Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5306-5310, November 1978 Biochemistry

Formation of protein micelles from amphiphilic membrane proteins (Semliki Forest virus/Sendai virus/membrane glycoproteins/penicillinase/Triton X-100) K. SIMONS*, A. HELENIUS*, K. LEONARD*, M. SARVAS*, AND M. J. GETHINGt *European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Germany; and t Imperial Cancer Research Fund Laboratories, London, United Kingdom Communicated by John C. Kendrew, August 10, 1978

ABSTRACT The membrane penicillinase (penicillin isolated, and labeled as described (14). Sendai virus from em- amido-f-lactamhydrolase, EC 3.5.2.6) from Bacillus licheni- bryonated eggs was purified as described (15). For radioactive formis, the Semliki Forest virus spike proteins, and the Sendai labeling, Sendai virus was grown in cultures of virus glycoproteins have each been isolated as soluble protein primary chicken embryo lung cells in Dulbecco's modified aggregates that are virtually free of and . The sedimentation coefficients of the complexes were 18 S, 29 S, and Eagle's medium and 10% fetal calf serum. One hour after in- 43 S, respectively. Mixed aggregates containing both the virus oculation with virus, the cells were washed with phosphate- glycoproteins and the penicillinase could also be formed. Such buffered saline and medium containing [3H]leucine was added protein micelles may serve a number of useful purposes in (modified Eagle's medium with 1/10th concentration of leucine membrane research. plus 100 ,uCi of [3H]leucine per ml). After 48 hr the medium was harvested and the virus was concentrated by centrifugation Membrane proteins that extend into the apolar phase of the at 120,000 X g for 1 hr at 40C, resuspended in phosphate-buf- can usually be solubilized by mild fered saline and purified by density gradient centrifugation without losing their function (1, 2). The protein-detergent (20-55%, wt/wt, sucrose in phosphate-buffered saline) at complexes can be isolated and characterized, but the presence 100,000Xgfor2hr. of detergent imposes severe limitations on further studies. These Preparation of Protein Micelles. The complex containing complexes cannot be used in studies involving living cells or membrane penicillinase and Triton X-100 (up to 1 mg of pro- biological membranes since the detergent will lyse the cells and tein in 0.2 ml) was layered onto a detergent-free density gra- solubilize the membrane. If the detergent is removed, the dient (13 ml of 20-50% sucrose, wt/wt, in 50 mM Tris-HCl, pH complexes usually precipitate, and this renders the proteins 7.4/0.1 M NaCl). Centrifugation was carried out in a Spinco useless for most purposes. Some amphiphilic proteins have been SW40 rotor at 40,000 rpm for 20 hr at 200C. Spike protein found to associate with each other in the absence of detergents micelles from Semliki Forest virus and Sendai virus were pre- to yield soluble aggregates, but these are exceptions (3-12). It pared by a single preparative step as described earlier for would be important to find a general way to make water-soluble Semliki Forest virus (9). To virus containing up to 1.5 mg of membrane protein aggregates. In this paper we have studied protein, Triton X-100 (4 times the weight of protein) was added. the formation of such complexes using three amphiphilic This mixture was layered onto a density gradient containing membrane proteins: the surface glycoproteins of Semliki Forest a zone of 0.3 ml of 1% Triton X-100 in 15% sucrose followed by virus and Sendai virus and the membrane penicillinase (peni- 12.5 ml of 20-50% sucrose (wt/wt) devoid of detergent in 50 cillin amido-,B-lactamhydrolase, EC 3.5.2.6) from Bacillus li- mM Tris-HCl, pH 7.4/0.1 M NaCl. cheniformis. When the detergent is removed from these pro- Electron Microscopy. Samples (dialyzed free of sucrose) in teins by sucrose gradient centrifugation they form