THE JOURNAL OF BIOLOGICAL CHEMISTRY GI 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol ,267, No. 2, Issue of January pp. 1375-1381,1992 Printed in U.S.A

Functional Reconstitution of Membrane Proteins in Monolayer Liposomes from Bipolar Lipids of Sulfolobus acidocaZdarius*

(Received for publication, July 26, 1991)

Marieke G . L. Elferink, Janny G. de Wit, Rudy DemelS, Arnold J. M. Driessen, and Wil N. Konings From the Department of Microbiology, University of Groningen, Kerklnnn 30, 9751 NN Haren and the $Center for Biomembranes and Lipid Enzymology and Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, Transitorium 3, Padualaan 8, 3854 CH Utrecht, The Netherlands

Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of un- usual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydro- phobic (& phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocal- darius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally re- constituted in these liposomes. In the presence of re- duced cytochrome c, a protonmotive force (Ap) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with mem- brane vesicles of Lactococcus lactis, the Ap generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous mem- brane proteins originating from a regular lipid bilayer.

Archaebacterial lipids differ considerably from conven- tional lipids in their structure and physicochemical properties. They are based on ether linkages instead of ester linkages and contain biphytanyl chains instead of fatty acyl chains. Mem- branes of Sulfolobus acidocaldarius mainly contain two classes of tetraethers, the glycerol dialkyl glycerol tetraethers (GDGTs)’ and the glycerol dialkyl nonitol tetraethers (GDNTs). Both lipid species contain two nonequivalent polar heads linked by two hydrophobic C40 phytanyl chains, with up to four cyclopentane rings per chain. GDGT contains two glycerol moieties, while in GDNT one of the glycerol moieties is substituted by nonitol, a poly01 with 9 carbon atoms. Most complex lipids occur as phosphoglycolipids in which a sugar residue and a phosphate group are linked to opposite sides of

*This research was supported by the Foundation for Technical Sciences (Stichting Technische Wetenschappen) with financial aid from the Netherlands Organization for Scientific Research (Neder- lands Organisatie voor Wetenschappelijk Onderzoek). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: GDGT, glycerol dialkyl glycerol tetraether lipid; GDNT, glycerol dialkyl nonitol tetraethers; COV, cytochrome c-oxidase vesicle; diSCa(5), 3,3-dipropylthiadicarbocy- anine iodide; Ap, protonmotive force; ApH, transmembrane pH gra- dient; A$, transmembrane electrical potential; TPP’, tetraphenyl- phosphonium ion; TMPD, N,N,N’,N”tetramethyl-p-phenylenedi- amine; TLC, thin-layer chromatography.

the tetraether molecules. It is generally accepted that each tetraether lipid molecule spans the entire archaebacterial membrane (1). Evidence has been presented that the sugar residues are directed to the outside of the cytoplasmic mem- brane (2). A review of the structure and composition of archaebacterial lipids has recently appeared (3).

S. acidocaldarius can grow at temperatures of up to 85 “C and a pH of 2-3. The archaebacterial membrane adapted to such extreme environmental conditions is assumed to be extremely stable. The rigidity of the membrane has been attributed to the monolayer organization of the membrane, the methyl side groups at the branch points in the biphytanyl chains, and the presence of a variable amount of cyclopentane rings per chain. In the tetraether lipids of Sulfolobus solfatar- icus and Thermoplasma acidophilum the degree of cyclization of the biphytanyl components increases with increasing growth temperature (4). The structural properties and stabil- ity of membranes composed of these lipids makes them very attractive for the construction of stable proteoliposomes.

In this paper we address the question whether sealed lipo- somes can be made of the tetraether lipids capable of sup- porting the activity of integral membrane proteins. Properties of the GDNT class of lipids of the extreme thermophilic archaebacterium Caldariella acidophila have been studied in black lipid membranes (5-8). In contrast, the more symmet- rical GDGT does not form stable black membranes, possibly as a result of the low polarity of the unsubstituted glycerol moieties (8). The construction of small unilamellar liposomes is possible only when lipids of C. acidophila are mixed with at least 25 mol% of egg phosphatidylcholine (9). Our work con- cerns the construction of closed liposomes composed of tetraether lipids of S. acidocaldarius. An extensive purification protocol to obtain a polar liposomal-forming tetraether lipid fraction was reported by Lo and Chang (10). Crude lipids were purified by subsequent reversed-phase chromatography, preparative TLC, and methanol precipitation. We adapted this protocol and obtained after reversed-phase chromato- graphy two different lipid fractions capable of forming closed vesicular structures in aqueous media without the need for further purification. Evidence is presented that the tetraether lipids span the entire membrane when arranged as a liposomal structure. Liposomes composed of these archaebacterial lipids retain their energy-conserving properties and allow the in vitro reconstitution of integral membrane proteins.

