169

Biochimica et Biophysica Acta, 471 (1977) 169--176 © Elsevier/North-Holland Biomedical Press

BBA 77856

STUDIES OF ASYMMETRIC MEMBRANE ASSEMBLY

CRAIG ZWIZINSKI and WILLIAM WICKNER

Department of Biological Chemistry and The Molecular Biology Institute, University of California at Los Angeles, Los Angeles, Calif. 90024 (U.S.A. (Received May 10th, 1977)

Summary The major capsid protein of M13 bacteriophage xs incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large ( > 6 0 0 0 h diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed.

Introduction

The asymmetric orientation of membrane components, particularly integral membrane proteins, is n o w well known [1]. The question of how this asymmetry is established is a central problem of membrane biosynthesis. One approach to this problem is to seek a simple model for membrane assembly. The (gene 8) capsid protein of the filamentous coliphage M13 is a 5280 dalton peptide with an acidic N-terminus, a basic C-terminus, and a hydrophobic center [1,2]. This protein is inserted in the host plasma membrane during infection [4--7]. Both this parental coat protein and the newly synthesized coat protein, which is also membrane-bound, are later utilized to produce progeny virus particles [7]. The parental and progeny coat proteins are oriented with their amino-termini exposed to the outer surface of the host plasma membrane [8]. It has been postulated that the basic carboxyl terminus is exposed on the

170 interior surface of the plasma membrane where it can bind the acidic viral DNA [9]. This orientation is also found when M13 coat protein assembles into phosphatidylcholine vesicles [10] prepared by the cholate-dilution technique of Racker et al. [ 11]. In this procedure, a clear, non-sedimentable solution of coat protein, lipid, and cholate is diluted below the critical micellar concentration of cholate, resulting in vesicle formation. When vesicles are prepared near the phase transition temperature {Tin) of the lipid fatty-acyl groups, the coat protein amino-terminus is found exposed on the exterior surface of the vesicle while the carboxy terminus is exposed to the interior space of the vesicle [12]. A previous communication from this laboratory [12] showed this orientation to be independent of such factors as pH, ionic strength, the identity of lipid polar head group or the presence of either polar portion of the coat protein sequence. It was suggested that orientation might be the result of hydrophobic interactions between the lipid hydrocarbon core and the hydrophobic protion of the coat protein during the assembly reaction. We now report the characterization of the molecular species participating in the vesicle-forming reaction and the kinetics of vesicle formation. The coat protein is found to exist in two general forms, a highly aggregated form and a smaller, possibly dimeric, form. The lipid is present as a single molecular weight species, probably in a mixed micellar structure with detergent. No interactions were found between lipid and protein prior to initiation of vesicle formation. Time course experiments indicated that after dilution there was a rapid incorporation of coat protein into structures in which the coat-protein C-terminal residues were resistent to proteolysis by chymotrypsin. After this reaction was complete, there was a slower formation of large (~>6000 h diameter) vesicles. Under similar conditions, the formation of closed vesicles was initially very rapid and then continued throughout the incubation period at a lower rate. We suggest that the coat protein is initially inserted asymmetrically into small vesicular structures. Initial asymmetry of the protein-lipid association is mediated by forces which are not well understood b u t which could include a graded h y d r o p h o b i c i t y of the protein's non-polar region and/or physical asymmetries in the protein or bilayer. Once the protein and lipid have formed small vesicles, fusion of these small vesicles, with preservation of protein asymmetry and internal volume, could produce large vesicles. Materials and Methods Materials. Bovine a-chymotrypsin was purchased from Sigma, phosphatidylcholine was from Calbiochem. 3H-Labelled and ~4C-labelled glucose was purchased from ICN Pharmaceuticals. [32P]¢X174 Virus and [32p]G4 phage were the generous gifts of Drs. Michael Farber, Jon Govner and Dan S. Ray of this Institute. Methods. Growth and isolation of M13 virus and purification of M13 coat protein from the virus were by previously published methods [8]. Coat protein was solubilized by incubation for 30 min at 37°C in either NaHCO3 (0.02 M, pH 8.5) with 0.92% sodium cholate, NaHCO3 (0.1 M, pH 9.5) with 2%

