, this activity is inhibited 90-95% by oligomycin. In
experiments not reported here, the capacity of this inner membrane subfraction to respire was barely detectable with most of the activity
(~30 nanoatoms of oxygen/min/mg of protein) recovered in the larger
membrane fragments that were discarded.
Characterization of the ATPase-enriched Inner Membrane Subfraction
by SDS-PAGE, N-terminal Sequence Analysis, Western Analysis, and
Electron Microscopy--
Further characterization of the
ATPase-enriched inner membrane fraction by SDS-PAGE (Fig.
1B) revealed 17 peptide components, 15 attributable to the
ATP synthase and 1 each to PIC and ANC. All were verified either by
N-terminal sequence or Western analysis and where indicated by both
methods. The only undetectable ATP synthase components were its two
regulatory proteins IF1, an inhibitor of ATP hydrolysis
(24), and Factor B, an activator of ATP synthesis (25). As both are
known to be loosely associated with the ATP synthase complex, they were
most likely depleted during preparation of the membranes.
Following the above studies, the purified cristae-like membrane
fraction containing the ATP synthase, PIC, and ANC was subjected to
both transmission and scanning electron microscopy (TEM and SEM,
respectively). Micrographs obtained by TEM of samples negatively stained with uranyl acetate (Fig. 1C) show vesicles with an
average diameter of about 120 nm that are densely packed with ATP
synthase molecules. These are distinctly evident from the typical
"lollipop" morphological features of those F1
headpieces projecting from the periphery. The micrograph obtained by
SEM (Fig. 1D) of samples fixed with glutaraldehyde and
stained with uranyl acetate, depict a more in depth "top" view of
the F1 headpieces projecting from the membrane surface.
These studies provided evidence that we had isolated a cristae-like
subfraction of the mitochondrial inner membrane and that this
subfraction contains in addition to the ATP synthase also PIC and
ANC.
Identification of Four Detergents That Readily Solubilize the
Cristae-like Membranes Containing the ATP Synthase, PIC, and ANC while
Retaining ATPase Activity Sensitive to Oligomycin--
To reduce the
task of identifying detergents meeting these criteria, we first set up
a multiwell screening assay using a 96-well microtiter plate (Fig.
2A and see "Methods").
Using this approach, we were able to screen 80 available
detergents (Table I), which could be
divided into five different categories, Types I-V (Fig. 2B). Of these, only Type I detergents that solubilize
cristae-like membranes with the least effect on ATPase activity, and
also preserve oligomycin sensitivity, were selected as "very
promising" for future work. The four detergents identified were
Cymal-5, n-decyl-
-D-thiomaltopyranoside, Hega-11, and n-tridecyl-
-D-maltopyranoside,
all of which are nonionic and exhibit similar volumes and surface areas
(Fig. 2C).

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Fig. 2.
A, schematic diagram illustrating
the use of a 96-well plate for screening detergents that solubilize the
cristae-like membranes containing the ATP synthase, PIC, and ANC. The
screening assay is described under "Methods." Each color
represents a different detergent. B, examples of the various
types of solubility and ATPase activity profiles that were obtained.
There were five types as exemplified by
n-tridecyl- -D-maltoside (Type I),
n-dodecyl- -D-maltoside (Type II), Chapso
(Type III), Fos-choline 10 (Type IV), and
cyclohexylpropyl- -D-glucoside (Type V). In this study
only Type I detergents were chosen for further study as they completely
solubilize cristae-like membranes while retaining ATPase activity
(62-95%) that is inhibited >80% by oligomycin. C, space
filling models of the four Type I detergents selected by the above
screening assay for solubilizing cristae-like membranes. The program
hyperChem version 7 was used. The detergents selected are nonionic and
exhibit similar volumes and surface areas.
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Table I
Results of a screen of 80 detergents to identify those that solubilize
cristae-like membranes containing the ATP synthase, PIC, and ANC
See "Methods" and Fig. 3 for screening details. Z = zwitterionic, N = neutral, C = cationic, and CMC = critical micelle concentration in mM. A plus (+) indicates
that oligomycin inhibits the remaining ATPase activity 80%. "Very
promising" means to solubilize 100% with >60% remaining ATPase
activity inhibited >80% by oligomycin, whereas "Promising" means
to solubilize 100% with >30% remaining ATPase activity inhibited
>80% by oligomycin. Other detergents (63 total) available from
Anatrace at the time of this study did not meet the above criteria.
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The ATP Synthase, PIC, and ANC, Localized in Cristae-like
Membranes, Sediment as a Single Species in Each of the Four "Type
I" Detergents That Disperse Them as Individual ATP Synthase-PIC-ANC
Complexes--
The cristae-like membrane fraction was solublized in
each of the four selected detergents and sedimented at 50,000 rpm in the Beckman LE 80K ultracentrifuge for 30 min at 4 °C. This resulted in the absence of a membrane pellet verifying the efficacy of the four
detergents in completely solubilizing the cristae-like membranes. The
clear fractions were then placed on a 25-ml bed of 25% sucrose and
centrifuged at 25,000 rpm for 10 h (see "Methods"). In each
case, the individual fractions formed a single sharp band at a distance
about one-third from the top. These bands were removed and assayed for
ATPase activity with and without oligomycin and also subjected to
SDS-PAGE and Western analysis using specific antibodies to the ATP
synthase
-subunit, PIC, and ANC. Fig.
