Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Summary

This protocol describes the purification of F₁-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Abstract

F₁-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F₁-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F₁-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F₁-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F₁-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F₁-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F₁-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F₁-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F₁-ATPase isolation from other organisms.

Introduction

The F-type ATP synthases are membrane-bound rotating multiprotein complexes that couple proton translocation across energy-transducing membranes of bacteria, mitochondria, and chloroplasts with the formation of ATP. Molecular details of the rotational mechanism of ATP synthesis are known mainly because of structural studies of purified bacterial and mitochondrial ATP synthases and their subcomplexes¹. F-type ATP synthase is organized into membrane-intrinsic and membrane-extrinsic moieties. The membrane-extrinsic part, known as F₁-ATPase, contains three catalytic sites, where the phosphorylation of adenosine diphosphate (ADP) to ATP or the reverse reaction occurs. F₁-ATPase can be released experimentally from the membrane-intrinsic moiety while retaining its ability to hydrolyze, but not synthesize, ATP. The membrane-bound sector, called F_o, mediates protein translocation, which drives the rotation of the central part of the enzyme. The F₁ and F_o sectors are connected by the central and peripheral stalks.

The first attempts to purify the F₁-ATPase from budding yeast and bovine heart mitochondria date back to the 1960s. These protocols used extracted mitochondria, which were disrupted by sonication, fractionated by ammonium or protamine sulfate precipitation, followed by optional chromatography step(s) and heat treatment^2,3,4,5,6. The purification was greatly improved and simplified by the use of chloroform, which readily releases the F₁-ATPase from the mitochondrial membrane fragments⁷. The chloroform extraction was then used to extract F₁-ATPases from various animal, plant, and bacterial sources (e.g., rat liver⁸, corn⁹, Arum maculatum¹⁰, and Escherichia coli¹¹). Further purification of the chloroform-released F₁-ATPase by affinity or size-exclusion chromatography (SEC) yielded a highly pure protein complex, which was suitable for high-resolution structure determination by X-ray crystallography, as documented by the structures of F₁-ATPase from bovine heart^12,13 and Saccharomyces cerevisiae¹⁴. F₁-ATPase structures were also determined from organisms that are difficult to cultivate and, thus, the amount of the initial biological material was limited. In this case, the F₁-ATPase subunits were artificially expressed and assembled into the complex in E. coli, and the whole heterologous enzyme was purified by affinity chromatography via a tagged subunit. Such approach led to the determination of F₁-ATPase structures from two thermophilic bacterial species, Geobacillus stearothermophilus¹⁵ and Caldalkalibacillus thermarum^16,17. However, this methodology is rather unsuitable for eukaryotic F₁-ATPases since it relies on the prokaryotic protheosynthetic apparatus, posttranslational processing, and complex assembly.

The chloroform-based extraction was previously used to isolate F₁-ATPases from unicellular digenetic parasites Trypanosoma cruzi¹⁸ and T. brucei¹⁹, important mammalian pathogens causing American and African trypanosomiases, respectively, and from monogenic insect parasite Crithidia fasciculata²⁰. These purifications led only to a simple description of the F₁-ATPases, since no downstream applications were used to fully characterize the composition, structure, and enzymatic properties of the complex. This article describes an optimized method for F₁-ATPase purification from the cultured insect life cycle stage of T. brucei. The method is developed based on the established protocols for isolation of bovine and yeast F₁-ATPases^21,22. The procedure yields highly pure and homogeneous enzyme suitable for in vitro enzymatic and inhibitory assays, detailed proteomic characterization by mass spectrometry²³, and structure determination²⁴. The purification protocol and the knowledge of the F₁-ATPase structure at the atomic level opens a possibility to design screens to identify small-molecule inhibitors, and aid in the development of new drugs against African trypanosomiases. Moreover, the protocol can be adapted to purify F₁-ATPase from other organisms.

