Fusion of Liposomes with Mitochondrial Inner Membranes (Membrane Phospholipid Enrichment/Protein Diffusion/Electron Transfer) HEINZ SCHNEIDER, JOHN J

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Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 442-446, January 1980 Cell Biology Fusion of liposomes with mitochondrial inner membranes (membrane phospholipid enrichment/protein diffusion/electron transfer) HEINZ SCHNEIDER, JOHN J. LEMASTERS, MATTHIAS HOCHLI, AND CHARLES R. HACKENBROCK Laboratories for Cell Biology, Department of Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 Communicated by Charles Tanford, October 22, 1979

ABSTRACT A procedure is outlined for the fusion of mixed with the lipid enrichment of the membrane bilayer. The pro- phospholipid liposomes (small unilamellar vesicles) with the cedure should be applicable to other cell membrane systems. mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was ini- tiated by manipulation of the pH of a mixture of freshly soni- METHODS cated liposomes and the functional inner membrane/matrix Mitochondria were isolated from the livers of male Sprague- fraction of rat liver mitochondria. During the pH fusion pro- Dawley rats in 70 mM sucrose/220 mM mannitol/2 mM cedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron Hepes/bovine serum albumin (0.5 mg/ml) brought to pH 7.4 microscopy. After the pH fusion procedure, a number of ultra- with KOH. Subsequent removal of the outer membrane and structural, compositional, and functional characteristics were purification of the inner membrane/matrix (mitoplast) fraction found to be proportionally related: the membrane surface area was carried out by use of a controlled digitonin incubation (9, increased; the lateral density distribution of intramembrane 10). The topographically complex inner membrane was con- particles proteins) in the plane of the membrane decreased whereas(integralthe particles remained random; the membrane verted to a simple spherical configuration by two washes in the became more buoyant; the ratio of membrane lipid phosphorus isolation medium diluted 1:7.5 (40 mosM) (9). to total membrane protein increased; the ratio of membrane Liposomes (small unilamellar vesicles) were prepared by lipid phosphorus to heme a of cytochrome c oxidase increased; sonicating 1.5 g of mixed phospholipids (asolectin) in 7.5 ml of and the rate of electron transfer between some interacting the diluted isolation medium with the microtip probe of a membrane oxidoreduction proteins decreased. These data reveal Branson sonifier (model W185) at 40 W for three 10-min cycles that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) at 0°C. The pH of the phospholipid dispersion was maintained and that integral membrane proteins diffused freely into the at 7.4 with KOH throughout sonication. For liposome-mem- laterally expanding bilayer. Furthermore, the data suggest that brane fusion, 2.5 ml of the freshly sonicated liposomes was the rate of electron transfer may be limited by the rate of lateral added to 7.5 ml of the spherical inner membranes (14 mg of diffusion of oxidoreduction components in the bilayer of the protein per ml) at 300C with constant stirring. The pH was then mitochondrial inner membrane. lowered to 6.5 with 0.01 M HCI. Two additional aliquots (2.5 ml) of sonicated liposomes were added at 15-min intervals; the Although a number of advances have been reported regarding pH was maintained at 6.5. After 45 min of incubation, the pH the theoretical aspects of membrane fusion generally and the was increased to 7.4 with 0.01 M KOH, and the liposome/ fusion of liposomes with plasma membranes specifically (1-5), membrane mixture was put on ice. The mixture was layered the evidence for liposome-membrane fusion appears to be more on a sucrose density gradient [0.6, 0.75, 1.0, and 1.25 M sucrose circumstantial than unequivocal (5). The essential difficulty in diluted (1:7.5) isolation medium] at 5 ml (17 mg of protein) in the assessment of fusion has been in the quantitation of the per gradient tube (40 ml) and centrifuged at 70,000 X g for 15 liposomal lipid fused with and incorporated into the membrane hr at 2°C. Four distinct density gradient membrane fractions bilayer. were collected. Recent observations in our laboratory on the lateral diffusion NADH, succinate, and cytochrome c oxidase activities were of integral proteins generally (6) and of cytochrome c oxidase determined polarographically with a Clark oxygen electrode specifically (7) in the mitochondrial energy-transducing (11). Reaction media were as follows: for NADH oxidase, 80 membrane prompted us to develop a liposome-membrane mM potassium phosphate, pH 7.4/2 mM NADH/1 ,M car- fusion procedure that can be utilized to