Eur. J. Biochem. 97, 175-181 (1979)
Trans-membrane Translocation of Proteins The Direct Transfer Model Gunnar VON HEIJNE and Clas BLOMBERG Research Group for Theoretical Biophysics, Department of Theoretical Physics, Royal Institute of Technology, Stockholm (Received December 15, 1978)
As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physicochemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans-membrane proteins. Globular proteins cannot in general penetrate biological membranes. The large number of charged groups on their surfaces forces them to stay in an aqueous environment ; specifically designed, highly hydrophobic chain segments are required if a protein is even to bind to the surface of a membrane. However, secreted and membrane-spanning proteins, the so-called ectoproteins [l 1, obviously manage to traverse membranes. A mass of experimental information relating to this process is now available [2], but the mechanisms involved are only partially understood. As far as one knows, ectoproteins are synthesized on membrane-bound ribosomes, and they thread through the membrane as they are being synthesized. Furthermore, the quasi-totality of all ectoproteins that have been sequenced to date have a transient, strongly hydrophobic N-terminal leader sequence of some 15 - 25 residues, the so-called prepiece, which apparently initiates the transfer process and which is proteolytically removed during or shortly after synthesis of the protein. Although most experiments have been performed on eucaryotic systems, it now seems that essentially identical events take place in procaryotes [3]. These observations led to the formulation of the ‘signal hypothesis’ [4], where it is assumed that the prepiece, once it has emerged from the ribosome, is recognized by a multimeric intrinsic membrane protein which binds to the ribosome, thereby forming a tunnel through the membrane, whereupon the nascent chain is extreded through this tunnel, the N-terminal end first, cf. Fig. 2 (I). After discharge of the completed chain, the ribosome is detached from the
membrane, and the tunnel is eliminated. To date, however, no demonstration of such tunnel-forming intrinsic membrane proteins has been published. It is the purpose of this article to discuss the actual passage of the nascent chain through the membrane from a physico-chemical point of view. Specifically, a detailed model that obviates the need for postulating tunnel-forming proteins will be suggested. The model is based on some simple considerations of the energetics of protein structure, and it will be shown to give a consistent picture of the transfer process, contradicted neither by the experimental data nor by considerations of physical plausibility. RESULTS AND DISCUSSION Elements of Membrane-Ribosome Interactions
Ectoproteins are synthesized on membrane-bound ribosomes, in contrast to cytoplasmic proteins [2]. Ribosomes can be attached to the membrane in at least two ways, either directly through a salt-labile interaction (loosely bound) or indirectly via the growing nascent chain (tightly bound). The large ribosomal subunit is implicated in both kinds of interaction [5,6]. In eucaryotic cells, loosely bound ribosomes bind to specific binding sites on the membrane. These binding sites are heat-sensitive and can be destroyed by protease treatment, indicating that some protein factor(s) on the membrane mediates binding. A binding constant of about lo7 M-’ at physiological salt concentration has been measured, corresponding to a free
176
Trans-membrane Translocation of Proteins
Pre-lysozym?
I441
K
Met-Arg-Ser-Leu-Leu-I
le-Leu-Val-Leu-Cys-Phe-Leu-Pro-Leu-Ala-Ala-~u~ly~L~s-Val-Phe~~-~g~
M e t . - A s ~ ~ ~ t - A r 9 - A l a - P r - A l a ~ , L n - I l e - P h e ~ l y - P h e - L e u - ~ u - L e u - L e u - P h e - P r o ~ l yle~~-~g~s~~
L chain
Pre-prm&umin
Met-Lys-T~Val-Tkr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser~ly-Ser-~a-Phe-Ser~Arg~ly-Val
8-Lactamase
~t-Ser-Ile~,Ln..His-Phe-Arg-Val-Ala-Leu-Ile-Pro-Phe-Phe-~a-Ala-Phe-Cys-Leu-P~-Val-Phe-~a~His-Pro
phage fl
~~t-Lys-~ys-Ser.-Leu-Val-Leu-Lys-Ala-Ser-val-Ala-Val-Ala~-Leu-Val-Pro-Met-Leu-Ser-Phe-Ald~~a~lu
E. C o l i l i p p r o t e i n
I
Phe-Arglu-Thr-
ly-Asp
M e t ~ - L y s - A l ~ ~ - L y s - L e u - V a l - L e u - G l y - A l a - V a l - I l e - L e u ~ l ~ - S e r ~ - L e u - ~ u - A l a ~ l y ~ - C y s - SSer-Asner
E4104E X, L chain
~ t - A l a ~ ~ I l e - S e r - ~ u - I l e - L e u - ~ - L e u - L e u - A l a - L e u - s f f - S e r ~ l ~ - A l a - I l e - S e ~ ~ l n -val-vali Ala
Pre-conaLbumin
~ ~ t - L y s - L e u - I l e - L e u ~ s ~ - V a l - L e u - S e r - L e u ~ l ~ - I l ~ - A l a - ~ a - V ~ ~ s - P h e - ~ a ~ProAl~-Pro
P-450 LM2
~tSlu-Phe-Sff-~u-Leu-~u-Leu-Leu-~a-Phe-Leu-Ala~ly-~u-Leu-Leu-Leu-Leu-Phe-
and cytochrome P-450 LM2 from Haugen et al. [54]
energy of binding AGb = - RT log K z - 42 kJ/mol [7]. Attempts to demonstrate a similar salt-labile interaction in procaryotes have failed so far, however (see Conclusion). The ribosome loses a considerable amount of entropy upon binding to a membrane, through the loss of three rotational and three translational degrees of freedom. The contribution to the free energy of the bound state from these degrees of freedom can be calculated [S]; for a spherical molecule with molecular weight of 4 . 5 lo6 ~ (91 it amounts to about + 176 kJ/mol. The free energy provided by the interaction between the large subunit and the binding site must thus compensate for this unfavourable term by providing an interaction free energy of some - 218 kJ/mol, corresponding to a buried surface area of approximately 20 nm2, close to the values found for dimeric proteins [lo]. Since the only obvious difference between a nascent ectoprotein and a cytoplasmic protein is the hydrophobic prepiece of the former, it seems natural to assume that this prepiece in some way influences the membrane-ribosome association. The prepiece was originally thought to constitute a specific ‘signal’ that could be recognized by an appropriate protein in the membrane [4]. However, it has become increasingly clear that there is no strong sequence homology among the different prepieces known. The only conspicious common feature is that they all are highly hydrophobic, and that they carry charged N-terminal ends (Fig. 1). The natural conclusion is that the prepiece does not recognize any particular binding protein in the membrane; rather it probably associates directly with the lipophilic core of the membrane by virtue of its high hydrophobicity, just as endoproteins [l], like cytochrome bs reductase with a hydrophobic C-terminus, spontaneously associate even with protein-free membranes [ l l ] .
Even a hydrophobic residue will be transferred only with some difficulty from an aqueous to an hydrophobic environment, however [12]. This is because two hydrogen bonds (H bonds) have to be broken for each peptide unit going into the hydrophobic phase, corresponding to a fairly large increase in free energy. This will force the residues in the membrane into some conformation minimizing the number of broken H bonds, the a-helix being the natural candidate. In fact, this is the conformation that one generally observes in the membrane-bound portions of membrane-associated proteins [l 1,13,14]. Moreover, the charged N-terminus will probably not go into or through the membrane, but will remain associated with the charged inner (cytoplasmic) surface, whereas the subsequent hydrophobic segment will go easily into the lipophilic phase (see below). A similar suggestion was recently made by DiRienzo et al. [3]. We thus envisage the first steps in the transfer process in the following way. Synthesis is initiated on a free ribosome. Once a sufficient portion (see below) of the prepiece has emerged from the approximately 30-residues-long protective groove or channel on the large subunit [15], it will bind to a membrane in the appropriate a-helical conformation, this binding being reinforced by the direct interaction between the ribosome and a ribosome-binding site. The situation is now as depicted in Fig. 2 (11,111); it is the situation from which the actual extrusion process starts.