3A shows that, for three of
the four detergents,
80% of the ATPase activity characteristic of
the solubilized cristae-like membranes put on the gradient is recovered
in the one sedimenting band. For the fourth detergent, the recovery of
70% is still quite good. There is also good retention of the capacity
of oligomycin to inhibit the recovered ATPase activity in each
case.

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Fig. 3.
A and B, results showing that
the single band obtained upon sedimenting cristae-like membranes
solubilized in each of the four selected detergents contains the ATP
synthase, PIC, and ANC. Cristae-like membranes were solubilized in the
detergents indicated (Cymal-5 (D1),
n-decyl- -D-thiomaltopyranoside
(D2), Hega-11 (D3),
tridecyl- -D-maltopyranoside (D4)) and
subjected to sedimentation in sucrose (see "Methods"). They were
then analyzed for ATPase activity and its sensitivity to oligomycin
(A) and analyzed also by Western analysis using antibodies
specific for the ATP synthase -subunit, PIC, and ANC (B).
Results obtained with all detergents used show that the ATP synthase,
PIC, and ANC comigrate, and that the ATP synthase remains active and
oligomycin sensitive. C, purification summary. Results shown
for protein and ATPase activity are the mean ± S.D. of five
different experiments. The ANC/ and PIC/ ratios are based on data
from Western analysis. Average values of four different experiments are
presented. Here, = the -subunit of ATP synthase.
D, TEM of cristae-like membranes containing the ATP
synthase-PIC-ANC complex following their solubilization in each of the
four selected detergents. The same samples noted above in which the ATP
synthase, PIC, and ANC comigrated as a single species were negatively
stained with uranyl acetate and visualized by TEM as described under
"Methods." Here, each of the selected detergents is shown to
disperse a large fraction of the total population into single molecular
species (scale bar = 120 nm). Inset,
individual ATP synthase-PIC-ANC complexes that have been further
magnified. The overall length of the particles (top to
bottom) of 230-240 Å is very close to that reported for
the purified bovine heart ATP synthase (26). However, the width of the
base piece (>100 Å in all cases measured) is significantly greater
than the value of only 84 Å obtained for the bovine enzyme, thus
accounting for the additional presence of PIC and ANC.
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Significantly, Western analysis presented in Fig. 3B shows
that the single sedimenting band also contains in each case both PIC
and ANC that are visualized just as clearly as the
-subunit of the
ATP synthase. Here, it is important to note from the summary table
presented in Fig. 3C that the PIC/
and the ANC/
ratios based on staining intensities remain nearly constant throughout the
purification (four experiments), consistent with the presence of a
native ATP synthase-PIC-ANC complex. In other data not presented, the
SDS-PAGE protein pattern of the single sedimenting band was in each
case nearly identical to that presented earlier in Fig. 1B,
lane 2, thus ruling out that one or more of the detergents causes some polypeptides to "fall off" the complex. Finally, when samples were subjected to negative staining and then electron microscopy (see "Methods"), a well dispersed set of single ATP synthase-PIC-ANC complexes with a tripartite structure (headpiece, basepiece, connecting stalk) was observed in all cases (Fig.
3D).
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DISCUSSION |
One of the greatest challenges in mitochondrial research remains
that of obtaining detailed structural information about the terminal
steps of oxidative phosphorylation, a complex process involving an ATP
synthase to make ATP from Pi and ADP, and two transporters,
PIC and ANC, to respectively allow the entrance of these two substrates
and the exit of ATP. Studies reported here provide evidence that both
the ATP synthase and its required transporters are localized in a
cristae-like subfraction of the mitochondrial inner membrane where they
form an ATP synthase-PIC-ANC complex. Other work involving an
exhaustive screen of 80 different detergents has identified four that
solubilize this complex intact and in dispersed form. Thus, these
studies have satisfied several important requirements essential for
future work that will focus on obtaining detailed structural
information about this "supercomplex" or "ATP synthasome."
As the complex that we have isolated in this study contains in addition
to the ATP synthase, also PIC and ANC, the basepiece (membrane sector)
is expected to be significantly larger than that characteristic of the
ATP synthase alone. This appears to be the case as the basepiece of the
bovine ATP synthase in a recent image reconstruction (26) has a width
of only 84 Å, whereas single ATP synthasomes reported here have
basepieces of greater than 100 Å. However, further analysis will be
necessary taking into consideration detergent and lipid content. As
yet, we do not know how "tight" the ATP synthase-PIC-ANC complex
is and cannot exclude the possible presence of one or more other
essential polypeptides.
Finally, the importance of the work described here deserves comment.
First, as it concerns the mechanism of oxidative phosphorylation in
mitochondria, these studies indicate that the substrates
(Pi and ADP) for ATP synthesis are delivered directly to
the ATP synthase and, following ATP synthesis, the product (ATP) is
delivered directly to ANC for export to the cytoplasm. Second, as it
concerns mitochondrial structure this work provides direct support for
the emerging view (27, 28) that the cristae represent a distinct
subcompartment of the inner membrane that harbors the terminal proteins
of oxidative phosphorylation. Third, these studies provide a method for
identifying an appropriate detergent to solubilize any membrane protein
and in the case of the ATP synthase-PIC-ANC complex set the stage for
structural studies of the complete terminal complex of oxidative phosphorylation in mitochondria.