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Chymotrypsi! F i g . 1. A m i n o a c i d s e q u e n c e o f t h e M 1 3 c o a t p r o t e i n . L y s i n e , w h i c h w a s t r i t i u m - l a b e l e d in the c u r r e n t s t u d y , is u n d e r l i n e d . P o i n t s o f c l e a v a g e b y c h y m o t r y p s i n are i n d i c a t e d b y a r r o w s .

sodium deoxycholate or NaHCO3 (0.1 M, pH 9.5) with 1% sodium cholate. Solutions were centrifuged (0°C, 30 min, 4 4 0 0 0 × g ) to remove any undissolved material. Unless otherwise noted, all preparations contained 0.5 mg protein per ml. Dimyristoyl phosphatidylcholine dispersions were formed by sonicating 40 mg dimyristoyl phosphatidylcholine in 1 ml of 0.1 M potassium phosphate, pH 7.0 at room temperature. Vesicles were produced by the cholate dilution technique of Racker et al. [11]. Typically, 5 parts protein-cholate solution and 2 p a r t s of 40 mg/ml dimyristoyl phosphatidylcholine were mixed and incubated 30 min at room temperature. This mixture was then diluted 30 fold with 0.1 M potassium phosphate, pH 7.0 at 20°C. The diluted mixture was incubated at 20°C for the time indicated. Incorporation of coat protein into lipid complexes was followed by observing the development of resistant of the C-terminal lysines to chymotrypsin digestion as described previously [10,12]. Briefly, M13 coat protein (Fig. 1) contains 5 lysine residues, one near the N-terminus and 4 near the C-terminus of the protein. Chymotrypsin digestion releases the N-terminal lysine and three of the C-terminal lysines from the detergent-solubilized coat protein [10,13]. Upon incorporation into vesicles, the C-terminal lysines become resistant to c h y m o t r y p t i c digestion [10]. Incorporation of coat protein into large vesicles was measured by sedimentation. Results

Physical state o f the lipid. A sonicated suspension of [3H]dimyristoyl phosphatidylcholine was mixed with sodium cholate in the absence of coat protein. The suspension clarified at once. It was analyzed by gel filtration in the same buffer and detergent. The lipid eluted in a single peak with Kay , 0.5 (Fig. 2) as would be expected for a mixed micelle of cholate and lipid. Physical state o f the protein was also measured by gel filtration. Detergentsolubilized M13 coat protein was found in two states of aggregation, one excluded from the resin and one eluting in a peak with Kay, 0.5 (Fig. 3A). A greater proportion of the protein was recovered in the included peak, shown by others to be a coat protein dimer [ 14], at alkaline pH and high detergent concentrations {Fig. 3B). The excluded species (not shown) and the dimer (Fig. 3C) gave single peaks with unchanged Kay upon second filtrations. Dimeric pro-