Chain Extrusion through a Membrane The interaction between the prepiece and the membrane is thus readily understood as a consequence of the hydrophobic character of the former. However, the overall amino acid composition of ectoproteins is not significantly different from that of cytoplasmic
G. von Heijne and C. Blomberg
177
J
Fig.2. Tlw f r m s f e r proccxs. ( I ) According to the signal hypothesis; ( I 1 - V I ) according to the direct transfer model. The feathered arrow indicates where the proteolytical processing of the prepiece takes place. The mRNA has been omitted for clarity
proteins [16], and it becomes a major problem to understand how charged or polar residues manage to traverse the lipophilic membrane core. One solution is of course to postulate the existence of a presumably water-filled tunnel, as is done in the signal hypothesis, but it should clearly be preferable if some less complicated process could be found. In this section we present a general outline of such a process where the nascent chain is extruded directly through the lipophilic portion of the membrane, the necessary driving force being provided by the direct, salt-labile membrane-ribosome interaction. Fig. 2 (111) shows the situation when the prepiece has just entered the membrane and the ribosome has bound to the binding site. If the next residue leaving the ribosome is hydrophilic, either of two things may happen: (a) the ribosome detaches from the binding site, thereby exposing the hydrophilic residue to the aqueous environment or (b) the hydrophilic residue is forced into the membrane (where it goes into the x-helix for the reasons discussed above) by virtue of the strong interaction between the ribosome and the binding site. The equilibrium constant between the two states depicted in Fig.2(II,III) is now much larger than lo7 M - * , since in Fig.2(11) the ribosome is already anchored to the membrane by the prepiece, i.e. it has already lost about two rotational and three translational degrees of freedom. Only one rotational degree of freedom, corresponding to about 34 kJ/mol, rerai n s to be frozen in going from state I1 to state 111 and the free energy difference between the two states is - 184 kJ/mol instead of the 42 kJ/mol gained when the ribosome was initially free. There is thus
+
~
considerable free energy available for forcing hydrophilic residues into the membrane. Moreover, once some residues begin to emerge on the extracytoplasmic side (Fig.2,IV) a kind of compensation effect is achieved since one residue always leaves the membrane as another one is forced into it. Ultimately, the protein folds up on the extracytoplasmic side, the prepiece is proteolytically removed by an enzymatic activity associated with the extracytoplasmic side of the membrane [17,18], and the protein detaches from the membrane as in Fig. 2 (V). Some proteins, the trans-membrane proteins, never detach from the membrane but fold up into native structures with hydrophilic domains on both sides of the membrane, connected by one or more hydrophobic, membrane-bound segment. Glycophorin is one such example. This is hard to understand within the framework of the signal hypothesis, unless one postulates the existence of a 'second signal' presumably being able to close the tunnel [l]. However, there is no difficulty involved in picturing the synthesis of such a protein in the context of the present model. Since the free energy available for forcing residues into the membrane is limited to about - 184 kJ/mol, situations can be envisaged where this free energy does not suffice. A highly hydrophobic segment, followed by a strongly polar or charged segment might well be enough to bring about the detachment of the ribosome from the binding site, confining the following parts of the chain to the cytoplasmic side (Fig. 2, VI).
Details and Quantijicution of'the Model In order to apply the scheme presented above to the real situation, it is necessary to develop it in some
178
detail. We start by a consideration of the free energies involved in the transfer of amino acid residues from an aqueous phase into a non-polar phase, followed by some model calculations on a few real proteins. The free energies of transfer of amino acids between an aqueous and a non-polar phase was studied by Nozaki and Tanford 1121. However, charged residues were not included in their measurements and, moreover, they measured the free energy of transfer of monomeric amino acids. Our problem here is to obtain an estimate of the free energy of transfer of a single residue in a polypeptide, from a random coil conformation in the aqueous phase to the a-helix conformation in the non-polar membrane interior, dGt,. Thus, instead of using the original Nozaki-Tanford data, we have calculated the hydrophobic contribution to AG,, from the accessible surface area values of Chothia 1191. Moreover, we have assumed that all residues are a-helical in the membrane, thus minimizing the number of broken H bonds. Each polar atom on a side chain has been assumed to form one H bond in water, this bond being broken upon transfer into the membrane. A broken H bond is assumed to increase the free energy by + 10.5 kJ/mol [20] (cf. Chothia 1211). The most difficult contribution to AGt, to estimate is that coming from a charge. Charges cannot be brought into non-polar environments without excessive increases in free energy [22]. Thus, we have assumed that a charged group must be neutralized before entering the lipophilic phase, either by adding or removing a proton. Under this assumption, the contribution from a charge can easily be calculated from the dissociation constant of the charged group 1231. These calculations are summarized in Table 1. Two minor points are worth mentioning. Proline is a strong helix-breaker 1241,since it necessarily breaks one H bond when placed in an m-helix. Its bulky side chain also introduces considerable steric strain in the structure. Model building indicates, however, that by breaking one more H bond this strain may be relieved without totally disrupting the helical structure. It is also possible that a compromise could be found between the weakening of the H bond and relieving the strain. The other point is the observation by Maxfield and Scheraga 1251 that charged, helical residues often have a neutralizing neighbouring residue of opposite charge four positions away on the helix. We thus omitted the charge contribution to AGt, when such 'Maxfield-Scheraga' pairs appeared in the proteins analyzed below. The lipophilic core of a biological membrane is about 3 nm thick [26]. Indeed, gramicidin A, a small helical polypeptide, forms an ion-penetrable transmembrane channel about 3.2 nm long 1271. Thus, the lipophilic part of a membrane should be spanned by an r-helix some 21 residues long, each residue advancing the helix 0.15 nm 1281.
Trans-membrane Translocation of Proteins Table 1. Estimated free energy dgference for ihe transfer ? f a residue .from a random coil conformation in water to an u-helical conformarion in a lipophilic phase Residue
Contributions to AG,, . -. .__ .... . hydroH bond phobic
AGtr ~~~
~~~~
charge
kJ/mol .
G ~ Y Ala Val Leu Ile Phe TYr TrP Ser Thr CYS Met Asn Gln Pro ASP Glu LY s Arg His
7.85 - 12.04 - 16.22 - 17.79 - 18.32 - 21.98 - 24.07 - 26.69 - 12.04 - 14.65 - 14.13 - 19.36 - 16.75 - 18.84 - 15.18 - 15.70 - 19.89 - 20.93 - 23.55 - 20.41 -
..- . ...