increase the surface area bonylcyanide m-chlorophenylhydrazone/13 ,uM cytochrome of the lipid bilayer of the functional membrane in a quantita- c; for succinate oxidase, 80 mM potassium phosphate, pH 7.4/5 tively controlled fashion. The need for such an approach has mM sodium succinate/5 AM rotenone/1 MM carbonylcyanide grown out of our interest in ascertaining the effects of the m-chlorophenylhydrazone/13 MM cytochrome c; for cyto- physical properties of the membrane bilayer lipid on diffu- chrome c oxidase, 10 mM potassium phosphate, pH 7.4/5 mM sional, collisional, and catalytic events that occur among the sodium ascorbate/2.7 MM N,N,N',N'-tetramethyl-p-phenyl- membrane oxidoreduction proteins and that are fundamental enediamine/1 ,M cytochrome c. Single determinations of the to the kinetics and mechanisms of electron transfer and oxi- heme a of cytochrome c oxidase were performed by mea- dative phosphorylation (8). surement of AA (605-630) after dithionite reduction, by using In the present report we outline a simple fusion procedure an extinction coefficient of 13.1 mM-1.cm-1 (12). for incorporating limited to massive quantities of liposomal Protein was determined by the Lowry method (13) and lipid lipids into the mitochondrial energy transducing membrane phosphorus, by the Bartlett method (14). and describe quantitative assessment of several ultrastructural, For freeze-fracture electron microscopy, unfixed density compositional, and functional changes that occur concurrently gradient membrane fractions were treated with 30% glycerol in diluted (1:7.5) isolation medium and frozen in liquid Freon The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- 22. Unglycerinated samples of the liposome/membrane mix- vertisement " in accordance with 18 U. S. C. §1734 solely to indicate ture in the process of fusion were propane-jet-frozen at this fact. 10,000°C/sec (15). Freeze-fracturing was carried out in a 442 Downloaded by guest on September 27, 2021 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 77 (1980) 443

Balzers BA 360 instrument equipped with electron guns (16). Electron micrographs were taken with a top-entry JEOL 100 CX electron microscope operated at 80 kV. RESULTS Ultrastructural Analysis. After the liposome-membrane pH fusion procedure, use of a discontinuous sucrose gradient routinely separated four distinct membrane fractions (Fig. 1). These fractions were designated: band 1, density between 1.080 and 1.098 g/cm3; band 2, 1.098-1.132 g/cm3; band 3, 1.132-1.167 g/cm3; and pellet, >1.167 g/cm3. An additional band, which did not penetrate into the gradient, contained free liposomes and no appreciable membrane protein. Independent analysis determined the density of control, untreated, inner membrane to be 1.188 g/cm3. With all other conditions held constant, incubation time was used to control the percentage yield of each membrane fraction: shorter incubations produced more pellet and less band 1, whereas longer incubations produced less pellet and more band 1. Owing to the uncertainties of chemical fixation of phospholipid vesicles, ultrastructural details of liposome-membrane interaction was assessed in nonfixed, freeze-fractured samples. Fig. 2 shows a typical sample of the liposome/membrane FIG. 2. Liposome/mitochondrial inner membrane mixture jet- mixture jet-frozen during incubation at pH 6.5. At this pH (and frozen without cryoprotection during incubation at pH 6.5. Small below), the small liposomes were observed in close apposition particle-free liposomes are observed in close apposition with a par- with and apparently sequestered by the inner membranes, in- ticle-rich membrane during the fusion process. (X69,000.) dicative of the fusion process. Freeze-fractured samples were also prepared from each of the four sucrose density gradient as the total surface area of the membranes and the average fractions (Fig. 3). Unlike samples frozen during the fusion lateral distance between particles increased. process, very few liposomes were observed adsorbed to the Compositional Analysis. If fusion occurs between liposomes surfaces of these membranes. As would be expected with lipo- and membranes, the membrane lipid-to-membrane protein some-membrane fusion, an average increase in membrane ratio must increase. Furthermore, it is important to determine surface area occurred progressively from pellet to band 1. In that such an increase does not occur as a result of loss of mem- addition, fracture faces invariably showed a progressive de- brane protein during the fusion process. To quantitate the crease in the lateral density distribution of intramembrane phospholipid incorporation during the liposome/membrane particles (i.e., number of particles per membrane unit surface incubation, the