aggregates the concentration range 10-20 ,ug of protein per ml, were that are homogeneous in size, soluble in aqueous media, and negatively stained with 1% aqueous uranyl acetate, pH 4.5, by free of lipid. Procedures have been devised that allow the the method of Valentine et al. (16). Micrographs were taken preparation of mixed aggregates containing two different with a Philips EM400 electron microscope, operating at 80 kV, membrane proteins. at magnifications of 55,000 or 70,000. Other Methods. Polyacrylamide gel electrophoresis in so- MATERIALS AND METHODS dium dodecyl sulfate (NaDodSO4) was carried out as described (9). The sedimentation coefficients of the proteins were de- Materials. 3H-Labeled leucine, isoleucine, and valine and termined according to Martin and Ames (17). As standards, the [35S]methionine were obtained from the Radiochemical Centre 29S complex of Semliki Forest virus spike protein, thyroglob- (Amersham). Ultrogel ACA34 was from LKB-Produkter, ulin, and immunoglobulin G were used. The Stokes radius was Sepharose 2B was from Pharmacia, and Triton X-100 was from estimated by gel filtration according to Laurent and Killander Rohm et Haas. 3H-Labeled Triton X-100 was kindly supplied (18). Ultrogel ACA34 or Sepharose 2B columns (1.5 X 85 cm) by W. R. Lyman of Rohm et Haas. were calibrated with the same standards as above. For precip- Membrane penicillinase and penicillinase labeled with itation of the protein aggregates, 20 Al of rabbit antisera was [35S]methionine were purified from B. licheniformis as a added to 20 Al of antigen. After 30 min of incubation at room complex with Triton X-100 (13). , the antibody-antigen complexes formed were Virus Preparations. Semliki Forest virus was produced, precipitated with 150 ,u of protein A (40 ,ug/ml) coupled to Sepharose 4B (Pharmacia). Triton X-100 was assayed by ra- The publication costs of this article were defrayed in part by page dioactivity using 3H-labeled Triton X-100 as described (9). charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: NaDodSO4, . 5306 Downloaded by guest on September 28, 2021 Biochemistry: Simons et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5307 RESULTS The membrane penicillinase of B. licheniformis (apparent Mr 31,000) is an amphiphilic membrane enzyme (19) with an amino-terminal hydrophobic peptide attaching it to the membrane (13). The membrane enzyme can be purified in the presence of Triton X-100 as an active, monomeric, and lipid- free form that has a sedimentation coefficient of 2.1 S and contains about 100 molecules of the detergent bound to the hydrophobic segment. The hydrophilic domain (Mr 29,500), possessing the enzyme activity, can be isolated after trypsin digestion of the membrane penicillinase (20). The 29,500 Mr 34 fragment is soluble as a monomer in aqueous solutions in the AM absence of detergent. When the detergent-solubilized enzyme was sedimented into Al a detergent-free sucrose gradient, protein complexes were formed which were recovered after 30 hr of centrifugation at Av 195,000 X g as a peak in the middle of the gradient (Fig. 1). The A, --- 216. Vff - ".1a complexes obtained had a sedimentation coefficient of 18 S, and 0 -9.G *--* a Stokes radius of 82 A. They contained less than 1% Triton S X-100 by weight. From the sedimentation coefficient, the Stokes radius, and the partial specific volume calculated on the basis of the amino composition, a Mr of 6.5 X 105 was ob- tained (see ref. 9). Electron microscopy after negative staining showed smooth particles with circular cross section and diam- eters in the range of 16-20 nm (Fig. 2c). c The spike glycoproteins from Semliki Forest virus have a three-chain structure containing one chain each of El (apparent Mr 49,000), E2 (Mr 52,000), and E3 (Mr 10,000) (21, 22). This glycoprotein spike monomer is attached to the membrane by a segment spanning the bilayer (23, 24). Triton X-100 solubilizes i-.01 the spike proteins from the virus as a monomer with a sedi- tl.. mentation coefficient of 4.5 S (25). When Semliki Forest virus was solubilized with Triton X-100 and centrifuged at 195,000 fit....: X g for 24 hr at 20'C through the detergent-free sucrose gra- dient, the nucleocapsid sedimented into the pellet and the spike ia.. proteins formed complexes containing eight spikes that were d virtually free of lipid and detergent (9). These have a Mr of 9.4 9%-. ..", X 105 and a sedimentation coefficient of 29 S (9). The 29S I.