~
-
-
-
-
-
-
-
-
+ 10.50 + 10.50 + 10.50 + 10.50 + 10.50 + 10.50 + 21.00 + 21.00 + 21.00 + 21.00 + 21.00 + 10.50 + 31.50 + 21.00
~~
7.85 12.04 - 16.22 - 17.79 - 18.32 - 21.98 - 1.51 - 16.19 - 1.54 - 4.15 + 3.95 - 8.86 + 4.25 + 2.16 + 5.82 + 23.22 + 16.81 + 9.71 + 39.23 + 6.28 -
+ 12.06 -
7.58
-
~~~
-
-
+
~~
+ 17.92 + 15.70 + 20.14 + 31.28 + 5.69
This fits well with the observed lengths of the prepieces (Fig. 1). It can also be estimated that the two helices that are formed initially (Fig.2,II) should be connected by about four residues in an extended conformation. The time scale for the various elementary events involved in the transfer process is also important. The rate constant for the elementary step of helix growth is about [29] and, although the membrane probably acts to slow these steps down, this kind of event should be much faster than the typical time for adding a residue to a growing polypeptide chain, which is of the order of 0.1 - 1 s [30]. Assuming that the association between the ribosome and a binding site on the membrane is diffusion-controlled, i.e. with an association rate of about 2 . lo9 M-' s-l 1311, and using the value for the equilibrium constant given above, we find that the mean life time of the bound complex should be s (attaching the ribosome to the membrane via the nascent chain should increase the association rate and leave the dissociation rate relatively unchanged). This is also short as compared with the elongation rate, and we may tentatively assume that, to a first approximation, equilibrium may be reached between each elongation step. We are now in a position that allows us to give a rough quantitative treatment of the transfer process. The free energies of the various possible states (i.e. one state with the ribosome bound to the mem-
179
G. von Heijne and C. Blomberg
brane, and all possible states with the ribosome detached and a varying number of residues on the cytoplasmic side) can be calculated for each step in the elongation process. The hydrophobic energy gained in binding a prepiece to the membrane as in Fig. 2(II) turns out to be around - 210 kJ/mol. This just about balances the free energy lost in initiating two helices, = 40 kJ/mol, and the contribution from the lost H bonds in the connecting and terminal residues E 170 kJ/mol. Stabilization increases as more residues are brought into the membrane, and in a situation as in Fig.2(IV) the total free energy provided by the ,chain-membrane interaction usually amounts to some - 200 kJ/mol for the proteins analysed. This is enough to compensate for the five degrees of freedom lost by the ribosome when the nascent chain binds to the membrane (cf. above). In Fig.3, the free energy difference between the state with the ribosome bound to the binding site and the detached state with lowest free energy is plotted for each step in the elongation process (in order to allow for some side-chain flexibility, the value given for position i was arrived at by averaging over positions i - 1 to i + 1). A negative value means that the bound state is the most stable, i.e. that there are no residues on the cytoplasmic side of the membrane in equilibrium. Two typical results are shown. The M-104E 21 L-chain (Fig. 3A) an antibody light chain, is a secreted protein, whereas the human erythrocyte glycophorin (Fig. 3B) is a trans-membrane protein [32-341. It is clear from this figure that, within the uncertainties inherent in the calculation (see below), one can predict just the behaviour observed in vivo, i.e. that the antibody light chain should be completely extruded (i.e. secreted), and that the transfer of the glycophorin should cease when residue 85 emerges on the outside with the detachment of the ribosome from the binding site. The remaining part of the glycophorin chain should be left on the cytoplasmic side, and the protein should adjust its position in relation to the membrane so as to minimize the free energy, i.e. residues 74-95 (give or take a few residues) should span the lipophilic core. Essentially similar results have been obtained for the other proteins analysed, viz. Bacillus lichenformis and Escherichia coli p-lactamase [35 - 371, proinsulin [38], phage fl coat protein [39], E. coli lipoprotein [3] and prelysozyme [40,41]. One interesting result concerns the phage fl coat protein, which apparently, in contrast to the other proteins analysed, spontaneously inserts itself even into artificial, protein-free membranes [42]. Our calculations for this protein shows that its sequence is such that no extra free energy from the ribosome-membrane interaction should be needed to transfer the relevant parts of its chain across the membrane, provided that the prepiece is intact (Fig. 4). This is what is observed experimentally.
+ +
Fig. 3 . Fsee energy diffiwnce hetween the state w,ith the rihosonie bound to the hinding site on the membrane and the detatched state with lowest free energy, ,for each step in the elongation process. (A) M-104E 2, L chain; (B) human erythrocyte glycophorin. A negative value means that the bound state is the most stable, and a value below the broken line at - 184 kJ/mol means that the free energy of the state with the ribosome bound to the binding site is less in that step than in any previous step. Step one refers to the situation when the first residue in the sequence (the prepiece not included) has just emerged on the outide. The glycophorin is assumed t o have a prepiece similar t o those in Fig. 1 I
'
-300 0
I
10
I
I
I
I
20 30 Step number
I
I
40
Fig. 4. Free energ]. for ('uch step in the trunsfkr of the completed phage f l coat pse-protein across a memhraizr. No membrane-ribosome interaction energy is included. Step i refers to the situation when residue i has just entered the lipophilic phase, and step zero refers to a situation with only the prepiece bound to the membrane. The helix-initiation free energy is assumed to be + 210 kJ/mol (see text). The minimum corresponds to the equilibrium state, with the protein partly extruded through the membrane
180
CONCLUSIOT\r’ The main motivation behind the undertaking of this work was the desire to see if a physically and biologically plausible model for the transfer process could be formulated without having to postulate any tunnel-forming intrinsic membrane protein. In a sense, postulating the existence of such a protein does not really solve the problem, since mechanisms both for the recognition of the signal and for the actual extrusion would have to be found that would not obstruct the transfer through the tunnel. Moreover, the tunnel-protein idea is unsatisfactory from an evolutionary point of view, since it apparently implies that primitive cells devoid of this protein should have been unable to produce ectoprotcins, the hypothetical tunnel protein itself probably Iiitving to be an ectoprotein. The direct transfer mechanism developed in this article rests on two basic assumptions, namely that the hydrophobic prepiece will bind in an a-helical conformation to the lipophilic core of any membrane, and that the subsequent binding of the ribosome to a binding site on the membrane (possibly a ribophorin [43,44]) will force even strongly hydrophilic residues into the lipophilic phase. If these assumptions are correct, i.e. if the nascent chain does indeed emerge from the large ribosomal subunit close to the part that interacts with the membraneous binding site, it also explains, at least in part, why ribosomes synthesizing cytoplasmic proteins do not bind to membranes, since in this case the emerging nascent chain would not go into the membrane in the first place and hence obstruct the binding of the ribosome. In addition to this effect, specific detachment factors may be involved [45]. The quantitative estimates of the energetics of the process indicate that not only can this process be used to secrete proteins, but that it can also be used to insert trans-membrane proteins. In this last case, the hydrophobic membrane-spanning sequence must be followed by a strongly hydrophilic part in order to detach the ribosome from the binding site on the membrane. Another prepiece-like, strongly hydrophobic sequence in the part of the protein being left on the cytoplasmic side could conceivably initiate a new round of transfer, thereby allowing for the synthesis and insertion of trans-membrane proteins with more than one membrane-spanning segment, such as the purple membrane protein [14]. The Semliki Forest virus membrane glycoproteins are perhaps inserted in a similar manner [46]. Moreover, the direct transfer mechanism seems reasonable from an evolutionary point of view, since trans-membrane proteins with particular sequences, such as the phage fl coat protein discussed above, could have been inserted even before specific binding sites on the membrane had evolved.
Trans-membrane Translocation of Proteins
In this context, it is important to consider the uncertainties in the quantitative estimates, since the fate of a particular protein will depend on whether or not the direct membrane-ribosome interaction will be strong enough to ensure complete transfer through the membrane. As was stressed above, the most uncertain values in Table 1 are those for charged residues, due to the lack of experimental data. Apart from this, we believe that we have somewhat overestimated the difficulties in transferring residues across the membrane since we have neglected to consider H bonds between polar side chains on the membrane-traversing helices (this would decrease the free energy of transfer for such residues). The folding of the nascent chain on the extracytoplasmic side is another factor that should serve to ‘pull’ residues through the membrane, i.e. it will tend to reduce their free energy on the extracytoplasmic side. This will hardly be important until stable domains can be formed by the extruded parts of the chain, i.e. until some 50-100 residues have emerged from the membrane (cf. Wetlaufer [47]). When such a domain has been formed, however, folding may contribute anything up to some 12 kJ/mol per residue, as estimated from the reduction in accessible surface areas upon folding [19], a fairly sizeable contribution. Stabilization through folding will also ensure that the C-terminal residues left in the membrane and on the cytoplasmic side after termination will be pulled to the extracytoplasmic side, provided that the C terminus does not carry too many charged residues. The failure to detect the tunnel proteins postulated by the signal hypothesis, as well as the physical plausibility demonstrated above for the direct transfer mechanism, supports our scheme. One clear prediction of this model is that the N-terminal prepiece residues should be found on the cytoplasmic side; this could possibly be checked experimentally. Another consequence of the model is that the hydrophobic membrane-spanning sequence in trans-membrane proteins must be followed by a stretch of highly hydrophilic, preferably charged residues. The salt-labile ribosome-membrane interaction referred to above, although well documented in eucaryotes, has so far not been demonstrated in procaryotes [48,49]. We d o not feel competent to judge whether the possibility of a direct ribosome-membrane interaction in procaryotes has thus been completely ruled out, but our feeling is that the many similarities relating to protein secretion observed between the two types of cells speak in favour of a close similarity between the underlying mechanisms. Although the suggested model can explain a number of phenomena, it is clear that more complicated schemes must be invoked to explain the transfer of proteins across double membranes such as the mitochondria1 membrane [50]. Ovalbumin, so far the
G. von Heijne and C. Blomberg
only example of a secreted protein lacking a prepiece [51], has recently been shown to compete for transfer with normal prepiece proteins [521. it stands, our scheme cannot explain this observation, but we hope to return to this question in a forthcoming paper. In any case, we submit that there is good reason to believe that the direct transfer mechanism elaborated in this should suffice to most Of the observations relating to protein secretion made so far, at least for eucaryotic cells.