EXPERIMENTAL PROCEDURES

Materials

S. acidocaldarius (DSM 639) cells were kindly supplied by Dr. G. Schiifer (Medical University of Lubeck). Escherichia coli L-a-phos- phatidylethanolamine (type IX) was obtained from Sigma and further purified by acetone-ether extraction as described (11). Lipids were

1375

1376 Reconstitution of Proteins in Membrane-spanning Lipids stored in chloroform under nitrogen at -20 "C. ~-[U-'~C]Leucine (312 mCi/mmol) was obtained from Du Pont-New England Nuclear (Dreieich, Federal Republic of Germany). TLC sheets (DC-Alufolien Kieselgel60) was from E. Merck (Darmstadt).

Methods Purification of Sulfolobus acidocaldarius Lipids-Freeze-dried S.

acidocaldarius cells (1.5 g) were soxhlet-extracted with 400 ml of chloroform/methanol (l:l, v/v) during 12 h. The crude lipid extract was dried with a rotary evaporator, resuspended in 20 ml of methanol/ water (l:l, v/v), and sonicated in a bath sonicator for about 1 h to facilitate dispersion. Homogenization was completed with a probe sonicator (MSE Scientific Instruments, West Sussex) under a NP atmosphere. Portions of 2 ml of this suspension was transferred to a Prep Sep Cl, column (Waters, Millipore, Milford, MA) and lipids were eluted stepwise as described by Lo et al. (10): fraction 1, 20 ml of methanol/water (l:l, v/v); fraction 2, 20 ml of chloroform/metha- nol/water (2:5:2, v/v/v); fraction 3, 20 ml of chloroform/methanol/ water (65:25:4, v/v). The three subsequent lipid fractions were dried with a rotary evaporator, resuspended in chloroform/methanol/water (65:25:4), and stored at 4 "C until use.

Preparation of Liposomes-E. coli lipids, S. acidocaldarius lipids, or a mixture of both was dried by rotary evaporation and suspended in 50 mM potassium phosphate, pH 7, at a concentration of 15-35 mg/ml. Liposomes were obtained by sonication using a probe-type sonicator (intervals of 15-s sonication and 45-s rest) a t 0 "C under a constant stream of nitrogen. Sonication was continued until a clear suspension was obtained. When S. acidocaldarius lipids were present the sample was not cooled during sonication. Liposomes were stored in liquid nitrogen. Before use a small sample was slowly thawed at room temperature and sonicated till clarity with a probe sonicator using intervals of 5 s.

Incorporation of Cytochrome c-oxidase in Liposomes-Beef heart cytochrome c-oxidase (2.25 nmol) suspended in 1.5% (w/v) sodi- umcholate was added to 1 ml of liposomes containing 15 mg of S. acidocaldarius lipid or 35 mg of E. coli lipid. The suspension was mixed extensively, and dialyzed twice for 3 h against 1 liter of 50 mM potassium phosphate, pH 7, at room temperature. Liposomes (COVs) were stored in liquid Nz before use, slowly thawed at room tempera- ture, and sonicated as described above.

Fusion of Cytochrome c-oxidase Proteoliposomes with Membrane Vesicles of Lactococcus lactis-Membrane vesicles of L. lactis ML, prepared as described by Otto et al. (12) were fused with COVs by a freeze-thaw/sonication technique (13, 14). COVs composed of E. coli or S. acidocaldarius lipid were mixed with L. &tis membrane vesicles in a ratio of 10 mg of lipid to 1 mg of vesicle protein. The suspension was rapidly frozen in liquid nitrogen, and then slowly thawed at room temperature. Hybrid membranes were sonicated with a probe-type sonicator until clarity using time intervals of 5 s.

Determination of the Protonmotive Force in Cytochrome c-oxidase Proteoliposomes and Hybrid Membranes-For the measurement of the transmembrane pH gradient (ApH, inside alkaline), COVs were loaded during the sonication step with 100 p~ of the fluorescence pH indicator pyranine (Mol. Probes, Eugene, OR). External pyranine was removed by chromatography using a Sephadex G-25 column (15 X 1 cm) (14). COVs in 50 mM potassium phosphate, pH 7, were energized by the addition of the electron donor system ascorbate (10 mM), horse heart cytochrome c (10 p ~ ) and N,N,N',N'-tetramethyl- p-phenylenediamine (TMPD) (100 p ~ ) . The internal pH of the liposomes was determined from the fluorescence of pyranine using excitation and emission wavelengths of 450 and 508 nm, respectively. Fluorescence measurements were performed at 25 "C using a Perkin- Elmer LS-50 spectrophotofluorometer. Pyranine fluorescence was calibrated by adjusting the external pH with NaOH and HCl in the presence of nigericin and valinomycin. ApH was calculated from the difference between the external and internal pH. A conversion factor Z of 59 at 25 "C was used to express ApH (-ZApH) in millivolts.