- .WI complexes were irregularly shaped, with small spikes projecting i4- -AW N from the periphery (Fig. 2a). The average diameter was 21 + it 2 nm. The 29S complexes remained intact if they were centri- fuged again in sucrose gradients containing 0.1% Triton X-100 or 10 mM deoxycholate (not shown). Jew '

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FIG. 2. Electron micrographs of (a) 29S complexes from Semliki 1 5 10 15 20 25 30 35 39 Forest virus, (b) mixed complexes formed from penicillinase and Fraction Semliki Forest virus glycoproteins, (c) 18S penicillinase complexes, (d) mixed complexes from penicillinase and Sendai virus glycopro- FIG. 1. Preparation of protein aggregates from membrane pen- teins, and (e) 43S complexes from Sendai virus. In d arrows indicate icillinase labeled with [35Simethionine. Sendai spikes. Downloaded by guest on September 28, 2021 5308 Biochemistry: Simons et al. Proc. Nati. Acad. Sci. USA 75 (1978) large spikes attached to a central core (Fig. 2e). The overall 2801 i diameter was about 32 ± 2 mm. If hydrophobic interactions provide the principal driving force for the formation of the membrane protein complexes, E100i it should be possible to produce mixed aggregates between E different membrane proteins. We tested this by centrifuging on penicillinase with either Semliki Forest virus or Sendai virus x in the same gradient. The membrane penicillinase was layered E onto a detergent-free sucrose gradient (Fig. 5 inset). On top of 0. this zone, two zones were layered: one containing 1% Triton 3~50 X-100 and another with the virus solubilized with Triton X-100. This arrangement was necessary to allow the faster sedimenting virus spike proteins to arrive at the same time as the more slowly sedimenting penicillinase into the detergent-free part of the gradient. Under these conditions, some membrane penicillinase cosedimented with either Semliki Forest virus or with Sendai 1 5 10 15 20 25 virus glycoproteins. Most of the penicillinase was found at the Fraction top of the gradient. The nucleocapsids of the viruses were in the FIG. 3. Preparation of spike protein aggregates from Sendai virus and labeled with [3H]leucine. Centrifugation was at 195,000 X g at 20°C pellet. If 35S-labeled membrane penicillinase 3H-labeled for 19 hr. Sedimentation towards the left. Semliki Forest virus were applied to the gradient (Fig. 5) in a protein ratio of 1:1 (wt/wt), the peak fractions 17-19 (Fig. 5) contained 21% penicillinase and 79% Semliki Forest virus spike The membrane spike glycoproteins of Sendai virus contain protein. If the ratio was 4:1, the peak fractions contained 43% three glycosylated polypeptides, HN, F1, and F2, with apparent penicillinase and 57% virus protein. If the virus and the peni- Mr of 69,000, 51,000, and 15,000, respectively (15, 26). The cillinase were mixed together in Triton X-100 and applied to subunit structure of the spikes is not known (12). When Sendai the gradient, no mixed complexes were formed. virus was solubilized with Triton X-100 and centrifuged into To prove that the complexes contained both protein species, a detergent-free sucrose gradient, a complex of the virus spike we used centrifugation in sucrose density gradients (5-20%), glycoproteins was formed (Fig. 3). The nucleocapsids and the electron microscopy, and antibody precipitation. In sucrose M protein were found