MITOCHONDRIAL ATP SYNTHASE - jbc.org · MITOCHONDRIAL ATP SYNTHASOME Cristae Enriched Membranes and A Multiwell Detergent Screening Assay Yield Dispersed Single Complexes Containing

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MITOCHONDRIAL ATP SYNTHASOME Cristae Enriched Membranes and A Multiwell Detergent Screening Assay Yield Dispersed

Single Complexes Containing The ATP Synthase and Carriers for Pi and ADP/ATP1

Young H. Ko2,4, Michael Delannoy3, Joanne Hullihen2, Wah Chiu5, and Peter L. Pedersen2,6

2Department of Biological Chemistry, 3Department of Cell Biology and Anatomy, and 4Russel

H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

and

5Program in Structural and Computational Biology and Molecular Biophysics, and National

Center for Macromolecular Imaging, Vern and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston Texas 77030

Running Title: Mitochondrial ATP Synthasome Key Words: ATP Synthase, Mitochondria, Cristae, Detergents, Electron Microscopy Footnotes: 1Supported by NIH Grant CA 10951 to PLP, and NIH grant P41RR02250 to WC for the National Center for Macromolecular Imaging 6To whom correspondence should be addressed: Phone: 410-955-3827: FAX: 410-614-1944; E. Mail: [email protected]

Abbreviations: F0F1, ATP synthase; PIC, phosphate carrier; ANC, adenine nucleotide carrier;

ATP Synthasome, ATP Synthase/PIC/ANC complex; TEM, transmission electron microscopy;

SEM, scanning electron microscopy; CMC, critical micelle concentration; CHAPS, (3-[3-

Cholamidopropyl) dimethylammononio]-1-propane sulfonate; CYMAL-5, cyclohexyl-pentyl-

β-D-maltoside; Hega 11, undecanoyl-N-hydroxyethylgluamide; PBS, phosphate buffered

saline; RCSB, Research Collaboratory for Structural Biology

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 30, 2003 as Manuscript C200703200 by guest on July 14, 2020

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SUMMARY

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP

synthase (F0F1) that works in close synchrony with the Pi and ADP/ATP carriers. Each carrier

consists of only a single polypeptide chain in dimeric form while the ATP synthase is highly

complex consisting in animals of 17 known subunit types and more than 30 total subunits.

Although structures at high resolution have been obtained for the ATP synthase’s water soluble

F1 part consisting of only 5 subunit types, such structures have not been obtained for either the

complete ATP synthase or the Pi and ADP/ATP carriers. Here, we report that all three proteins

are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the

mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4

nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single

soluble complexes or “ATP synthasomes” that contain the ATP synthase in association with the

Pi and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of

oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed

to visualize in atomic detail the entire complex involved. They also provide evidence that the

cristae are a subcompartment of the inner membrane.

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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).

The latter two events are closely synchronized as each ATP molecule that is made on the ATP

synthase’s F1 unit 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 70S 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

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obtaining 2 or 3 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 RCSB database, 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.

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 from

Labsystems and Flow Laboratories. Oligomycin was from Sigma, PVDF membranes from

Millipore, Western blot reagents from Amersham Pharmacia Biotech, and Coomassie dye from

Pierce. An antibody to ANC was from Santa Cruz Biotechnology whereas antibodies to the rat

ATP synthase β-subunit (Walker A region), δ subunit, and PIC (residues 302-312) were from our

own stocks. Antibodies to other ATP synthase subunits were from Drs.Y.Hatefi and Akemi

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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 oligomycin/mg protein) and

respiration (oxygen consumption) were monitored as previously described (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 previously described (20).

Cristae-like Inner Membrane Vesicles. Each preparation commenced by preparing from

4 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 Buffer A (300 mM KPi, 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 οC. The IMF was then slowly thawed, made up to 65 ml with Buffer B (300 mM KPi, 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 70.1 Ti rotor at 50,000 rpm in a Beckman LE

80K ultracentrifuge. The combined pellets in the multiple tubes were suspended in 65 ml Buffer

C (300 mM KPi, 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 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

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pellets were suspended in 16 ml of the same buffer and centrifuged. Then pellets from the 2

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 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 Buffer C and centrifuged in the SS-24

rotor as before. The supernatants were saved, suspended in 16 ml 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 Buffer A and centrifuged for 15 min at 20,000 rpm. The resultant supernatants were

centrifuged then for 1 hr at 50,000 rpm. Finally, the supernatants were discarded and the pellets

suspended in 32 ml Buffer A and centrifuged again for 1 hr 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 οC 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

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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 οC 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 + 0.1M 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 4 different detergents at 12

different concentrations ranging from 0 to 2X CMC (in mM) were tested at 4 οC. For example,

detergent “X” and ∼ 1 mg 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, one 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,

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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 οC 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.