The transmembrane electrical potential (A+, inside negative) was estimated from the distribution of the lipophilic cation tetraphenyl- phosphonium (TPP+) measured with a TPP+ electrode. Hybrid mem- branes were diluted 40-fold in 50 mM potassium phosphate, pH 7, and energized by the addition of the electron donor system as de- scribed above. Measurements were performed at 25 "C using a TPP+ concentration of 4 p ~ . Nigericin was added to a final concentration of 100 nM. The magnitude of A+ was calculated with the Nernst equation. A correction for concentration dependent TPP+ binding was applied (15, 16) assuming symmetric binding of TPP' to both

membrane surfaces. A fluorescent technique was used as a qualitative assay for the generation of a A+, inside negative. The fluorescence of the dye 3,3-dipropylthiadicarbocyanine iodide [diSCa(5)] (Molecular Probes Inc., Eugene, OR) was monitored at an excitation and emis- sion wavelength of 643 and 666 nm, respectively.

Protonmotive Force-driven Transport of Leucine in the Hybrid Membranes-Leucine uptake driven by a Ap generated by cytochrome c oxidase activity was assayed as follows: hybrid membranes were diluted 10-fold in 50 mM potassium phosphate, pH 7, and 5 mM MgCI,. Energization was initiated by the addition of 10 mM potassium ascorbate, 100 p~ TMPD, and 10 p~ cytochrome c. At the start of the experiment, 3 p~ leucine (312 mCi/mmol) was added. The final volume of the suspension was 100 pl. At different time intervals, transport was terminated by dilution of the sample into 2 ml of ice- cold 0.1 M LiCl, and immediate filtration over cellulose nitrate mem- brane filters (450 nm, BA 85, Schleicher and Schuell). Filters were washed once with 2 ml of 0.1 M LiCl. Filters were transferred to scintillation vials containing 4 ml of Packard liquid scintillation TM299 and counted.

Electron Microscopy-Freeze-etch electron micrographs were pre- pared from liposomes frozen in liquid nitrogen ("N, slush") in 50 mM potassium phosphate, pH 7. Freeze-etch replicas were prepared with a Leybold 2005 bio-etch unit (Leybold GmbH, Koln, Germany). Freeze-fracture and freeze-etch replicas were obtained using an etch- ing time of 0 and 100 s, respectively. For etching, a temperature difference of 50 "C was applied. The replicas were examined in a Philips EM-300 electron microscope. For ultrathin sections, lipo- somes were fixed with 1% (w/v) osmiumtetroxide for 1 h at room temperature. Fixed liposomes were stained with uranylacetate, em- bedded in epon, and examined in a Philips CM-10 electron micro- scope.

Lipid Monolayer Experiments-Tetraether lipids were spread from a solution in chloroform on distilled water. The surface pressure of the tetraether lipids was measured at 22 "C with use of the Wilhelmy method (17-19).

RESULTS

Formation of Liposomes from Archaebacterinl Lipids-Lip- ids from S. acidocaldarius were extracted from freeze-dried cells and partially purified as described under "Experimental Procedures." Three different lipid fractions with decreasing polarity were obtained. Each of the fractions represented a mixture of archaebacterial lipids as different spots were ob- served upon TLC-analysis (Fig. 1). The lipid fractions were tested for their ability to form liposomes alone or in combi- nation with E. coli lipid. As a first indication for the formation

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FIG. 1. One-dimensional TLC of lipid fractions 1, 2, and 3 from S. acidocaldarius. 20 p1 of each fraction containing about 15 mg of lipid/ml were spotted on TLC at the level indicated by the lower line. Fraction 1 was spotted after removing the oily layer on top of fraction 1. Development was in the direction of the arrow with chloroform/methanol/water (65:25:4, v/v). Spots were detected in iodine vapor. The hatched spots were the most prominent ones.