at the bottom of the gradient (Fig. 4). The gradients the 3H and a5S labels cosedimented (Fig. 6A). The sedimentation coefficient of the Sendai virus glycoprotein sedimentation coefficient depended on the relative amounts complex was 43 S. In Sepharose 2B this protein complex was of the two proteins. It varied between that of the pure 29S and eluted with a Kd (see ref. 18) of 0.32, ahead of the 29S Semliki 18S complexes. The morphology of the complexes depended Forest virus complexes (Stokes radius 80 A), which had a Kd Of on which protein was predominant. Semliki Forest virus spikes 0.49. Electron micrographs of the Sendai virus and Semliki could be seen projecting from typical smooth-surfaced peni- Forest virus spike complexes showed differences in size and cillinase complexes (Fig. 2b). Moreover, antibody precipitations structure. The Sendai complexes had a radial arrangement of confirmed that the complexes contained both proteins. With antiserum against Semliki Forest virus spike protein, both spike protein and penicillinase were precipitated. Antiserum against penicillinase also brought down both the proteins (Table 1). When Sendai virus was added to membrane penicillinase in a protein ratio of 1:1 (wt/wt), some of the 35Slabeled mem- -P brane penicillinase sedimented with the 3H-labeled Sendai virus -HN - glycoproteins (not shown). The cosedimenting fractions were 4_ NC pooled and analyzed by centrifugation in sucrose gradients. -F1 Most of the two labels cosedimented at about 20 S (Fig. 6B). NaDodSO4 gel electrophoresis showed that the fractions con- tained penicillinase and Sendai virus spike proteins (not shown). That the penicillinase and the Sendai virus glycoproteins were associated was seen in electron micrographs (Fig. 2d). One or more Sendai virus spikes could be seen as projections in particles which otherwise looked like the penicillinase 18S complexes. Precipitation with antibodies to penicillinase confirmed that the complexes contained both penicillinase and Sendai virus spike protein (Table 1). DISCUSSION

A B C Preparation of the protein aggregates is schematically depicted in Fig. 7. The proteins are first solubilized from the membrane as Triton X-100 protein complexes. Subsequent removal of the FIG. 4. NaDodSO4polyacrylamide gel electrophoresis of: (gel detergent leads to the association of the proteins to water-soluble A) fractions 5-10, (gel B) fractions 1-2, and (gel C) pellet from gra- aggregates. It is important that no are present during dient in Fig. 3. (Stain, Coomassie blue.) Proteins HN (Mr 69,000) and detergent removal; otherwise formation of protein-lipid F1 (Mr 51,000) are seen. Protein F2 is not seen because it stains poorly. structures will interfere with protein association. Sargent and The internal proteins of Sendai virus (gels B and C) are the poly- merase (P, Mr 75,000), the nucleocapsid protein (NC, Mr 60,000), and Lampen (27) have earlier shown that heterogeneous complexes the matrix protein (M, Mr 38,000). of membrane penicillinase are formed when detergent is re- Downloaded by guest on September 28, 2021 Biochemistry: Simons et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5309

500-AT

400 _e ~20-50%0 300 sucrose 20 x x E iE &1 200

5 10 15 20 25 30 35 40 45 Fraction FIG. 5. Preparation of mixed complexes from penicillinase and Semliki Forest virus glycoproteins. (Inset) On a gradient (12.4 ml) of 20-50% sucrose devoid of detergent, three zones were layered: one layer (A) of 0.3 ml of membrane penicillinase (0.3 mg) labeled with [35S]methionine. in 1% Triton X-100 and 16% sucrose, a second layer (B) of 0.15 ml of 12% sucrose in 1% Triton X-100, and a third layer (C) of 0.16 ml containing Semliki Forest virus (0.3 mg) labeled with 3H-labeled isoleucine, leucine, and valine in 3% Triton X-100. Centrifugation was in a Spinco SW40 rotor at 40,000 rpm at 20'C for 30 hr. 0, Semliki Forest virus; 0, penicillinase.