Protocol

1. Buffers and Solutions

1. Prepare the solutions listed below. Degas all buffers for liquid chromatography. Add ADP, benzamidine, and protease inhibitors just before use.
   1. Prepare buffer A: 50 mM Tris buffer with hydrochloric acid (Tris-HCl) pH 8.0, 0.25 M sucrose, 5 mM benzamidine, 5 mM aminocaproic acid (ACA), and protease inhibitors (10 µM amastatin, 50 µM bestatin, 50 µM pepstatin, 50 µM leupeptin, and 50 µM diprotin A).
   2. Prepare buffer B: 50 mM Tris-HCl pH 8.0, 0.25 M sucrose, 4 mM ethylenediaminetetraacetic acid (EDTA), 5 mM benzamidine, 5 mM ACA, 1 mM ADP, and protease inhibitors (10 µM amastatin, 50 µM bestatin, 50 µM pepstatin, 50 µM leupeptin, and 50 µM diprotin A).
   3. Prepare Q-column buffer: 20 mM Tris-HCl pH 8.0, 4 mM EDTA, 10 mM MgSO₄, 5 mM benzamidine, 5 mM ACA, and 1 mM ADP.
   4. Prepare Q-column elution buffer: Q-column buffer with 1 M NaCl.
   5. Prepare SEC buffer: 20 mM Tris-HCl pH 8.0, 10 mM MgSO₄, 100 mM NaCl, 1 mM ADP.
   6. Prepare chloroform saturated with 2 M Tris-HCl pH 8.5. Mix chloroform with 2 M Tris-HCl pH 8.5 in approximately 1:1 ratio in a screw-cap bottle, shake, let the organic and aqueous phases separate, and measure pH in the upper aqueous layer with a strip of pH-indicator paper. Store at room temperature. Just before use, shake again and let the phases separate. Use the lower chloroform layer.
      CAUTION: Chloroform is volatile and irritating to eyes and skin. Work in a fume hood. Use safety spectacles when shaking.

2. Preparation of sub-mitochondrial Particles

1. Resuspend mitochondrial vesicles (mitoplasts) isolated by hypotonic lysis²⁵ from 1 x 10¹¹ to 2 x 10¹¹ cells of procyclic T. brucei in 5 mL of ice-cold buffer A. Keep the sample chilled until step 3.2.
2. Determine the protein concentration in the suspension by the bicinchoninic acid (BCA) protein assay²⁶ according to the manufacturer's instructions.
   1. Use a bovine serum albumin (BSA) dilution series in ultrapure water to construct the standard curve. Dilute a small amount of sample 20 - 100 times with ultrapure water to fit into the range of BSA standards.
   2. Calculate the total protein amount in the sample and bring the protein concentration to 16 mg/mL by diluting it with additional buffer A.
3. Fragment mitoplasts into inverted vesicles and membrane pieces by sonication of the suspension 7x for 15 s with a total energy of 70 to 100 J per impulse with a microtip with a diameter of 3.9 mm. If the ultrasonic homogenizer does not display the energy output, start the optimization at 50% of the maximal power. Incubate the sample on ice for 30 s between impulses. After the sonication, the suspension becomes slightly darker.
4. Sediment the membrane fragments by ultracentrifugation at 54,000 x g for 16 h or at 98,000 x g for 5 h at 4 °C. Decant the supernatant and proceed with the chloroform extraction, or flash-freeze the sediment in liquid nitrogen and store it at -80 °C.

3. Release of F₁-ATPase from Membrane by Chloroform

1. Resuspend the pellet of mitochondrial membranes in buffer B with the aid of a small Dounce homogenizer. Calculate the volume of buffer B based on the total amount of buffer A used in steps 2.1 and 2.2 using the following formula: volume (buffer B) = volume (buffer A) x 12/21. Transfer the suspension to a 15- or 50-mL conical tube.
2. Remove the sample from ice and, from now on, keep the sample and all solutions to be used at room temperature.
3. Add chloroform saturated with 2 M Tris-HCl pH 8.5; the volume of chloroform to be added equals half the volume of the suspension. Close the cap tightly. Shake it vigorously for exactly 20 s. Centrifuge it immediately at 8,400 x g for 5 min at room temperature.
4. Transfer the upper cloudy aqueous phase to 1.6-mL microtubes. Add protease inhibitors (10 µM amastatin, 50 µM bestatin, 50 µM pepstatin, 50 µM leupeptin, and 50 µM diprotin A) to replace the inhibitors removed by the chloroform treatment. Centrifuge the samples at 13,000 x g for 30 min at room temperature. Transfer the supernatant to fresh microtubes and repeat the centrifugation to remove any insoluble material.

4. Anion-exchange Chromatography

1. Equilibrate the 5-mL anion exchange (Q) column attached to a fast-protein liquid chromatography system with the Q-column buffer at a flow rate of 5 mL/min until the absorbance at 280 nm and the conductivity stabilize (approximately 50 mL of buffer).
2. Load the supernatant from step 3.3 on the equilibrated column at a flow rate of 1 mL/min. Wait until the absorbance at 280 nm stabilizes at the background. Apply a 25-mL linear gradient of the Q-column elution buffer from 0% to 100% at a flow rate of 0.5 mL/min. Collect 1-mL fractions.
3. Assay the individual fractions corresponding to the major elution peak for ATP hydrolytic activity by the Pullman ATPase assay² at pH 8.0. Use 10 µL of each fraction per 1 mL of reaction mixture.Pool the fractions that exhibit ATPase activity. Optionally, separate 10 µL of each fraction on sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS-PAGE) and stain the gel by Coomassie Blue to visualize individual F₁-ATPase subunits and contaminating proteins.
4. Concentrate the pooled sample by membrane ultrafiltration using a spin column with a 100,000 MWCO PES filter to 200 - 500 µL. Proceed to SEC or store the sample overnight at room temperature.