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. 1 A). 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 (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.

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 µmoles ATP hydrolyzed/min/mg protein), 5-6 fold higher than the mean value of

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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 natoms

oxygen/min/mg 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.

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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, Methods).

Using this approach, we were able to screen 80 available detergents (Table 1), which could be

divided into 5 different categories, Type 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 4 detergents

identified were CYMAL-5, n-Decyl-β-D-thiomaltopyranoside, HEGA-11, and n-Tridecyl-β-D-

maltopyranoside, all of which are nonionic and exhibit similar volumes and surface areas (Fig.

2C).

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 4 selected detergents and sedimented at 50, 000 rpm in the Beckman LE

80K ultracentrifuge for 30 min at 4 οC. This resulted in the absence of a membrane pellet

verifying the efficacy of the 4 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 (Methods). In each case, the individual fractions formed a single sharp band at a

distance about 1/3 from the top. These bands were removed and assayed for ATPase activity

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with and without oligomycin, and also subjected to SDS-PAGE and Western analysis using

specific antibodies to the ATP synthase β subunit, PIC, and ANC. Fig. 3A shows that, for 3 of

the 4 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 4th detergent,

the recovery of 70% is still quite good. There is also good retention of oligomycin’s capacity to

inhibit the recovered ATPase activity in each case.

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 β

subunit of the ATP synthase. Here, it is important to note from the summary table presented in

Fig. 3C that the PIC/β and the ANC/β ratios based on staining intensities remain nearly constant

throughout the purification (4 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 (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).

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

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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 4 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. Secondly, 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.

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TABLE I

Results of a Screen of Eighty 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.

Detergent (Type) Solubility, % at

( x CMC)

Retention of ATPase

Activity, %

Retention of Inhibitor

Sensitivity

Candidate for Structural

Studies CHAPS (Z) 100 (1.6) 43 + Promising

Cymal-5 (N) 100 (1.6) 62 + Very Promising C-Hega-9 (N) 100 (1.1) 45 + Promising

n-Nonyl-β-D-glucopyranoside (N) 100 (1.5) 31 + Promising Hega-9 (N) 100 (1.1) 33 + Promising

Hega-10 (N) 100 (1.8) 33 + Promising Hega-11 (N) 100 (1.9) 95 + Very Promising

n-Decyl-N,N-dimethylglycine (Z) 100 (1.2) 38 + Promising n-Dodecyl-N,N-dimethylglycine (Z) 100 (1.3) 51 + Promising n-Undecyl-β-D-maltopyranoside (N) 100 (1.9) 53 + Promising n-Dodecyl-β-D-maltopyranoside (N) 100 (1.5) 49 + Promising

n-Tridecyl-ββββ-D-maltopyranoside (N) 100 (18) 80 + Very Promising Mega-9 (N) 100 (1.1) 32 + Promising

n-Octyl-β-D-thiomaltopyranoside (N) 100 (1.7) 33 + Promising n-Nonyl-β-D-thiomaltopyranoside (N) 100 (1.4) 50 + Promising

n-Decyl-ββββ-D-thiomaltopyranoside (N) 100 (1.6) 63 + Very Promising Triton X-100 (N) 100 (34) 47 + Promising

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FIGURE LEGENDS

FIGURE 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 (Methods). The ATPase specific activity (Methods) was 14.8 ± 0.32 µmoles ATP

hydrolyzed/min/mg 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 (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. & D. Electron Microscopy of the Cristae-like Membranes. Sample

preparation and electron microscopy were carried out as described (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.

FIGURE 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

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17

Were Obtained. There were 5 types as exemplified by n-Tridecyl-β-D-maltoside (Type I), n-

Dodecyl-β-D-maltoside (Type II), Chapso (Type III), Fos-Choline 10 (Type IV), and

Cyclohexyl-propyl-β-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.