moved by gel filtration in the presence of lipids. We know from in the upper part of the sucrose gradient. This centrifugation our studies that association of lipids with proteins will take place method is probably generally applicable to the preparation of already at detergent concentrations slightly below the critical water-soluble aggregates from amphiphilic membrane proteins. micellar concentration of the detergent (unpublished). In the The optimal centrifugation times and gradient designs will vary method we used here, the lipids remained with the detergent depending on the sedimentation coefficients of both of the protein-detergent complexes and of the protein aggregates. The protein aggregates obtained resemble detergent micelles in their structure. The monomers forming the micelle are probably arranged so that the surface is polar and the interior - apolar. Hydrophobic interactions provide the driving force for their formation. If the hydrophobic domain is removed by proteolytic cleavage, micelles can no longer be formed. Little specificity seems to be involved. Membrane proteins from such E diverse sources as B. licheniformis and eukaryotic viruses can El0 Qn form mixed aggregates. So far, the membrane proteins that Um have been shown to form micelles all have fairly large hydro- philic domains outside the membrane and small hydrophobic portions in the lipid bilayer (3, 4, 6, 10). The properties of the protein aggregates probably depend on the size and shape of the hydrophobic and the hydrophilic moieties of the protein monomers. It would be surprising if membrane proteins like bacteriorhodopsin (28), which are almost fully embedded in the lipid bilayer, could self-associate to form protein micelles. ) . - Table 1. Precipitation of protein complexes with antibodies I- 0 % of material E Material Antibody added precipitated cJ

C . I- SFV spike Anti-SFV 99 ._ Ic (29S complex) Anti-Pen 12 Penicillinase Anti-SFV 8 (18S complex) Anti-Pen 64 Sendai virus spike (43S complex) Anti-Pen 3 1 5 10 15 20 25 30 SFV spike- Anti-SFV 96 of spike Fraction Pen complex* 70 of Pen FIG. 6. Sedimentation velocity centrifugation of mixed aggregates Anti-Pen 91 of spike in 13-ml, 5-20% sucrose gradients in 50 mM Tris-HCl/0.1 M NaCl, 98 of Pen pH 7.4. (A) Mixed complexes of (0) penicillinase and (0) Semliki Sendai virus spike- Anti-Pen 98 of spike Forest virus glycoproteins (fractions 17-19, Fig. 5). Centrifugation Pen complext 98 of Pen was in a Spinco SW40 rotor for 6 hr at 195,000 X g at 20°C. (B) Mixed complexes of (0) penicillinase and (0) Sendai virus glycoproteins. SFV, Semliki Forest virus; Pen, penicillinase. Centrifugation was for 3 hr at 195,000 X g at 20°C. Penicillinase was * Fractions 17-19, Fig. 5. measured enzymatically (13). t Fractions 18-26, Fig. 6B. Downloaded by guest on September 28, 2021 5310 Biochemistry: Simons et al. Proc. Natt. Acad. Sci. USA 75 (1978)

Membrane Protein --deterg en t 'P rotei n m icel les' reading of the manuscript. M.S. was supported by grants from the complexes Sigrid Juselius Foundation, Helsinki. 1. Helenius, A. & Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79. 2. Tanford, C. & Reynolds, J. (1976) Biochim. Biophys. Acta 457, 133-170. 3. Laver, W. G. & Valentine, R. C. (1969) Virology 38, 105-119. *....e NS 4. Ito, A. & Sato, R. (1968) J. Biol. Chem. 243,4922-4923. N 5. Spatz, L. & Strittmatter, P. (1971) Proc. Nati. Acad. Sci. USA 68, 1042-1046. 6. Spatz, L. & Strittmatter, P. (1973) J. Biol. Chem. 248, 793- -- L I/ 799. 7. Calabro, M. A., Katz, J. T. & Holloway, P. W. (1976) J. Biol. ,~~~~ Chem. 251,2113-2118. 8. Nelson, N. & Racker, E. (1972) J. Biol. Chem. 247, 3848- 3853. 