Reconstitution of Proteins in Membrane-spanning Lipids

of closed vesicular structures in aqueous media, clearing of the lipid suspensions after sonication was inspected. E. coli lipid was mixed with increasing amounts of the individual S. acidocaldarius lipid fractions in organic solvent and dried with a rotary evaporator. The lipid mixture was then suspended in 50 mM potassium phosphate, pH 7, and sonicated. The frac- tion with the highest polarity (fraction 1) could be sonicated till clarity when mixed with E. coli lipids. The suspension remained turbid even after prolonged sonication when the fractional amount of fraction 1 lipids exceeded 50%. At room temperature, organic suspensions of fraction 1 lipids slowly segregated leaving an oily layer on top of the solution. When this oily layer was carefully removed, it was possible to obtain a clear suspension in aqueous media even in the absence of E. coli lipid. The fraction with intermediate polarity (fraction 2) could be sonicated till clarity in the absence and in the presence of E. coli lipid. In a previous report by Lo and Chang (lo), tetraether lipids of this fraction could form multilamellar liposomes in aqueous media only after further purification via TLC and methanol precipitation. Finally, the fraction with the lowest polarity (fraction 3) yielded a clear suspension only when at least 90% E. coli lipid was present.

Archaebacterial Lipids form Sealed Liposome Structures- To test whether the liposomes indeed form closed vesicles, their ability to maintain a valinomycin-induced K+-diffusion potential was evaluated using a fluorescent assay. Liposomes composed of S. acidocaldarius lipid fraction 2 were prepared in 50 mM potassium phosphate, pH 7.0, and diluted 100-fold into 50 mM sodium phosphate, pH 7.0. Upon imposition of an outwardly directed K+-diffusion gradient by the addition of valinomycin, the fluorescence intensity of diSC3(5) de- creased (Fig. 2C). After the addition of nigericin, the fluores- cence returned to its initial level. Similar results were obtained with liposomes composed of E. coli lipids (Fig. 2 A ) , a mixture of S. acidocaldarius lipid fraction 2 with E. coli lipids (Fig. 2B) , and each of the other membrane preparations that became transparent after sonication (data not shown). In a control experiment, liposomes were diluted in 50 mM potas- sium phosphate, pH 7 (Fig. 20 ) . Valinomycin and nigericin did not affect the fluorescence of diSCs(5) in the absence of a K+-diffusion gradient. These results demonstrate that the bipolar tetraether lipids of S. acidocaldarius form closed li- posomes in aqueous medium without the necessity to mix these lipids with exogenous phospholipid. For the reconsti- tution experiments, liposomes were used composed of fraction 2 archaebacterial lipids.

Reconstitution of Membrane Proteins into the Archuebacte- rial Liposomes-Cytochrome c-oxidase from beef heart mito- chondria can be used as a Ap generator in liposomes and other membrane systems (13, 14, 20). The activity of cyto- chrome c-oxidase only leads to the generation of a Ap if the protein is reconstituted in the same transmembrane configu- ration as in uiuo. To evaluate if an exogenous membrane protein can be functionally reconstituted into archaebacterial lipids, cytochrome c-oxidase was reconstituted in fraction 2 lipids. Reconstitution was achieved by dialysis followed by freeze-thawing and sonication. This procedure normally leads to a random orientation of the protein (14). However, the use of the membrane-impermeable electron donor cytochrome c only permits activity of those oxidase molecules with the cytochrome c bindings site facing to the outside. This guar- antees the formation of a Ap with a right-side out polarity, i.e. A$, inside negative and ApH, inside alkaline.

After reconstitution of cytochrome c-oxidase in the lipo- somes of archaebacterial lipid, both a A$ and a ApH could be measured when cytochrome c-oxidase was activated by re-

100

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FIG. 2 . Generation of a K+-diffusion potential in liposomes composed of E. coli phospholipids ( A ) , mixture of E. coli phospholipids and S. acidocaldarius lipids, 25:75 (w/w) ( B ) , and S. acidocaldarius lipids alone (C, D ) . Liposomes were pre- pared in 50 mM potassium phosphate, pH 7, and diluted 100-fold into 50 mM sodium phosphate, pH 7 (A-C) or 50 mM potassium phosphate, pH 7 (D). The fluorescence of diSCa(5), present at a concentration of 3 PM, was monitored as described under “Experimental Proce- dures.’’ Valinomycin (0.25 FM) and nigericin (1 /IM) were added as indicated by arrows 1 and 2. Lipids of S. acidocaldarius were from fraction 2.

duced cytochrome c (Fig. 3). The magnitude of ApH was measured with the fluorescent pH indicator pyranine en- trapped by the liposomes. After the addition of ascorbate, cytochrome c and TMPD, the internal pH increased slowly (Fig. 3A). A rapid increase in internal pH was observed in the presence of valinomycin reaching a steady-state ApH of 1.3 pH units. ApH was dissipated by the addition of nigericin. A$ was monitored with the fluorescent dye diSCa(5) (Fig. 3B). Nigericin was included in the assay to prevent the generation of a ApH. When TMPD, cytochrome c, and ascorbate were added, the fluorescence of diSC3(5) was rapidly quenched. This effect was reversed by the ionophore valinomycin that collapses A$. The results demonstrate that beef heart cyto- chrome c-oxidase can be functionally reconstituted into lipo- somes composed of bipolar tetraether lipids.