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Fig. 3. Gel f i l t r a t i o n of M 1 3 c o a t p r o t e i n . (A), C o a t p r o t e i n (0.5 m g / m l , 1.5 - 106 c p m ) was solubilized in 0 . 5 m l o f 0 . 9 2 % s o d i u m c h o l a t e in 0 . 0 2 M N a H C O 3 , p H 8.5 as d e s c r i b e d in Materials a n d M e t h o d s . I n o r g a n i c H 3 2 p o 4 (3 • 105 c p m ) was a d d e d as a c o l u m n v o l u m e m a r k e r a n d t h e s o l u t i o n was a p p l i e d t o a 1 X 13 c m S e p h a d e x G - 1 5 0 S F c o l u m n e q u i l i b r a t e d w i t h the s a m e b u f f e r . F r a c t i o n s (0.2 m l ) w e r e coll e c t e d a n d 15-/~1 a h q u o t s w e r e a s s a y e d f o r 3H (o) a n d 3 2 p (e). (B), c o a t p r o t e i n (0.5 m g / m l , 1.5 • 105 c p m ) w a s s o l u b i l i z e d in 0 . 5 m l o f 2% s o d i u m d e o x y c h o l a t e in 0.1 M N a H C O 3 , p H 9 . 5 as d e s c r i b e d as a b o v e . 5 pl of H 3 2 p o 4 in 0.1 M p o t a s s i u m p h o s p h a t e , p H 7.0 w a s a d d e d as a c o l u m n v o l u m e m a r k e r . This was a p p l i e d to a 1 × 13 c m S e p h a d e x G - 1 5 0 S F c o l u m n e q u i l i b r a t e d w i t h 1% s o d i u m c h o l a t e in 0.1 M N a H C O 3 , p H 9.5 f r a c t i o n s (0.2 m l ) w e r e c o l l e c t e d a n d 15-pl was a s s a y e d for 3H (o) a n d 32p Co); (C), i n c l u d e d p e a k c o a t p r o t e i n was p r e p a r e d b y p o o l i n g t h e a p p r o p r i a t e f r a c t i o n s d e s c r i b e d in 3B a b o v e . ! 0 0 - p l of this s o l u t i o n (4.7 • 104 c p m ) was m i x e d w i t h 4 pl e a c h of H 3 2 p o 4 ( 1 6 0 0 c p m ) a n d 3 2 p - G 4 b a c t e r i o p h a g e ( 2 0 0 0 c p m ) a c t i n g as c o l u m n a n d v o i d v o l u m e m a r k e r s r e s p e c t i v e l y . This was a p p l i e d to a 1 X 13 c m S e p h a d e x G - 1 5 0 S F c o l u m n e q u i l i b r a t e d w i t h 1% s o d i u m c h o l a t e in 0.1 M N a H C O 3 , p H 9.5. F r a c t i o n s (0.2 m l ) w e r e c o l l e c t e d a n d a s s a y e d f o r 3H (o) a n d 3 2 p (o); (D), 50/~1 of i n c l u d e d p e a k m a t e r i a l was i n c u b a t e d f o r 30 rain a t r o o m t e m p e r a t u r e w i t h 16 pl o f d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e s o l u t i o n ( 4 0 m g / m l i n 0.1 M p o t a s s i u m p h o s p h a t e , p H 7.0). 3 2 p - G 4 a n d H 3 2 P O 4 w e r e a d d e d as a b o v e a n d the solution was a p p l i e d to a 1 X 13 c m c o l u m n e q u i l i b r a t e d w i t h five p a r t s 1% s o d i u m c h o l a t e in 0.1 M N a H C O 3 , p H 9.5 a n d 2 p a r t s 0.1 M p o t a s s i u m p h o s p h a t e , p H 7.0. F r a c t i o n s (0.2 m l ) w e r e c o l l e c t e d a n d a s s a y e d f o r 3H (o) a n d 3 2 p (o).

173

tein from the experiment shown in Fig. 3B was mixed with dimyristoyl phosphatidylcholine and again analyzed by gel filtration (Fig. 3D). There was no detectable change in the position of protein elution, indicating a failure of protein and lipid to form vesicles in the presence of high detergent concentrations. Both aggregated and dimeric species were 50--80% incorporated into vesicles upon dilution of the detergent-protein-lipid solution (data not shown). Kinetics of membrane assembly. Protection of [3H]lysyl residues from chymotrypsin was used to assay incorporation of coat protein into closed structures. Both before and after initiation of vesicle formation by dilution, aliquots were heated to 37 ° C, where coat protein will not be incorporated into vesicles [12]. Chymotrypsin digestion at 37°C was followed by measurement of nonsedimentable 3H that was included in Sephadex G150, representing lysinebearing unprotected portions of coat protein, and 3H that was either sedimentable or was nonsedimentable but excluded from the gel, representing the protected coat protein. Prior to dilution, chymotrypsin released four-fifths of the lysines (Fig. 4A), consistant with complete accessibility of coat protein [13], if21 kJ t(D