REFERENCES I . Rothman, J. E. Sr Lenard, J . (1977) Science (Wash. D.C.) 195, 743 - 753. 2. Shore. G . C. Sr Tata. J. R. (1977) Biochim. Biuphys. Acta, 472, 197-236. 3. DiRienzo, J. M., Nakamurd, K. & Inouye, M. (1978) Annu. Rev. Biochcm. 47, 481 -532. 4. Blobel, G. Sr Dobberstein, B. (1975) J . Cell. B i d . 67, 835-851. 5. Sabatini, D . D., Tashiro, Y. Sr Palade, G. E. (1966) J . hfol. B i d . 19, 503 - 524. 6. Unwin, P. N . T. (1977) Nature (Land.) 269, 118-122. 7. Borgese, N.,Mok, W.. Kreibich, G. Sr Sabatini, D . D. (1974) J . Mol. B i ~ l 88, . 559 - 580. 8. Janin, J. Sr Chothia, C. (1978) Biochemistry, 17, 2943-2948. 9. Hamilton, H. G.. Pavlovee, A. Sr Peterman, H. L. (1971) Biocliemi.ctry, 10, 3424- 3427. 10. Chothia, C. & Janin, J. (1975) Narure (Lond.) 256, 705-708. 11. Mihara, K., Sato, R., Sakakibara, R. Sr Wada, H. (1978) Biochemistry, 17, 2829 - 2834. 12. Nozaki. Y. Sr Tanford. C . (1971) J . Biol. Chem. 246, 22112217. 13. Lenard, J. Sr Singer, D. J. (1 966) Proc. Nut1 Acad. Sci. U.S.A. 56. 1828-1835. 14. Henderson, R. Sr Unwin, P. N . T. (1975) Nature (Lond.) 257, ’8 - 32. 15. Malkin, L. I. Sr Rich, A. (1967) J . Mol. B i d . 26, 329-346. 16. Segrest. J. P. Sr Feldman, R. J. (1974) J . hfol. Biol. 87, 853858. 17. Jackson, R. C. & Blobel, G. (1977) Proc. “it1 Acad. Sci. U.S.A. 74, 5598-5602. 18. Nan Chang, C., Blobel, G . Sr Model, P. (1978) Proc. Nut1 Accid. Sci. U.S.A. 75, 361 -365. 19. Chothia, C . (1976) J . Mol. B i d . 105, 1 - 14. 20. Volkenstein, M. V. (1977) in Molecular Biophysics, p. 191, Academic Press, London. 21. Chothia, C. (1974) Nururc, (L,ond.) 248, 338-339. 22. Parsegian, A. (1969) Nature (Land.) 221, 844-846. 23. Bohinski, R . C . (1973) in Modern Cuncep.pt.s in Biochemistry, pp. 61 -63, Allyn and Bacon, Boston. 24. Chou. P. Sr Fasman, G. D. (1974) Biochemistry, I S , 222-245.
181 25. Maxfield, F. R. Sr Scheragd, H. A. (1975) Macromolecules, 8 , 491 -493. 26. Tanford, C. (1978) SCienc’C’ (Wash. D.C.) 200, 1012-1018. 27. Koeppe, R. E., Hodgson, K. 0. & Stryer, L. (1978) J . hfol. Biol. 121. 41 - 54. 28. Dickerson, R. E. & Geis, I . (1969) in The Structure and ~ c t i o n uf Proteins, p. 28. Harper & Row. London. 29. Edna, R. (1975) Biopolymers, 14, 2425-2428. 30. Palmiter, R. D. (1975) Cell, 4, 189- 197. 31. Daniels, F, & Alberty, R , A. (1955) in pI1y,yjcu[ Chem;,s/ry, D. 505. John Wilev Sr Sons. New York 32. Birstein; Y. Sr Schechter. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 716-720. 33. Appella, E. (1971) Proc. Nut1 Acad. Sci. U.S.A. 68, 590-594. 34. Tomita, M . & Marchesi, V. T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 2964- 2968. 35. Thatcher, D . R. (1975) Biochem. J . 147. 313-326. 36. Yamamoto, S. 91 Lampen, J. 0. (1976) Proc. Nut1 Acad. %i. U . S . A . 73. 1457-1461. 37. Sutcliffe, J . G . (1978) Proc,. Nut/ Acad. Sci. U.S.A. 75, 37373741 38. Chance, R. E., Ellis, R. M. & Bromer, W. W. (1968) Science, ( WUdl. D.C.) 161, 165-167. 39. Sugimoto, K., Sugisaki, H., Okamoto, T. Sr Takanami, M. (1977) J . Mol. Biol. 110, 487-507. 40. Jolles, J., Jauregui-Adell, J., Bernier, I. & Jolles, P. (1963) Biochim. Biopl7y.s. Acts, 78, 668 - 689. 41. Strauss, A. W., Bennett, C. D., Donohue, A. M., Rodkey. J. A. Sr Alberts, A. W. (1977) J . Biol. Clirm. 252, 6846-6855. 42. Wickner, W., Mandel, G., Zwizinski, C.. Bates, M. Sr Killick, T. (1978) PVW. Nut/ Acad. Sci. U.S.A. 75, 1754-1758. 43. Kreibich, G., Ulrich, B. L. & Sabatini, D. D. (1978) J . CPIl B i d . 77, 464-487. 44. Kreibich, G., Freienstein, C. M., Pereyra, B. N., Ulrich. B. L. Sr Sabatini, D. D. (1978) J . Cell B i d . 77, 488- 506. 45. Blobel, G. (1976) Biochem. Biuphys. Res. C‘ommun. 68, 1-7. 46. Garoff, H., Simons, K. Sr Dobberstein, B. (1978) J . M u / . B i d . 124, 587 - 600. 47. Wetlaufer, D. B. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 697 - 701. 48. Smith, W. P., Tai, P-C. & Davis, B. D. (1978) Proc. Nut1 Acud. Sci. U.S.A. 75, 814-817. 49. Smith, W. P., Tai, P-C. & Davis, B. D. (1979) Biochemistry. 18, 198-202. 50. Hallermayer, G., Zimmerman, R. & Neupert, W. (1977) Eur. J . Biochem. 81. 523 - 532. 51. Palmiter, R. D . , Gagnon, J. & Walsh, K . A. (1978) Proc. Natl Acad. Sci. U.S.A . 75, 94 - 98. 52. Lingappa, V. R.. Shields, D., Woo, S. L. C. 9i Blobel, G. (1978) J . Ci.11 Biol.79. 567 - 572. 53. Thibodeau, S. N . , Lee. D. C. Sr Palmiter. R. D. (1978) J . B i d . ( % c m 253, 3771 -3774. 54. Haugen, D. A., Armes, L. G., Yasunobu, K . T. Sr Coon, M. J. (1977) Biochem. Biophys. Res. Commun. 77, 967-973.
i.