Figure 3. A. & 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 (D1, Cymal-5; D2, n-Decyl-β-D-thiomaltopyranoside; D3, Hega 11; D4, Tridecyl-β-D-

maltopyranoside) and subjected to sedimentation in sucrose (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 β 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 5 different experiments. The ANC/β and

PIC/β ratios are based on data from Western analysis. Average values of 4 different experiments

are presented. Here, β = the β 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

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18

visualized by TEM as described in 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 basepiece

(>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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A. B.

C. D.

Figure 1

KDa50

25

20

37

Livers

Mitochondria

Digitonin

Mitoplasts

Lubrol

Cristae-Like Membranes(ATP Synthase Enriched)

Inner Membrane

SubfractionationExtensive

( S.A. = ~15 µmoles ATP hydrolyzed/min/mg; 90-95 % inhibition by oligomycin )

MWSTDs

αβ

a, boscp

δF6, fe

A6L

εc

Picγ, ANC

d, (g)2

Cristae-LikeMembranes

IdentificationMethods

Western & N-SeqWestern & N-Seq

Western Western & N-Seq

Western (a)Western & N-Seq (b)Western & N-SeqWestern (d, g)

Western & N-Seq Western & N-Seq (F6, e)Western (f )

Western Western & N-SeqWestern & N-Seq

7.5(Usually stains weakly)

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1 2 3 4 5 6 7 8 9 10 11 12A

A'

C

C'

E

E'

G

G'

Increase in Solubility

Detergent "A" + IM

Detergent "A", No IM

Detergent "C" + IM

Detergent "C", No IM

Detergent "E" + IM

Detergent "E", No IM

Detergent "G" + IM

Detergent "G", No IM

[Detergent Conc.] = (CMC ) X 0 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Figure 2

Surface Area= 489.29 (Å)2

Volume=1261.85 (Å)3

Surface Area= 586.11 (Å)2

Volume=1311.05 (Å)3

Surface Area= 651.20 (Å)2

Volume=1151.69 (Å)3

Surface Area= 697.86 (Å)2

Volume=1485.30 (Å)3

A.

B.

C.

020406080

100120

0 1.0 1.2 1.4 1.6 1.8 2.00

20406080

100120

0 1.0 1.2 1.4 1.6 1.8 2.0 0 1.0 1.2 1.4 1.6 1.8 2.00

20406080

100120

020406080

100120

0 1.0 1.2 1.4 1.6 1.8 2.00

20406080

100120

0 1.0 1.2 1.4 1.6 1.8 2.0

TYPE I TYPE II TYPE III

TYPE IV TYPE V

x [CMC]x [CMC] x [CMC]

x [CMC] x [CMC]

ATPa

se A

ctiv

ity,

%

o

r

So

lub

ility

, %

ATPa

se A

ctiv

ity,

%

o

r

So

lub

ility

, %

ATPase Activity, %

Solubility, %

Cymal 5 n-Decyl-β-thiomaltoside Hega11 n-Tridecyl-β-thiomaltoside

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Figure 3

Total Activity before Detergent Treatment = 150 µmoles ATP hydrolyzed/min

Total ActivityRecovered in the Single Band, %

Inhibition by Oligomycin, %

D1 D2 D3 D4

80 85 88 70

93 95 93 79

A.

C.

B. β - Ab PIC - Ab ANC - Ab

D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4

D.

Crude Memb.

ATP Synthasome

D3-SolubilizedATP Synthasome

from a Sucrose Gradient Peak

Protein,Sp. Activity,

Total Activity, Yield,*Units/mg(*Units=µmoles/min) Units

138 ± 3 381

351

260

100

92

68

ANC/β,Arbitrary

Units

0.38

0.44

0.41

PIC/β,Arbitrary

Units

1.1

1.3

1.0

23.7 ± 3.5

20.1 ± 3.5

2.76 ± 0.61

14.8 ± 0.32

13.0 ± 0.30

%mg

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Young H. Ko, Michael Delannoy, Joanne Hullihen, Wah Chiu and Peter L. Pedersensynthase and carriers for Pi and ADP/ATP1

detergent screening assay yield dispersed single complexes containing the ATP Mitochondrial ATP synthasome: Cristae enriched membranes and a multiwell

published online January 30, 2003J. Biol. Chem. 

  10.1074/jbc.C200703200Access the most updated version of this article at doi:

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