To investigate whether the archaebacterial lipids support the activity of solute transport systems, membrane vesicles of L. &tis were fused with COVs composed of S. acidocaldarius lipid in a ratio of 1 mg vesicle protein to 10 mg of lipid. Fusion was induced by the freeze-thaw/sonication technique (13,14). The ability of these hybrid membranes to accumulate solutes depends on the formation of a Ap by cytochrome c-oxidase. This approach ensures that transport only takes place in the hybrid membranes, where both cytochrome c-oxidase and the leucine carrier are present. We have previously shown that the activity of the leucine transport system is determined by

1378 Reconstitution of Proteins in Membrane-spanning Lipids

30 t t

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FIG. 3. Generation of a ApH (A) and A* ( B ) in cytochrome c-oxidase proteoliposomes composed of S. acidoculdarius lipid. Beef heart cytochrome c-oxidase was reconstituted into S. acidocaldarius fraction 2 lipids as described under “Experimental Procedures.” COVs were suspended in 50 mM potassium phosphate and energized by the addition of 10 mM ascorbate, 10 p~ cytochrome c and 100 p M TMPD. Cytochrome c and TMPD were present prior to the start of the experiment. A, ApH was measured with the fluorescent dye pyranine. At the arrows, the following compounds were added in sequential order: ascorbate (10 mM), valinomycin (50 nM), and nigericin (1 p ~ ) . B, A+ was monitored with the fluorescent probe diSC3(5). Ascorbate (10 mM) and nigericin (25 nM) were added at arrow 1, and valinomycin (250 nM) was added at arrow 2. The fluorescence is plotted as arbitrary units.

the nature of lipid species introduced into the L. lactis mem- brane vesicles during the fusion procedure (21, 22). In the absence of a suitable lipid matrix leucine transport activity in the fused membranes is almost negligible (21). Hybrid mem- branes containing the archaebacterial lipids rapidly accumu- lated leucine upon the addition of reduced cytochrome c (Fig. 4, closed squares). The steady state of accumulation reached in about 10 min was approximately 98-fold. As a control, L. lactis membrane vesicles were fused with COVs composed of E. coli lipid. In these hybrid membranes, the steady-state accumulation ratio was 205-fold (Fig. 4, closed circles). In the absence of cytochrome c, no accumulation of leucine could be measured in either preparation (Fig. 4, open symbols). Al- though both the rate and extent of leucine transport were lower with S. acidocaldarius lipids compared to E. coli lipids, the results demonstrate that the archaebacterial lipids form a suitable matrix for the function of eubacterial transport systems.

The lower transport activity in the presence of 8. acidocal- darius lipids compared to E. coli lipids could be caused by a lower steady-state level of the Ap which acts as a driving force for the uptake of leucine. Therefore, the magnitude of A+ was estimated from the transmembrane distribution of the lipo- philic cation tetraphenylphosphonium (TPP’) (Fig. 5 ) . Upon energization with ascorbate, cytochrome c and TMPD, TPP’ disappeared from the external medium indicating the forma- tion of a A+, inside negative (Fig. 5A). Upon addition of nigericin, the level of TPP+ uptake increased further. Though a steady state was not attained after 16 min, hybrid mem- branes containing S. acidocaldarius lipids reached a A+ of -79 mV. The magnitude of the A+ in hybrid membranes contain- ing E. coli lipids was -146 mV (Fig. 5B) . These data indicate that the steady-state A+ in the archaebacterial lipids is of

v

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FIG. 4. Ap-driven leucine transport in cytochrome c-oxi- dase proteoliposomes fused with L. Zuctis membrane vesicles. Membrane vesicles of L. lactis were fused with COVs composed of S. acidocaldarius or E. coli lipids. Hybrid membranes were energized by the addition of 10 mM ascorbate, 10 p M cytochrome c, and 100 p M TMPD (closed symbols). In control experiments cytochrome c was left out (open symbols). After 30 s, leucine (3 p ~ ) was added and uptake was followed during 10 min. L. lactis membrane vesicles fused with COVs containing E. coli (0,O) or S. acidocaldarius lipid (H, 0).