G. von Heijne and C . Blomberg, Forskningsgruppen for Teoretisk Biofysik, Institutionen for Teoretisk Fysik, Kungliga Tekniska Hogskolan, S-100 44 Stockholm, Sweden
Trans-membrane Translocation of Proteins The Direct Transfer Model Gunnar VON HEIJNE and Clas BLOMBERG Research Group for Theoretical Biophysics, Department of Theoretical Physics, Royal Institute of Technology, Stockholm (Received December 15, 1978)
As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physicochemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans-membrane proteins. Globular proteins cannot in general penetrate biological membranes. The large number of charged groups on their surfaces forces them to stay in an aqueous environment ; specifically designed, highly hydrophobic chain segments are required if a protein is even to bind to the surface of a membrane. However, secreted and membrane-spanning proteins, the so-called ectoproteins [l 1, obviously manage to traverse membranes. A mass of experimental information relating to this process is now available [2], but the mechanisms involved are only partially understood. As far as one knows, ectoproteins are synthesized on membrane-bound ribosomes, and they thread through the membrane as they are being synthesized. Furthermore, the quasi-totality of all ectoproteins that have been sequenced to date have a transient, strongly hydrophobic N-terminal leader sequence of some 15 - 25 residues, the so-called prepiece, which apparently initiates the transfer process and which is proteolytically removed during or shortly after synthesis of the protein. Although most experiments have been performed on eucaryotic systems, it now seems that essentially identical events take place in procaryotes [3]. These observations led to the formulation of the ‘signal hypothesis’ [4], where it is assumed that the prepiece, once it has emerged from the ribosome, is recognized by a multimeric intrinsic membrane protein which binds to the ribosome, thereby forming a tunnel through the membrane, whereupon the nascent chain is extreded through this tunnel, the N-terminal end first, cf. Fig. 2 (I). After discharge of the completed chain, the ribosome is detached from the
membrane, and the tunnel is eliminated. To date, however, no demonstration of such tunnel-forming intrinsic membrane proteins has been published. It is the purpose of this article to discuss the actual passage of the nascent chain through the membrane from a physico-chemical point of view. Specifically, a detailed model that obviates the need for postulating tunnel-forming proteins will be suggested. The model is based on some simple considerations of the energetics of protein structure, and it will be shown to give a consistent picture of the transfer process, contradicted neither by the experimental data nor by considerations of physical plausibility. RESULTS AND DISCUSSION Elements of Membrane-Ribosome Interactions
Ectoproteins are synthesized on membrane-bound ribosomes, in contrast to cytoplasmic proteins [2]. Ribosomes can be attached to the membrane in at least two ways, either directly through a salt-labile interaction (loosely bound) or indirectly via the growing nascent chain (tightly bound). The large ribosomal subunit is implicated in both kinds of interaction [5,6]. In eucaryotic cells, loosely bound ribosomes bind to specific binding sites on the membrane. These binding sites are heat-sensitive and can be destroyed by protease treatment, indicating that some protein factor(s) on the membrane mediates binding. A binding constant of about lo7 M-’ at physiological salt concentration has been measured, corresponding to a free
176
Trans-membrane Translocation of Proteins
Pre-lysozym?
I441
K
Met-Arg-Ser-Leu-Leu-I
le-Leu-Val-Leu-Cys-Phe-Leu-Pro-Leu-Ala-Ala-~u~ly~L~s-Val-Phe~~-~g~
M e t . - A s ~ ~ ~ t - A r 9 - A l a - P r - A l a ~ , L n - I l e - P h e ~ l y - P h e - L e u - ~ u - L e u - L e u - P h e - P r o ~ l yle~~-~g~s~~
L chain
Pre-prm&umin
Met-Lys-T~Val-Tkr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser~ly-Ser-~a-Phe-Ser~Arg~ly-Val
8-Lactamase
~t-Ser-Ile~,Ln..His-Phe-Arg-Val-Ala-Leu-Ile-Pro-Phe-Phe-~a-Ala-Phe-Cys-Leu-P~-Val-Phe-~a~His-Pro
phage fl
~~t-Lys-~ys-Ser.-Leu-Val-Leu-Lys-Ala-Ser-val-Ala-Val-Ala~-Leu-Val-Pro-Met-Leu-Ser-Phe-Ald~~a~lu
E. C o l i l i p p r o t e i n
I
Phe-Arglu-Thr-
ly-Asp
M e t ~ - L y s - A l ~ ~ - L y s - L e u - V a l - L e u - G l y - A l a - V a l - I l e - L e u ~ l ~ - S e r ~ - L e u - ~ u - A l a ~ l y ~ - C y s - SSer-Asner
E4104E X, L chain
~ t - A l a ~ ~ I l e - S e r - ~ u - I l e - L e u - ~ - L e u - L e u - A l a - L e u - s f f - S e r ~ l ~ - A l a - I l e - S e ~ ~ l n -val-vali Ala
Pre-conaLbumin
~ ~ t - L y s - L e u - I l e - L e u ~ s ~ - V a l - L e u - S e r - L e u ~ l ~ - I l ~ - A l a - ~ a - V ~ ~ s - P h e - ~ a ~ProAl~-Pro
P-450 LM2
~tSlu-Phe-Sff-~u-Leu-~u-Leu-Leu-~a-Phe-Leu-Ala~ly-~u-Leu-Leu-Leu-Leu-Phe-
and cytochrome P-450 LM2 from Haugen et al. [54]
energy of binding AGb = - RT log K z - 42 kJ/mol [7]. Attempts to demonstrate a similar salt-labile interaction in procaryotes have failed so far, however (see Conclusion). The ribosome loses a considerable amount of entropy upon binding to a membrane, through the loss of three rotational and three translational degrees of freedom. The contribution to the free energy of the bound state from these degrees of freedom can be calculated [S]; for a spherical molecule with molecular weight of 4 . 5 lo6 ~ (91 it amounts to about + 176 kJ/mol. The free energy provided by the interaction between the large subunit and the binding site must thus compensate for this unfavourable term by providing an interaction free energy of some - 218 kJ/mol, corresponding to a buried surface area of approximately 20 nm2, close to the values found for dimeric proteins [lo]. Since the only obvious difference between a nascent ectoprotein and a cytoplasmic protein is the hydrophobic prepiece of the former, it seems natural to assume that this prepiece in some way influences the membrane-ribosome association. The prepiece was originally thought to constitute a specific ‘signal’ that could be recognized by an appropriate protein in the membrane [4]. However, it has become increasingly clear that there is no strong sequence homology among the different prepieces known. The only conspicious common feature is that they all are highly hydrophobic, and that they carry charged N-terminal ends (Fig. 1). The natural conclusion is that the prepiece does not recognize any particular binding protein in the membrane; rather it probably associates directly with the lipophilic core of the membrane by virtue of its high hydrophobicity, just as endoproteins [l], like cytochrome bs reductase with a hydrophobic C-terminus, spontaneously associate even with protein-free membranes [ l l ] .
Even a hydrophobic residue will be transferred only with some difficulty from an aqueous to an hydrophobic environment, however [12]. This is because two hydrogen bonds (H bonds) have to be broken for each peptide unit going into the hydrophobic phase, corresponding to a fairly large increase in free energy. This will force the residues in the membrane into some conformation minimizing the number of broken H bonds, the a-helix being the natural candidate. In fact, this is the conformation that one generally observes in the membrane-bound portions of membrane-associated proteins [l 1,13,14]. Moreover, the charged N-terminus will probably not go into or through the membrane, but will remain associated with the charged inner (cytoplasmic) surface, whereas the subsequent hydrophobic segment will go easily into the lipophilic phase (see below). A similar suggestion was recently made by DiRienzo et al. [3]. We thus envisage the first steps in the transfer process in the following way. Synthesis is initiated on a free ribosome. Once a sufficient portion (see below) of the prepiece has emerged from the approximately 30-residues-long protective groove or channel on the large subunit [15], it will bind to a membrane in the appropriate a-helical conformation, this binding being reinforced by the direct interaction between the ribosome and a ribosome-binding site. The situation is now as depicted in Fig. 2 (11,111); it is the situation from which the actual extrusion process starts.