5. Size-exclusion Chromatography

1. Equilibrate the SEC column attached to a liquid chromatography system with at least 48 mL (two column volumes) of the SEC buffer at a flow rate of 0.5 mL/min.
2. Apply the sample on the column and run chromatography at a flow rate of 0.25 mL/min. Collect 0.25-mL fractions.
3. Run 10 µL of the fractions that correspond to the peaks of the UV_280nm absorbance trace on SDS-PAGE and stain them by Coomassie Blue. The first major peak contains the F₁-ATPase. Assay the fractions corresponding to this peak for the ATP hydrolytic activity and azide sensitivity by the Pullman ATPase assay. Determine the protein concentration by the BCA assay.
4. Keep the purified F₁-ATPase at room temperature and use it within 3 d after purification for downstream applications. Alternatively, concentrate the sample using a spin column with a 100,000 MWCO PES filter to > 1.5 mg/mL, precipitate it by mixing it with saturated ammonium sulfate adjusted to pH 8.0 (1.2x the volume), and store it at 4 °C.

Results

A typical purification (Figure 1) starts with mitochondrial vesicles (mitoplasts) isolated on the Percoll gradient from hypotonically lysed 1 x 10¹¹ to 2 x 10¹¹ procyclic T. brucei cells²⁵ cultured in standard glucose-rich SDM-79 medium²⁷. The mitoplasts are fragmented by sonication, spun, and the matrix-containing supernatant is discarded. Mitochondrial membranes are treated with ...

Discussion

The protocol for F₁-ATPase purification from T. brucei was developed based on previously published methods for the isolation of F₁-ATPase complexes from other species^13,14. The method does not require any genetic modification (e.g., tagging) and yields a fully active complex with all subunits present. The crucial step is the chloroform-facilitated release of the F₁-ATPase from the membrane-attached part of the en...

Materials

Name	Company	Catalog Number	Comments
Chemicals
Adenosin Diphosphate Disodium Salt (ADP)	Applichem	A0948
Amastatin Hydrochloride	Glantham Life Sciences	GA1330
Aminocaproic Acid	Applichem	A2266
BCA Protein Assay Kit	ThermoFischer Scientific/Pierce	23225
Benzamidine Hydrochloride	Calbiochem	199001
Bestatin Hydrochloride	Sigma Aldrich/Merck	B8385
Chloroform	Any supplier
cOmplete Tablets, Mini EDTA-free	Roche	4693159001	Protease inhibitor cocktail tablets
Ethylenediaminetetraacetic Acid (EDTA)	Any supplier
Hydrochloric Acid	Any supplier		For pH adjustment
Ile-Pro-Ile	Sigma Aldrich/Merck	I9759	Alias Diprotin A
Leupeptin	Sigma Aldrich/Merck	L2884
Magnesium Sulfate Heptahydrate	Any supplier
Pepstatin A	Sigma Aldrich/Merck	P5318
Protein Electrophoresis System	Any supplier
Sodium Chloride	Any supplier
Sucrose	Any supplier
Tris	Any supplier
Name	Company	Catalog Number	Comments
Consumables
Centrifuge Tubes for SW60Ti, Polyallomer	Beckman Coulture	328874
DounceTissues Homogenizer 2 mL	Any supplier
Glass Vacuum Filtration Device	Sartorius	516-7017	Degasing solutions for liquid chromatography
HiTrap Q HP, 5 mL	GE Healthcare Life Sciences	17115401	Anion exchange chromatography column
Regenaretad Cellulose Membrane Filters, pore size 0.45 μm, diameter 47 mm	Sartorius	18406--47------N	Degasing solutions for liquid chromatography
Superdex 200 Increase 10/300 GL	GE Healthcare Life Sciences	29091596	Size-exclusion chromatography column
Vivaspin 6 MWCO 100 kDa PES	Sartorius	VS0641
Name	Company	Catalog Number	Comments
Equipment
AKTA Pure 25	GE Healthcare Life Sciences	29018224	Or similar FPLC system
Spectrophotometer Shimadzu UV-1601	Shimadzu		Or similar spectrophotometer with kinetic assay mode
Ultracentrifuge Beckman Optima with SW60Ti Rotor	Beckman Coulture		Or similar ultracentrifuge and rotor
Ultrasonic Homogenizer with Thin Probe, Model 3000	BioLogics	0-127-0001	Or similar ultrasonic homogenizer