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Formation of Protein Micelles from Amphiphilic Membrane Proteins (Semliki Forest Virus/Sendai Virus/Membrane Glycoproteins/Penicillinase/Triton X-100) K

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Formation of Protein Micelles from Amphiphilic Membrane Proteins (Semliki Forest Virus/Sendai Virus/Membrane Glycoproteins/Penicillinase/Triton X-100) K 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 monolayer 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 lipid and detergent. 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- lipid bilayer can usually be solubilized by mild detergents 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). temperature, 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, sodium dodecyl sulfate. 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 acid 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 ' ^.~ ~ ~ ~ ~ ~ 4 ae 3: F}{ s "44k 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.
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