9. Helenius, A. & von Bonsdorff, C. H. (1976) Biochim. Biophys. Acta 436,895-899. FIG. 7. Schematic presentation of protein micelle formation. 10. Kuchel, P. W., Campbell, D. G., Barclay, A. N. & Williams, A. F. (1978) Biochem. J. 169,411-417. 11. Scheid, A., Caliguiri, L. A., Compans, R. W. & Choppin, P. W. Such proteins probably cannot aggregates (1972) Virology 50,640-652. form spherical with 12. Shimizu, K., Shimizu, Y. K., Kohama, T. & Ishida, N. (1974) a surface sufficiently polar to remain soluble. Virology 62,90-101. Association of amphiphilic proteins has, in some cases, been 13. Simons, K., Sarvas, M., Garoff, H. & Helenius, A. (1978) J. Mol. observed in the presence of detergent. In sucrose solutions, Biol., in press. Triton X-100 solubilizes the Semliki Forest virus spike proteins 14. Kaariiinen, L., Simons, K. & von Bonsdorff, C. H. (1969) Ann. as monomers with 75 molecules of bound Triton X-100 (25). Med. Exp. Fenn. 47,235-248. The monomer form associates reversibly to octamers with 260 15. Gething, M. J., White, J. M. & Waterfield, M. D. (1978) Proc. molecules of bound Triton X-100 when sucrose is removed. Nati. Acad. Sci. USA 75,2737-2740. With octylglucoside, another nonionic detergent, monomers 16. Valentine, R. C., Shapiro, B. M. & Stadman, E. R. (1968) Bio- chemistry 7,2143-2152. and protein are in oligomers of the spike solubilized equilibrium 17. Martin, R. G. & Ames, B. N. (1961) J. Biol. Chem. 236, 1372- with each other (unpublished data). Protein association of this 1379. type may cause complications during the purification of 18. Laurent, T. C. & Killander, J. (1964) J. Chromatog. 14, 317- membrane proteins. The apparent size of the protein may 330. change depending on the conditions, and the protein to be 19. Sawai, T. & Lampen, J. 0. (1974) J. Biol. Chem. 249, 6288- isolated may associate with contaminating membrane proteins. 6294. It is a well-known fact that most membrane proteins are diffi- 20. Yamamoto, S. & Lampen, J. 0. (1976) J. Biol. Chem. 251, cult to isolate in high yields. 4102-4110. Protein micelles may serve a number of useful purposes in 21. Garoff, H., Simons, K. & Renkonen, 0. (1974) Virology 61, membrane research. They can be used as multivalent probes 493-504. 22. for receptors on cell surfaces. We have been able to and Ziemiecki, A. & Garoff, H. (1978) J. Mol. Biol. 122,259-269. identify 23. Utermann, G. & Simons, K. (1974) J. Mol. Biol. 85,569-587. characterize the receptors for Semliki Forest virus in the host- 24. Garoff, H. & Simons, K. (1974) Proc. Nati. Acad. Sci. USA 71, cell plasma membranes using the 29S complexes (29). The 3988-3992. protein micelles can be used for structural studies. Since the 25. Simons, K., Helenius, A. & Garoff, H. (1973) J. Mol. Biol. 80, micelles seem quite homogeneous, it may even be possible tc 119-133. crystallize them. The virus glycoprotein micelles can also be 26. Scheid, A. & Choppin, P. W. (1974) Virology 57,475-490. used as vaccines The Semliki Forest virus spike protein micelles 27. Sargent, M. G. & Lampen, J. 0. (1970) Arch. Biochem. Biophys. are very powerful immunogens and induce an efficient pro- 136, 167-177. tection of mice against the encephalitis caused by the virus 28. Henderson, R. & Unwin, P. N. T. (1975) Nature (London) 257, (unpublished data). 28-32. 29. Helenius, A., Morein, B., Fries, E., Simons, K., Robinson, P., We thank Hilkka Virta for excellent technical assistance, Talmon Schirrmacher, V., Terhorst, C. & Strominger, J. (1978) Proc. Nati. Arad for electron microscopy, and Graham Warren for a critical Acad. Sci. USA 75,3846-3850. Downloaded by guest on September 28, 2021