lipid phosphorus-to-protein ratio was deter- area) from pellet to band 1 (Figs. 3 and 4a). However, the rel- mined in the four sucrose density gradient fractions. Membrane ative distribution of the particles in the two halves of the bilayer lipid phosphorus was found to increase progressively from pellet remained unchanged (Fig. 4a). Furthermore, the particles to band 1 fractions (Table 1). Notably, the most buoyant of the maintained a random distribution in the plane of the membrane four membrane fractions (i.e., band 1) contained an apparent 6-fold increase in phospholipid compared to the least buoyant Sucrose 0.0 yield of (i.e., pellet fraction). density membrane Cytochrome c oxidase was quantitated in order to assess the at 2cCg cm3 Fraction protein possibility of membrane protein loss during the fusion process. Cytochrome c oxidase, a heme a-containing, oxidoreduction protein, is completely transmembranous in the mitochondrial Liposomes inner membrane (17); it can be quantitated by the difference between its reduced and oxidized spectra. Heme a content per mg of total membrane protein remained constant in all four 1.0803 sucrose density fractions (Table 1), indicating that loss of Band 1 6-9 membrane protein did not occur during lipid enrichment of the inner membrane bilayer. An apparent large increase in 1.0987 Band 2 9-11 Table 1. Composition of liposome-mitochondrial inner - Aii6 membrane fusion product % increase 1.1322 Lipid P, Lipid P/ in Band 3 15-20 Mimol/ Heme a, nmol/ heme a lipid bilayer Fraction mg protein* mg protein* (mol/mol) surface area Control 0.24 ± 0.01 0.27 + 0.01 900 1.1674 Pellet 0.61 ± 0.05 0.51 + 0.02 1200 30 "" RFI1, Pellet 60-70 Band 3 0.90 ± 0.09 0.57 ± 0.03 1600 80 Band 2 1.65 ± 0.13 0.54 + 0.03 3100 240 FIG. 1. Sucrose density gradient of a liposome/mitochondrial ± inner membrane mixture incubated at pH 6.5 for 40 min at 30°C. The Band 1 3.76 0.29 0.52 + 0.02 7200 700 gradient was centrifuged at 70,000 X g for 15 hr at 2°C. * Results are expressed as mean ± SEM for six to eight experiments. Downloaded by guest on September 27, 2021 444 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 77 (1980) cytochrome c oxidase between the control inner membrane and , , . ;> ...AT~ E the four sucrose density gradient membrane fractions occurred *~~ owing to the loss of soluble mitochondrial matrix protein during ,s s sb~.'\:. 4 the fusion process and subsequent washing steps. The membrane lipid phosphorus-to-heme a molar ratio revealed that the lipid bilayer of the four sucrose density gradient fractions increased by as little as 30% in the pellet fraction to as much as 700% in band 1. Consistent with true fusion, the decrease in the A_V density distribution of intramembrane particles in the plane of the membrane was proportional to the increase in the molar ratio of membrane lipid phosphorus to heme a (Fig. 4a). 9, 8! *, , " Functional Analysis. The observation that the distribution of the intramembrane particles decreased in density but remained random as the membrane became increasingly enriched with phospholipid indicated that integral proteins can diffuse independently of one another in the plane of the inner membrane bilayer. We therefore examined directly the possibility that specific electron-transferring oxidoreduction proteins integral to the membrane normally diffuse randomly and independently of one another in the plane of the bilayer. Should this be so, electron transfer activity between two or more of these interacting proteins might be decreased in the lipid- enriched membranes because such proteins would be required to diffuse laterally over greater distances prior to collision and electron transfer. Such a decrease in activity would be consistent with a rate-limiting diffusional process by electron-transferring components in the membrane. It was determined that the rate of electron transfer through the entire sequence of oxidoreduction proteins, from NADH dehydrogenase through cytochrome c oxidase (NADH oxidase) and from succinate dehydrogenase through cytochrome c oxidase (succinate oxidase), decreased in proportion to the increase in lipid enrichment of the membrane bilayer and the increase in the average distance between integral proteins represented by intramembrane particles (Fig. 4). That the newly incorpo- zC rated lipid did not simply inhibit oxidoreduction proteins generally was suggested by the finding that electron-transfer activity by cytochrome c oxidase, which does not require collisional interaction with other integral proteins in the bilayer, did not decrease but rather increased slightly (Fig. 4b). It should be noted that after the fusion process the membranes did not show a membrane potential (AI) and were permeable to NADH.