4 A I

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Time ( min FIG. 5. Uptake of tetraphenylphosphonium ion by L. lactis

membrane vesicles fused with cytochrome c-oxidase proteo- liposomes composed of ( A ) S. ucidocaldurius and ( B ) E. coli lipids. Uptake of tetraphenylphosphonium ion (TPP+) by hybrid membranes was monitored in a 1-ml vessel with an ion-selective TPP’ electrode using a TPP+ concentration of 4 pM. Membranes were energized by the addition of 10 mM ascorbate, 10 PM cytochrome c, and 10 p M TMPD. The arrows indicate: I , addition of membranes; 2, ascorbate, cytochrome c, and TMPD; 3, nigericin (100 nM); and 4, valinomycin (500 nM).

considerably lower magnitude compared to hybrid membranes with E. coli lipids.

Liposomes Composed of Archaebacterial Lipids Are Arranged in a Monolayer-The structure of the liposomes composed of tetraether lipids was studied with different electron micro- scopic techniques. Ultrathin sections were prepared after fix- ation of the liposomes with osmiumtetroxide and staining

Reconstitution of Proteins in Membrane-spanning Lipids 1379

with uranylacetate (Fig. 6). With this method the polar head groups are stained. The tetraether monolayer appears as a normal bilayer with two dense lines separated by a light band. Often three dense lines were observed. This is presumably due to the close interaction of two membranes as a goniomet- ric analysis (data not shown) of a single liposome indicated that it is surrounded by two dense lines only (Fig. 6, arrow). The diameter of the two dense lines is 2 nm and the light band in between is approximately 3.5 nm. This amounts to 7.5 nm for diameter of the stained membranes. These values are in the same range as found for phospholipid bilayers with dense lines ranging from 1 to 2 nm and light bands of 2.5 to 3 nm.

With freeze-fracture electron microscopy a pronounced dif- ference between the tetraether lipid monolayer and the phos- pholipid bilayer was observed (Fig. 7). Cytochrome c-oxidase containing liposomes of both S. acidocaldarius lipids and of E. coli lipids were examined. In freeze-fracturing the prefer-

FIG. 6. Electron micrograph of an ultrathin section of lipo- somes composed of S. acidocaldarius lipid. The bar indicates 200 nm. At the arrow, a cross-section of a tetraether lipid monolayer is shown stained at the polar head groups.

FIG. 7. Freeze-fracture (A, C) and freeze-etch (B, D ) rep- licas of COVs of s. acidocaldarius and of E. coli lipid. Freeze- fracture of 5'. acidocaldarius ( A ) and E. coli lipid (B). Freeze-etching of S. acidocaldarius (C) and E. coli lipid (D). The arrow indicates the direction of shadowing. Bar = 200 nm.

entia1 fracture plane is the middle of the phospholipid bilayer. With the tetraether lipid monolayer no fracture plane was found and only cross-fracturing of the total membrane was observed (Fig. 7A) . In contrast, E. coli phospholipid showed two fracture faces of the bilayer membrane: A convex half and a concave half, both containing intramembranous parti- cles (Fig. 7C). In order to visualize the intramembranous particles in the two different membrane preparations freeze- etch replicas were prepared after freeze-fracturing. This en- ables the observation of the lipid membrane from the outside (Fig. 7, B and D). At the outside of the liposomes, intramem- branous particles were hardly visible and no difference was observed between S. acidocaldarius (Fig. 7 B ) and E. coli (Fig. 7 0 ) cytochrome c-oxidase liposomes. These results demon- strate that s. acidocaldarius lipids form vesicular structures and confirm the membrane-spanning nature of the tetraether lipids.

Membrane components like phospholipids are oriented on an air-water interphase as a monomolecular film. The polar head groups are in contact with the water-phase while the fatty acid chains are exposed to the air. Since the archaebac- terial membrane-spanning lipids have polar head groups on both sides of the molecule it was of interest to analyze the orientation of these lipids on a water-air interphase. A compression curve of the archaebacterial lipids was estab- lished with the aid of the Wilhelmy method (Fig. 8). At the maximum level of the surface pressure, the surface area of one single lipid molecule corresponds to 0.82 nm2 assuming an average molecular mass of the lipids of 1800. The average surface area found is in the same order of magnitude as observed before for purified tetraether lipids (23). These re- sults indicate that the mixed tetraether lipids are arranged as a monomolecular film at the water-air interphase, and further

Molecular Area 1 nm'l molecule )

FIG. 8. Pressure-area isotherm for tetraether lipids from S. acidocaldarius spread on distilled water at 22 "C. At maximum compressibility of the monolayer, the mean surface area per lipid molecule was 0.82 nm'.