Chain Extrusion through a Membrane The interaction between the prepiece and the membrane is thus readily understood as a consequence of the hydrophobic character of the former. However, the overall amino acid composition of ectoproteins is not significantly different from that of cytoplasmic
G. von Heijne and C. Blomberg
177
J
Fig.2. Tlw f r m s f e r proccxs. ( I ) According to the signal hypothesis; ( I 1 - V I ) according to the direct transfer model. The feathered arrow indicates where the proteolytical processing of the prepiece takes place. The mRNA has been omitted for clarity
proteins [16], and it becomes a major problem to understand how charged or polar residues manage to traverse the lipophilic membrane core. One solution is of course to postulate the existence of a presumably water-filled tunnel, as is done in the signal hypothesis, but it should clearly be preferable if some less complicated process could be found. In this section we present a general outline of such a process where the nascent chain is extruded directly through the lipophilic portion of the membrane, the necessary driving force being provided by the direct, salt-labile membrane-ribosome interaction. Fig. 2 (111) shows the situation when the prepiece has just entered the membrane and the ribosome has bound to the binding site. If the next residue leaving the ribosome is hydrophilic, either of two things may happen: (a) the ribosome detaches from the binding site, thereby exposing the hydrophilic residue to the aqueous environment or (b) the hydrophilic residue is forced into the membrane (where it goes into the x-helix for the reasons discussed above) by virtue of the strong interaction between the ribosome and the binding site. The equilibrium constant between the two states depicted in Fig.2(II,III) is now much larger than lo7 M - * , since in Fig.2(11) the ribosome is already anchored to the membrane by the prepiece, i.e. it has already lost about two rotational and three translational degrees of freedom. Only one rotational degree of freedom, corresponding to about 34 kJ/mol, rerai n s to be frozen in going from state I1 to state 111 and the free energy difference between the two states is - 184 kJ/mol instead of the 42 kJ/mol gained when the ribosome was initially free. There is thus
+
~
considerable free energy available for forcing hydrophilic residues into the membrane. Moreover, once some residues begin to emerge on the extracytoplasmic side (Fig.2,IV) a kind of compensation effect is achieved since one residue always leaves the membrane as another one is forced into it. Ultimately, the protein folds up on the extracytoplasmic side, the prepiece is proteolytically removed by an enzymatic activity associated with the extracytoplasmic side of the membrane [17,18], and the protein detaches from the membrane as in Fig. 2 (V). Some proteins, the trans-membrane proteins, never detach from the membrane but fold up into native structures with hydrophilic domains on both sides of the membrane, connected by one or more hydrophobic, membrane-bound segment. Glycophorin is one such example. This is hard to understand within the framework of the signal hypothesis, unless one postulates the existence of a 'second signal' presumably being able to close the tunnel [l]. However, there is no difficulty involved in picturing the synthesis of such a protein in the context of the present model. Since the free energy available for forcing residues into the membrane is limited to about - 184 kJ/mol, situations can be envisaged where this free energy does not suffice. A highly hydrophobic segment, followed by a strongly polar or charged segment might well be enough to bring about the detachment of the ribosome from the binding site, confining the following parts of the chain to the cytoplasmic side (Fig. 2, VI).
Details and Quantijicution of'the Model In order to apply the scheme presented above to the real situation, it is necessary to develop it in some
178
detail. We start by a consideration of the free energies involved in the transfer of amino acid residues from an aqueous phase into a non-polar phase, followed by some model calculations on a few real proteins. The free energies of transfer of amino acids between an aqueous and a non-polar phase was studied by Nozaki and Tanford 1121. However, charged residues were not included in their measurements and, moreover, they measured the free energy of transfer of monomeric amino acids. Our problem here is to obtain an estimate of the free energy of transfer of a single residue in a polypeptide, from a random coil conformation in the aqueous phase to the a-helix conformation in the non-polar membrane interior, dGt,. Thus, instead of using the original Nozaki-Tanford data, we have calculated the hydrophobic contribution to AG,, from the accessible surface area values of Chothia 1191. Moreover, we have assumed that all residues are a-helical in the membrane, thus minimizing the number of broken H bonds. Each polar atom on a side chain has been assumed to form one H bond in water, this bond being broken upon transfer into the membrane. A broken H bond is assumed to increase the free energy by + 10.5 kJ/mol [20] (cf. Chothia 1211). The most difficult contribution to AGt, to estimate is that coming from a charge. Charges cannot be brought into non-polar environments without excessive increases in free energy [22]. Thus, we have assumed that a charged group must be neutralized before entering the lipophilic phase, either by adding or removing a proton. Under this assumption, the contribution from a charge can easily be calculated from the dissociation constant of the charged group 1231. These calculations are summarized in Table 1. Two minor points are worth mentioning. Proline is a strong helix-breaker 1241,since it necessarily breaks one H bond when placed in an m-helix. Its bulky side chain also introduces considerable steric strain in the structure. Model building indicates, however, that by breaking one more H bond this strain may be relieved without totally disrupting the helical structure. It is also possible that a compromise could be found between the weakening of the H bond and relieving the strain. The other point is the observation by Maxfield and Scheraga 1251 that charged, helical residues often have a neutralizing neighbouring residue of opposite charge four positions away on the helix. We thus omitted the charge contribution to AGt, when such 'Maxfield-Scheraga' pairs appeared in the proteins analyzed below. The lipophilic core of a biological membrane is about 3 nm thick [26]. Indeed, gramicidin A, a small helical polypeptide, forms an ion-penetrable transmembrane channel about 3.2 nm long 1271. Thus, the lipophilic part of a membrane should be spanned by an r-helix some 21 residues long, each residue advancing the helix 0.15 nm 1281.
Trans-membrane Translocation of Proteins Table 1. Estimated free energy dgference for ihe transfer ? f a residue .from a random coil conformation in water to an u-helical conformarion in a lipophilic phase Residue
Contributions to AG,, . -. .__ .... . hydroH bond phobic
AGtr ~~~
~~~~
charge
kJ/mol .
G ~ Y Ala Val Leu Ile Phe TYr TrP Ser Thr CYS Met Asn Gln Pro ASP Glu LY s Arg His
7.85 - 12.04 - 16.22 - 17.79 - 18.32 - 21.98 - 24.07 - 26.69 - 12.04 - 14.65 - 14.13 - 19.36 - 16.75 - 18.84 - 15.18 - 15.70 - 19.89 - 20.93 - 23.55 - 20.41 -
..- . ...