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fatty aeyl chain (lauryl or myristoyl). When this assembly occurred at temperatures which were physiological in being near the lipid fatty acyl Tin, the coat protein spanned the bilayer with the physiological orientation. At lower temperatures, that protein which was incorporated had both ends exposed to the vesicle exterior. In contrast to this pronounced effect of the physical state of the h y d r o p h o b i c phase, polar interactions seemed to have little role in orientation. We therefore proposed [12] that the asymmetry of assembly in this model reaction was governed by hydrophobie forces. In this report, we have examined in greater detail the physical state of the reactants prior to vesicle assembly and the kinetics of this assembly. After vesiele formation was initiated by dilution, there was rapid formation of internal volume (defined experimentally as a space which can trap labelled glucose). Sinee turbidity (absorbanee at 600 nm) developed over a longer period of incubation, the initial internal volume was presumably in small vesicles. Another event oceuring soon after dilution is the protection of the lysinerich C-terminus of coat protein from protease. We propose that this is a result of protein incorporation into these small vesicles during their formation. Vesicles then fuse during subsequent incubation with preservation of internal volume and protein orientation. This working hypothesis is shown schematically in Fig. 5. Such a model is, at best, an oversimplification of the assembly process. Nevertheless, understanding this "simple" asymmetric assembly may provide a foundation for studying the more complex cellular processes. Acknowledgment This work was supported by grants from the National Institutes of Health, the National Science Foundation and the National Foundation. C.Z. is a predoctoral trainee of the National Cancer Institute, DHEW Grant No. CA09056.

176

References 1 Singer, S . J . ( 1 9 7 4 ) A n n u . R e v . B i o c h i m . 4 3 , 8 0 5 - - 8 3 3 2 A s b e c k , V . F . , B e y r e u t h e r , K., K o h l e r , H . , v o n W e t t s t e i n , G. a n d B r a u n i t z e r , G. ( 1 9 6 9 ) H o p p e - S e y l e r ' s Z. P h y s i o l . C j e m . 3 5 0 , 1 0 4 7 - - 1 0 6 6 3 N a k a s h i m a , Y. a n d K o n i g s b e r g , W. ( 1 9 7 4 ) J . Mol. Biol. 8 8 , 5 9 8 - - 6 0 0 4 T r e n k n e r , E., B o n h o e f f e r , F. a n d G i e r e r , A. ( 1 9 6 7 ) B i o c h e m . B i o p h y s . Res. C o m m . 28, 9 3 2 - - 9 3 9 5 S m i l o w i t z , H . , C a r s o n , J . a n d R o b b i n s , P.W. ( 1 9 7 2 ) J. S u p r a m o l . S t r u c t . 1, 8 - - 1 8 6 M a r c o , R . , J a z w i n s k i , S.M. a n d K o r n b e r g , A. ( 1 9 7 4 ) V i r o l o g y 6 2 , 2 0 9 - - 2 2 3 7 Smilowitz, H. (1974) J. Virol. 13, 94--99 8 W i c k n e r , W., ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 7 2 , 4 7 4 9 - - 4 7 5 3 9 M a r v i n , D . A . a n d W a c h t e l , E.J. ( 1 9 7 5 ) N a t u r e 2 5 3 , 1 9 - - 2 3 1 0 W i c k n e r , W. ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci. U.S. 7 3 , 1 1 5 9 - - 1 1 6 3 11 R a c k e r , E., C h i e n , T . F . a n d K a n d r a c h , A . ( 1 9 7 5 ) F E B S L e t t . 5 7 , 1 4 - - 1 8 1 2 W i c k n e r , W., ( 1 9 7 7 ) B i o c h e m i s t r y 1 6 , 2 5 4 - - 2 5 8 1 3 W o o l f o r d , J r . , J . L . a n d W e b s t e r , R . E . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 4 3 3 3 - - 4 3 3 9 1 4 M a k i n o , S., W o o l f o r d , J r . , J . L . T a n f o r d , C. a n d W e b s t e r , R . E . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 4 3 2 7 - - 4 3 3 2 1 5 S h o r t , S . A . , K a b a c k , H . R . a n d K o h n , L . D . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 4 2 9 1 - - 4 2 9 6 1 6 E n o c h , H . G . , C a t a l a , A. a n d S t r i t t m a t t e r , P. ( 1 9 7 6 ) J . Biol. C h e m . 2 5 1 , 5 0 9 5 - - 5 1 0 3 1 7 E y t a n , G . D . , M a t h e s o n , J . J . a n d R a c k e r , E. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 6 8 3 1 - - 6 8 3 7