DISCUSSION The liposome-membrane fusion procedure presented here grew out of an interest in enriching the mitochondrial energy-transducing membrane with bulk exogenous phospholipid for purposes of studying the diffusional, collisional, and catalytic activities of the electron-transfer and energy-transducing in-

tegral membrane proteins which function in the synthesis of .1 ATP by oxidative phosphorylation. The method requires the manipulation of pH and utilizes a class of liposomes designated "small unilamellar vesicles." The pH fusion procedure is technically uncomplicated, can be controlled with reasonable precision, and does not require the addition of divalent cations.

Although the rate and quantity of fusion can be controlled .L '¶%. by incubation time, temperature, type of exogenous lipid, and w.,... .. the concentration ratio of liposomes to inner membranes, the most important variable is pH. When all other conditions are optimal but pH is maintained at neutrality, essentially no fusion occurs between asolectin liposomes and inner membranes. For fusion of asolectin liposomes the pH optimum is 6.5. The re- covery of membranes enriched with precise quantities of ex- FIG. 3. (Legend appears at the bottom of thenextpage). ogenous lipid is further controlled.by selection through sucrose FIG. 3. (Legend appears at the bottom of the next page.) Downloaded by guest on September 27, 2021 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 77 (1980) 445 density gradient centrifugation. Buoyant-distinct membrane of nonfused liposomes to the inner membrane surface which fractions enriched by a few percent to as much as 10-fold or gave spurious density gradient and quantitative results. more with exogenous lipid can be obtained routinely. Whether or not digitonin is bound to the inner membrane Although the mechanism by which liposome inner mem- during removal of the outer membrane and whether or not such brane fusion occurs is unknown, a minimal liposome size ap- bound digitonin would support the low pH fusion process are pears to be an important criterion. A minimal radius of curva- unknown. ture is thought to strain the packing order of the phospholipids The pH method is not limited to fusion with asolectin lipo- in the liposome bilayer (18). This, together with optimal pro- somes. Using the procedure as described, we have recently tonation to decrease electrostatic repulsion between the lipo- determined that small unilamellar vesicles composed of bovine some and membrane surfaces, most likely supports the spon- heart cardiolipin, egg phosphatidylethanolamine, and egg taneous fusion. Divalent cations do not play a significant role phosphatidylcholine (1:2:2) and also vesicles of cholester( l and in the pH method because fusion occurred routinely in Ca2+_ asolectin (1:1) fuse with mitochondrial membranes as dily free media in the presence of 1 mM EDTA. Previous attempts as do asolectin vesicles. Use of the pH method for the 1I I en- to fuse inner membranes with small unilamellar vesicles com- richment of other cell membranes will be reportt else- posed of either asolectin or phosphatidylglycerol/phosphati- where. dylcholine in the presence of 0.5-10 mM Ca2+ at pH 7.4 gave That liposome-membrane fusion occurred in our studies is significant levels of fusion. However, we found the major dif- supported by several ultrastructural, compositional, and ficulty with Ca2+-supported fusion to be an irreversible functional characteristics that were determined to be propor- aggregation of inner membranes and an irreversible adsorption tionally related: (i) the membrane surface area increased; (ii)

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FIG. 3. Freeze-fracture convex faces ofmitochondrial inner membranes from four sucrose density gradient fractions after fusion with liposomes. (X69,000.) (a) Pellet fraction; (b) band 3 fraction; (c) band 2 fraction; (d) band 1 fraction. Membrane surface area and average distance between intramembrane particles (= integral proteins) increase progressively from pellet to band 1. Downloaded by guest on September 27, 2021 446 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 77 (1980)