~
-
-
-
-
-
-
-
-
+ 10.50 + 10.50 + 10.50 + 10.50 + 10.50 + 10.50 + 21.00 + 21.00 + 21.00 + 21.00 + 21.00 + 10.50 + 31.50 + 21.00
~~
7.85 12.04 - 16.22 - 17.79 - 18.32 - 21.98 - 1.51 - 16.19 - 1.54 - 4.15 + 3.95 - 8.86 + 4.25 + 2.16 + 5.82 + 23.22 + 16.81 + 9.71 + 39.23 + 6.28 -
+ 12.06 -
7.58
-
~~~
-
-
+
~~
+ 17.92 + 15.70 + 20.14 + 31.28 + 5.69
This fits well with the observed lengths of the prepieces (Fig. 1). It can also be estimated that the two helices that are formed initially (Fig.2,II) should be connected by about four residues in an extended conformation. The time scale for the various elementary events involved in the transfer process is also important. The rate constant for the elementary step of helix growth is about [29] and, although the membrane probably acts to slow these steps down, this kind of event should be much faster than the typical time for adding a residue to a growing polypeptide chain, which is of the order of 0.1 - 1 s [30]. Assuming that the association between the ribosome and a binding site on the membrane is diffusion-controlled, i.e. with an association rate of about 2 . lo9 M-' s-l 1311, and using the value for the equilibrium constant given above, we find that the mean life time of the bound complex should be s (attaching the ribosome to the membrane via the nascent chain should increase the association rate and leave the dissociation rate relatively unchanged). This is also short as compared with the elongation rate, and we may tentatively assume that, to a first approximation, equilibrium may be reached between each elongation step. We are now in a position that allows us to give a rough quantitative treatment of the transfer process. The free energies of the various possible states (i.e. one state with the ribosome bound to the mem-
179
G. von Heijne and C. Blomberg
brane, and all possible states with the ribosome detached and a varying number of residues on the cytoplasmic side) can be calculated for each step in the elongation process. The hydrophobic energy gained in binding a prepiece to the membrane as in Fig. 2(II) turns out to be around - 210 kJ/mol. This just about balances the free energy lost in initiating two helices, = 40 kJ/mol, and the contribution from the lost H bonds in the connecting and terminal residues E 170 kJ/mol. Stabilization increases as more residues are brought into the membrane, and in a situation as in Fig.2(IV) the total free energy provided by the ,chain-membrane interaction usually amounts to some - 200 kJ/mol for the proteins analysed. This is enough to compensate for the five degrees of freedom lost by the ribosome when the nascent chain binds to the membrane (cf. above). In Fig.3, the free energy difference between the state with the ribosome bound to the binding site and the detached state with lowest free energy is plotted for each step in the elongation process (in order to allow for some side-chain flexibility, the value given for position i was arrived at by averaging over positions i - 1 to i + 1). A negative value means that the bound state is the most stable, i.e. that there are no residues on the cytoplasmic side of the membrane in equilibrium. Two typical results are shown. The M-104E 21 L-chain (Fig. 3A) an antibody light chain, is a secreted protein, whereas the human erythrocyte glycophorin (Fig. 3B) is a trans-membrane protein [32-341. It is clear from this figure that, within the uncertainties inherent in the calculation (see below), one can predict just the behaviour observed in vivo, i.e. that the antibody light chain should be completely extruded (i.e. secreted), and that the transfer of the glycophorin should cease when residue 85 emerges on the outside with the detachment of the ribosome from the binding site. The remaining part of the glycophorin chain should be left on the cytoplasmic side, and the protein should adjust its position in relation to the membrane so as to minimize the free energy, i.e. residues 74-95 (give or take a few residues) should span the lipophilic core. Essentially similar results have been obtained for the other proteins analysed, viz. Bacillus lichenformis and Escherichia coli p-lactamase [35 - 371, proinsulin [38], phage fl coat protein [39], E. coli lipoprotein [3] and prelysozyme [40,41]. One interesting result concerns the phage fl coat protein, which apparently, in contrast to the other proteins analysed, spontaneously inserts itself even into artificial, protein-free membranes [42]. Our calculations for this protein shows that its sequence is such that no extra free energy from the ribosome-membrane interaction should be needed to transfer the relevant parts of its chain across the membrane, provided that the prepiece is intact (Fig. 4). This is what is observed experimentally.
+ +
Fig. 3 . Fsee energy diffiwnce hetween the state w,ith the rihosonie bound to the hinding site on the membrane and the detatched state with lowest free energy, ,for each step in the elongation process. (A) M-104E 2, L chain; (B) human erythrocyte glycophorin. A negative value means that the bound state is the most stable, and a value below the broken line at - 184 kJ/mol means that the free energy of the state with the ribosome bound to the binding site is less in that step than in any previous step. Step one refers to the situation when the first residue in the sequence (the prepiece not included) has just emerged on the outide. The glycophorin is assumed t o have a prepiece similar t o those in Fig. 1 I
'
-300 0
I
10
I
I
I
I
20 30 Step number
I
I
40
Fig. 4. Free energ]. for ('uch step in the trunsfkr of the completed phage f l coat pse-protein across a memhraizr. No membrane-ribosome interaction energy is included. Step i refers to the situation when residue i has just entered the lipophilic phase, and step zero refers to a situation with only the prepiece bound to the membrane. The helix-initiation free energy is assumed to be + 210 kJ/mol (see text). The minimum corresponds to the equilibrium state, with the protein partly extruded through the membrane
180
CONCLUSIOT\r’ The main motivation behind the undertaking of this work was the desire to see if a physically and biologically plausible model for the transfer process could be formulated without having to postulate any tunnel-forming intrinsic membrane protein. In a sense, postulating the existence of such a protein does not really solve the problem, since mechanisms both for the recognition of the signal and for the actual extrusion would have to be found that would not obstruct the transfer through the tunnel. Moreover, the tunnel-protein idea is unsatisfactory from an evolutionary point of view, since it apparently implies that primitive cells devoid of this protein should have been unable to produce ectoprotcins, the hypothetical tunnel protein itself probably Iiitving to be an ectoprotein. The direct transfer mechanism developed in this article rests on two basic assumptions, namely that the hydrophobic prepiece will bind in an a-helical conformation to the lipophilic core of any membrane, and that the subsequent binding of the ribosome to a binding site on the membrane (possibly a ribophorin [43,44]) will force even strongly hydrophilic residues into the lipophilic phase. If these assumptions are correct, i.e. if the nascent chain does indeed emerge from the large ribosomal subunit close to the part that interacts with the membraneous binding site, it also explains, at least in part, why ribosomes synthesizing cytoplasmic proteins do not bind to membranes, since in this case the emerging nascent chain would not go into the membrane in the first place and hence obstruct the binding of the ribosome. In addition to this effect, specific detachment factors may be involved [45]. The quantitative estimates of the energetics of the process indicate that not only can this process be used to secrete proteins, but that it can also be used to insert trans-membrane proteins. In this last case, the hydrophobic membrane-spanning sequence must be followed by a strongly hydrophilic part in order to detach the ribosome from the binding site on the membrane. Another prepiece-like, strongly hydrophobic sequence in the part of the protein being left on the cytoplasmic side could conceivably initiate a new round of transfer, thereby allowing for the synthesis and insertion of trans-membrane proteins with more than one membrane-spanning segment, such as the purple membrane protein [14]. The Semliki Forest virus membrane glycoproteins are perhaps inserted in a similar manner [46]. Moreover, the direct transfer mechanism seems reasonable from an evolutionary point of view, since trans-membrane proteins with particular sequences, such as the phage fl coat protein discussed above, could have been inserted even before specific binding sites on the membrane had evolved.
Trans-membrane Translocation of Proteins
In this context, it is important to consider the uncertainties in the quantitative estimates, since the fate of a particular protein will depend on whether or not the direct membrane-ribosome interaction will be strong enough to ensure complete transfer through the membrane. As was stressed above, the most uncertain values in Table 1 are those for charged residues, due to the lack of experimental data. Apart from this, we believe that we have somewhat overestimated the difficulties in transferring residues across the membrane since we have neglected to consider H bonds between polar side chains on the membrane-traversing helices (this would decrease the free energy of transfer for such residues). The folding of the nascent chain on the extracytoplasmic side is another factor that should serve to ‘pull’ residues through the membrane, i.e. it will tend to reduce their free energy on the extracytoplasmic side. This will hardly be important until stable domains can be formed by the extruded parts of the chain, i.e. until some 50-100 residues have emerged from the membrane (cf. Wetlaufer [47]). When such a domain has been formed, however, folding may contribute anything up to some 12 kJ/mol per residue, as estimated from the reduction in accessible surface areas upon folding [19], a fairly sizeable contribution. Stabilization through folding will also ensure that the C-terminal residues left in the membrane and on the cytoplasmic side after termination will be pulled to the extracytoplasmic side, provided that the C terminus does not carry too many charged residues. The failure to detect the tunnel proteins postulated by the signal hypothesis, as well as the physical plausibility demonstrated above for the direct transfer mechanism, supports our scheme. One clear prediction of this model is that the N-terminal prepiece residues should be found on the cytoplasmic side; this could possibly be checked experimentally. Another consequence of the model is that the hydrophobic membrane-spanning sequence in trans-membrane proteins must be followed by a stretch of highly hydrophilic, preferably charged residues. The salt-labile ribosome-membrane interaction referred to above, although well documented in eucaryotes, has so far not been demonstrated in procaryotes [48,49]. We d o not feel competent to judge whether the possibility of a direct ribosome-membrane interaction in procaryotes has thus been completely ruled out, but our feeling is that the many similarities relating to protein secretion observed between the two types of cells speak in favour of a close similarity between the underlying mechanisms. Although the suggested model can explain a number of phenomena, it is clear that more complicated schemes must be invoked to explain the transfer of proteins across double membranes such as the mitochondria1 membrane [50]. Ovalbumin, so far the
G. von Heijne and C. Blomberg
only example of a secreted protein lacking a prepiece [51], has recently been shown to compete for transfer with normal prepiece proteins [521. it stands, our scheme cannot explain this observation, but we hope to return to this question in a forthcoming paper. In any case, we submit that there is good reason to believe that the direct transfer mechanism elaborated in this should suffice to most Of the observations relating to protein secretion made so far, at least for eucaryotic cells.