1380 Reconstitution of Proteins in Membrane-spanning Lipids

verify the membrane-spanning nature of the lipids used for the reconstitution experiments.

DISCUSSION

In this report we demonstrate the functional reconstitution of membrane proteins into liposomes of bipolar tetraether lipids of the extreme thermophilic archaebacterium S. acido- caldarius. Although the chemical structure of these bipolar lipids differs considerably from the structure of conventional phospholipids, they appear to be functionally comparable to E. coli lipids. Beef heart cytochrome c-oxidase (eukaryotic orgin) as well as the leucine transport system of L. lactis (eubacterial orgin) were active after incorporation in the archaebacterial lipid matrix. Like conventional phospholipid bilayers the archaebacterial monolayer forms a competent matrix for the function of these membrane proteins. More- over, this data represents the first in vitro evidence for func- tionality of an integral membrane protein of eubacterial orgin when reconstituted into archaebacterial lipids.

Beef heart cytochrome c-oxidase was reconstituted into liposomes via dialysis followed by freeze-thaw and sonication. The leucine carrier was introduced into these COVs by fusion with membrane vesicles of L. lac& Purified cytochrome c- oxidase contains a tightly bound cardiolipin essential for activity (24). After reconstitution of cytochrome c-oxidase, the exogenous lipid introduced together with the enzyme is nearly negligible. In contrast, the fusion procedure used to evaluate the activity of the leucine transport system leads to the introduction of approximately 10% L. lactis lipid in the archaebacterial liposomes. However, this residual level of endogenous lipids cannot account for the observed transport activity. Previous experiments have demonstrated that the activity of leucine transport system is determined by the bulk lipid composition of the liposomes used for fusion (21, 22). Our results suggest that the steady-state level of the Ap generated by cytochrome c-oxidase in hybrid archaebacterial liposomes is lower than that with E. coli lipids (Fig. 5). Similar results were obtained when the liposomes were not fused with L. lactis membrane vesicles. Taking this lower Ap level into account, the data suggest that the archaebacterial lipids sup- port a substantial leucine transport activity (Fig. 4). In addi- tion, the specific lipid requirement of the leucine transport system may account for the lower activity observed with archaebacterial lipids compared to E. coli lipids (21).

The structure of the tetraether lipids, two different polar head groups at opposite ends, suggests that each molecule can span the entire membrane. The absence of a preferential fracture plane upon freeze-fracturing of the liposomes com- posed of S. acidocaldarius strongly suggests that these lipids indeed form a monomolecular layer (Fig. 7A). The liposomes cross-fracture perpendicularly and no inner and outer mem- brane face is observed. The same phenomenon was recognized earlier in freeze-fracture preparations of tetraether lipid con- taining Thermoplasma and Sulfolobus cells (25, 26). A mono- molecular organization of the tetraether lipids was also ob- served at the water-air interphase (Fig. 8). The surface be- havior of some purified bipolar ether lipids have been studied before (23). Purified bipolar GDGT and GDNT lipid species with unsubstituted polar groups appear to be unstable at the water-air interphase. This was attributed to an increasing number of molecules adhering with both ends to the water surface and/or a less sharply curved configuration of mole- cules which touches the water phase with both ends already. Lipids with sugar residues substituted at the polar ends form a stable monolayer. Mixed lipids of S. acidocaldarius are

expected to behave as the latter class of lipids since in Sulfo- lobus the lipids with glycosidic linkages on one end of the polar heads constitute about 92% of the total complex lipids (3). Two complex derivatives, glycolipid B and phospholipid 11, occupy 0.75 and 0.85 nm2 of surface area per molecule at the maximum level of surface pressure, respectively (23). The value obtained for our mixed tetraether lipid preparation, 0.82 nm', is in the same order.

The liposomes constructed of the archaebacterial lipids offer attractive possibilities for the study of the (thermo)stability of these lipids in relation to its structure. Since membrane proteins can be functionally incorporated in the liposomes it is also possible to study the (therm0)stability of proteins after reconstitution in different liposome prepa- rations. In additions, these liposomes may provide a useful vehicle for studies on lipid-protein interactions and the inser- tion of proteins into biological membranes. Recently, a gene was identified in the archaebacterium Methanococcus vannei- Eii bearing strong homology with the SecY protein that me- diates protein export in E. coli (27). Moreover, electron trans- port components have been isolated from S. acidocaldarius and evidence has been presented for a classical chemiosmotic mechanism of energy coupling in these cells (28, 29). The construction of the archaebacterial liposomes provides the basis for the study of these membrane proteins in their natural environment.