0 CN _ CN r gral proteins of the inner mitochondrial membrane are free to D 'a a) 'D = C C C = C C C diffuse randomly in the plane of the membrane independently 0 I-) a. Gm m m m m m of one another is supported by the observation that, during the tF-tt t to fusion process, a decrease occurred in the density distribution NE of intramembrane particles. This decrease was proportional to 6 2000 the increase in lipid phosphorus-to-heme a ratio. Related to c these findings were the decreases in electron-transfer activities of the interacting oxidoreduction proteins making up the cCu NADH oxidase and succinate oxidase sequences. Significantly, 'a 1000 these decreases were proportional to the increase in membrane .2 lipid enrichment as well as to the increase in the average distance between intramembrane particles. These results suggest that the average distances between some of the functionally 0.5 0 interacting oxidoreduction components in the electron-transfer (P/heme a)-' X 103 sequence increase during lipid enrichment and surface area FIG. 4. (a) Density distribution of intramembrane particles expansion of the bilayer. Most likely, this results in a greater (integral proteins) as a function of lipid phosphorus-to-heme a ratio diffusion distance for such oxidoreduction components prior (P/heme a). Particle densities on the convex (0) and concave (0) to collision, thus limiting the overall rate of electron transfer. fracture faces are shown as mean ± SEM. (b) Activities of NADH oxidase (A), succinate oxidase (-), and cytochrome c oxidase (v) as We thank Linda Fuller and Phil Ives for their expert technical as- a function of P/heme a. Relative activities of oxidases are shown as investigation was by Research Grants means of three experiments. sistance. This supported BMS75-02372 and PCM77-20689 from the National Science Foun- dation to C.R.H. and research fellowships to M.H. from the Swiss National Foundation and The Muscular Dystrophy Association of the lateral density distribution of intramembrane particles America. (integral proteins) in the plane of the membrane decreased whereas the particles remained random; (iii) the membranes 1. Gregoriadis, G. & Ryman, B. E. (1972) Biochem. J. 129, 123- 133. more ratio phos- became buoyant; (iv) the of membrane lipid 2. Tyrrell, D. A., Heath, T. D., Colley, C. M. & Ryman, B. E. (1976) phorus to total membrane protein increased; (v) the ratio of Biochim. Biophys. Acta 457, 259-302. membrane lipid phosphorus to heme a of cytochrome c oxidase 3. Post, G., Papahadjopoulos, D. & Vail, W. J. (1976) in Methods increased; (vi) the rate of electron transfer between some in- in Cell Biology, ed. Prescott, D. M. (Academic, New York), Vol. teracting membrane oxidoreduction proteins decreased. The 14, p. 3371. observation that the decrease in density distribution of in- 4. Pagano, R. E. & Weinstein, J. N. (1978) Annu. Rev. Biophys. tramembrane particles was proportional to the increase in the Bioeng. 7, 435-468. molar ratio of membrane lipid phosphorus to heme a empha- 5. Post, G. & Papahadjopoulos, D. (1978) Ann. N.Y. Acad. Sci. 308, sizes that the new membrane lipid was incorporated into the 164-181. bilayer and not simply adsorbed to the membrane surface. In 6. Hochli, M. & Hackenbrock, C. R. (1976) Proc. Natl. Acad. Sci. USA 73, 1636-1640. a routine that adds to the structural evi- addition, observation 7. Hochli, M. & Hackenbrock, C. R. (1979) Proc. Natl. Acad. Sci. dence for fusion was the close apposition and sequestering of USA 76, 1236-1240. liposomes by the membrane that occurred during the fusion 8. Hackenbrock, C. R. (1976) in Structure ofBiological Membranes: process as determined in unfixed, jet-frozen, freeze-fractured 34th Nobel Foundation Symposium, eds. Abrahamson, S. & samples. Pasher, I. (Plenum, New York), pp. 199-234. Although real fusion must result in an increase in the mem- 9. Hackenbrock, C. R. (1973) J. Cell Biol. 53, 450-465. brane lipid-to-protein ratio, it is necessary to assess the possibility 10. Schnaitman, C. & Greenwalt, J. W. (1968) J. Cell Biol. 38, that this increase might be due to the loss of membrane protein 158-175. rather than the enrichment of membrane lipid. In this regard 11. Estabrook, R. W. (1967) Methods Enzymol. 10, 41-47. 175-178. it was determined that the ratio of the heme a of cytochrome 12. Vanneste, W. H. (1966) Biochim. Biophys. Acta 114, 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. c oxidase, a major integral protein, to the total membrane (1951) J. Biol. Chem. 193, 265-275. constant process whereas protein remained during the fusion 14. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. the lipid phosphorus-to-heme a ratio increased dramatically. 15. Moor, H., Kistler, J. & Muller, M. (1976) Experientia 32, 805. Finally, real fusion can be expected to alter some catalytic 16. Moor, H. (1969) Int. Rev. Cytol. 23,391-412. functions of the membrane owing to the newly incorporated 17. Hackenbrock, C. R. & Miller, H. K. (1975) J. Biol. Chem. 250, lipid, especially those functions requiring diffusional or colli- 9185-9197. sional mobility between two or more proteins integral to the 18. Sheetz, M. P. & Chan, S. I. (1972) Biochemistry 11, 4573- membrane. That some of the 30 or so catalytically active inte- 4581. Downloaded by guest on September 27, 2021