REFERENCES I . Rothman, J. E. Sr Lenard, J . (1977) Science (Wash. D.C.) 195, 743 - 753. 2. Shore. G . C. Sr Tata. J. R. (1977) Biochim. Biuphys. Acta, 472, 197-236. 3. DiRienzo, J. M., Nakamurd, K. & Inouye, M. (1978) Annu. Rev. Biochcm. 47, 481 -532. 4. Blobel, G. Sr Dobberstein, B. (1975) J . Cell. B i d . 67, 835-851. 5. Sabatini, D . D., Tashiro, Y. Sr Palade, G. E. (1966) J . hfol. B i d . 19, 503 - 524. 6. Unwin, P. N . T. (1977) Nature (Land.) 269, 118-122. 7. Borgese, N.,Mok, W.. Kreibich, G. Sr Sabatini, D . D. (1974) J . Mol. B i ~ l 88, . 559 - 580. 8. Janin, J. Sr Chothia, C. (1978) Biochemistry, 17, 2943-2948. 9. Hamilton, H. G.. Pavlovee, A. Sr Peterman, H. L. (1971) Biocliemi.ctry, 10, 3424- 3427. 10. Chothia, C. & Janin, J. (1975) Narure (Lond.) 256, 705-708. 11. Mihara, K., Sato, R., Sakakibara, R. Sr Wada, H. (1978) Biochemistry, 17, 2829 - 2834. 12. Nozaki. Y. Sr Tanford. C . (1971) J . Biol. Chem. 246, 22112217. 13. Lenard, J. Sr Singer, D. J. (1 966) Proc. Nut1 Acad. Sci. U.S.A. 56. 1828-1835. 14. Henderson, R. Sr Unwin, P. N . T. (1975) Nature (Lond.) 257, ’8 - 32. 15. Malkin, L. I. Sr Rich, A. (1967) J . Mol. B i d . 26, 329-346. 16. Segrest. J. P. Sr Feldman, R. J. (1974) J . hfol. Biol. 87, 853858. 17. Jackson, R. C. & Blobel, G. (1977) Proc. “it1 Acad. Sci. U.S.A. 74, 5598-5602. 18. Nan Chang, C., Blobel, G . Sr Model, P. (1978) Proc. Nut1 Accid. Sci. U.S.A. 75, 361 -365. 19. Chothia, C . (1976) J . Mol. B i d . 105, 1 - 14. 20. Volkenstein, M. V. (1977) in Molecular Biophysics, p. 191, Academic Press, London. 21. Chothia, C. (1974) Nururc, (L,ond.) 248, 338-339. 22. Parsegian, A. (1969) Nature (Land.) 221, 844-846. 23. Bohinski, R . C . (1973) in Modern Cuncep.pt.s in Biochemistry, pp. 61 -63, Allyn and Bacon, Boston. 24. Chou. P. Sr Fasman, G. D. (1974) Biochemistry, I S , 222-245.
181 25. Maxfield, F. R. Sr Scheragd, H. A. (1975) Macromolecules, 8 , 491 -493. 26. Tanford, C. (1978) SCienc’C’ (Wash. D.C.) 200, 1012-1018. 27. Koeppe, R. E., Hodgson, K. 0. & Stryer, L. (1978) J . hfol. Biol. 121. 41 - 54. 28. Dickerson, R. E. & Geis, I . (1969) in The Structure and ~ c t i o n uf Proteins, p. 28. Harper & Row. London. 29. Edna, R. (1975) Biopolymers, 14, 2425-2428. 30. Palmiter, R. D. (1975) Cell, 4, 189- 197. 31. Daniels, F, & Alberty, R , A. (1955) in pI1y,yjcu[ Chem;,s/ry, D. 505. John Wilev Sr Sons. New York 32. Birstein; Y. Sr Schechter. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 716-720. 33. Appella, E. (1971) Proc. Nut1 Acad. Sci. U.S.A. 68, 590-594. 34. Tomita, M . & Marchesi, V. T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 2964- 2968. 35. Thatcher, D . R. (1975) Biochem. J . 147. 313-326. 36. Yamamoto, S. 91 Lampen, J. 0. (1976) Proc. Nut1 Acad. %i. U . S . A . 73. 1457-1461. 37. Sutcliffe, J . G . (1978) Proc,. Nut/ Acad. Sci. U.S.A. 75, 37373741 38. Chance, R. E., Ellis, R. M. & Bromer, W. W. (1968) Science, ( WUdl. D.C.) 161, 165-167. 39. Sugimoto, K., Sugisaki, H., Okamoto, T. Sr Takanami, M. (1977) J . Mol. Biol. 110, 487-507. 40. Jolles, J., Jauregui-Adell, J., Bernier, I. & Jolles, P. (1963) Biochim. Biopl7y.s. Acts, 78, 668 - 689. 41. Strauss, A. W., Bennett, C. D., Donohue, A. M., Rodkey. J. A. Sr Alberts, A. W. (1977) J . Biol. Clirm. 252, 6846-6855. 42. Wickner, W., Mandel, G., Zwizinski, C.. Bates, M. Sr Killick, T. (1978) PVW. Nut/ Acad. Sci. U.S.A. 75, 1754-1758. 43. Kreibich, G., Ulrich, B. L. & Sabatini, D. D. (1978) J . CPIl B i d . 77, 464-487. 44. Kreibich, G., Freienstein, C. M., Pereyra, B. N., Ulrich. B. L. Sr Sabatini, D. D. (1978) J . Cell B i d . 77, 488- 506. 45. Blobel, G. (1976) Biochem. Biuphys. Res. C‘ommun. 68, 1-7. 46. Garoff, H., Simons, K. Sr Dobberstein, B. (1978) J . M u / . B i d . 124, 587 - 600. 47. Wetlaufer, D. B. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 697 - 701. 48. Smith, W. P., Tai, P-C. & Davis, B. D. (1978) Proc. Nut1 Acud. Sci. U.S.A. 75, 814-817. 49. Smith, W. P., Tai, P-C. & Davis, B. D. (1979) Biochemistry. 18, 198-202. 50. Hallermayer, G., Zimmerman, R. & Neupert, W. (1977) Eur. J . Biochem. 81. 523 - 532. 51. Palmiter, R. D . , Gagnon, J. & Walsh, K . A. (1978) Proc. Natl Acad. Sci. U.S.A . 75, 94 - 98. 52. Lingappa, V. R.. Shields, D., Woo, S. L. C. 9i Blobel, G. (1978) J . Ci.11 Biol.79. 567 - 572. 53. Thibodeau, S. N . , Lee. D. C. Sr Palmiter. R. D. (1978) J . B i d . ( % c m 253, 3771 -3774. 54. Haugen, D. A., Armes, L. G., Yasunobu, K . T. Sr Coon, M. J. (1977) Biochem. Biophys. Res. Commun. 77, 967-973.
i.
G. von Heijne and C . Blomberg, Forskningsgruppen for Teoretisk Biofysik, Institutionen for Teoretisk Fysik, Kungliga Tekniska Hogskolan, S-100 44 Stockholm, Sweden