Acknowledgments-We would like to thank Dr. G. Schafer for supplying us with S. acidocaldarius cells. W e also thank J. Zagers for the preparation of the freeze-fracture and freeze-etch replicas and K. Sjollema who provided us with the electron micrograph of the ultra- thin section.

REFERENCES 1. De Rosa, M., Gambacorta, A. & Gliozzi, A. (1986) Microbwl. Reu.

2. De Rosa, M., Gambacorta, A. & Nicolaus, B. (1983) J. Membrane Sci. 16,287-294

3. De Rosa, M., Trincone, A., Nicolaus, B. & Gambacorta, A. (1991) in Life Under Extreme Conditions (di Prisco, G., ed) pp. 61-87,

4. De Rosa, M. & Gambacorta, A. (1988) Prog. Lipid. Res. 27,153- Springer Verlag, Berlin

175 5. Gliozzi, A., Paoli, G., De Rosa, M. & Gambacorta, A. (1983)

Biochim. Biophys. Acta 735 , 234-242 6. Gliozzi, A., Rolandi, R., De Rosa, M. & Gambacorta, A. (1982)

Biophys. J. 37 , 563-566 7. Gliozzi, A., Rolandi, R., De Rosa, M. & Gambacorta, A. (1983) J.

Membrane Biol. 76,45-56 8. Gliozzi, A., Rolandi, R., De Rosa, M., Gambacorta, A. & Nicolaus,

B. (1982) in Transport in Bwmembranes: Model Systems and Reconstitution (Antonili, A., Gliozzi, A., and Jorio, A., eds) pp. 39-47, Raven Press, New York

9. Lelkes, P. I., Goldenberg, D., Gliozzi, A., De Rosa, M., Gamba- corta, A. & Miller, I. R. (1983) Biochim. Biophys. Acta 732 ,

10. Lo. S.-L. & Chane. E. L. (1990) Biochim. Bzbhvs. Res. Commun.

50,70-80

714-718 Yl

i67,238-243 . . "

11. Viitanen. P.. Newman. N. J.. Foster. D. L.. Hastines Wilson. T. & Kaback: H. R. (1986) Methods Enzymol. 125, i29-452 '

J. Bacteriol. 149 , 733-738

Natl. Acad. Sci. U. S. A. 82, 7555-7559

in press

Biochim. Biophys. Acta 681,85-94

(1983) Eur. J. Biochem. 130 , 287-292

12. Otto, R., Lageveen, R. C., Veldkamp, H. & Konings, W . N. (1982)

13. Driessen, A. J. M., de Vrij, W . & Konings, W . N. (1985) Proc.

14. Driessen, A. J. M. & Konings, W . N. (1991) Methods Enzymol.,

15. Lolkema, J. S., Hellingwerf, K. J. & Konings, W . N. (1982)

16. Lolkema, J. S., Abbing, A., Hellingwerf, K. J. & Konings, W . N.

17. Demel, R. A. (1974) Methods Enzymol. 32,539-545 18. Verger, R. & Pattus, F. (1982) Chem. Phys. Lipids 30 , 189-227

Reconstitution of Proteins in Membrane-spanning Lipids 1381

19. Demel, R. A. (1982) in Membranes and Transport (Martonosi, A. N., ed) Vol. 1, pp. 159-164, Plenum Publishing Co., New York

20. Driessen, A. J. M., Hellingwerf, K. J. & Konings, W. N. (1987) Microbiol. Sci. 4, 173-180

21. Driessen, A. J. M., Zheng, T., In 't Veld, G., Op den Kamp, J. A. F. & Konings, W. N. (1988) Biochemistry 27,865-872

22. In 't Veld, G., Driessen, A. J. M., Op den Kamp, J. A. F. & Konings, W. N. (1991) Biochim. Biophys. Acta 1065, 203-212

23. Rolandi, R., Schindler, H., De Rosa, M. & Gambacorta, A. (1986) Eur. Biophys. J. 14, 19-27

24. Yu, C., Yu, L. & King, T. E. (1975) J. Biol. Chern. 250, 1383- 1392

25. Langworthy, T. A. (1985) in The Bacteria (Woese, C. R., and Wolfe, R. S., eds) Vol. 8, Academic Press, New York

26. Langworthy, T. A. (1977) Biochim. Biophys. Acta 487,37-50 27. Auer, J., Spicker, G. & Bock, A. (1991) Biochimie 73,683-688 28. Anemuller, S. & Schafer, G. (1990) Eur. J . Biochern. 191, 297-

29. Schafer, G., Anemuller, S., Moll, R., Meyer, W. & Lubben, M. 305

(1990) FEMS Microbiol. Reu. 75, 335-348