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On the insertion of proteins into membranes
BIOCHIMIE, 1983, 65, 325-338.
On the insertion of proteins into membranes. (Refu le 20-4-1983, acceptd le 27-4-1983).
Unitd de Programmation Moldculaire et Toxicologie Gdndtique ( C N R S L A 271, I N S E R M U 163). lnstitut Pasteur, 28, rue du Docteur-Roux, 75015 Paris.
R6sum6.
Summary.
Les donndes rdcentes concernant la structure primaire et l'interaction entre les protdines et les membranes suggkrent l'existence d e deux classes de protdines intdgraIes de membrane. Dans la premiere Classe, hi Chatne polypeptidique traverse une seule lois la membrane. Le segment transmembranaire est particuli~rement hydrophobe et est limitd du c6t~ C-terminal par plusieurs charges positives. Dans la seconde classe, la protdine est replide de fafon complexe au sein de la membrane et la seule connaissance de sa structure primaire ne permet pas de pr~dire son mode d'interaction avec la membrane.
Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins. In the first class, the polypeptide chain crosses the membrane only once. The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border. In the second class, the protein is Jolded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.
Mots-el6s :
Key-words :
Mots-el6s : prot~ines int~grales de membrane / polypeptides hydrophobes / interactions prot~ines-membrane.
Key-words : integral membrane proteins / hydrophobie polypeptides / protein-membrane interactions.
Jean-Marie CLI~MENT.
Introduction.
Membrane proteins are usually divided into two classes [1] : Peripheral membrane proteins are not deeply associated with the phospholipid bilayer and can be released from the membrane using mild treatments such as increasing the ionic strength. Integral membrane proteins cross the phospholipid bilayer at least once and can be released only through disruption of the membrane (use of detergents) or mild proteolytic treatment. Integral membrane proteins are generally very hydrophobic and are therefore difficult to handle. This probably explains why, until recently tittle was known which could explain their localization. However technical advances in the miniaturization of protein sequencing and the development of gene cloning and sequencing have given some insight into the nature of the attachment of protein to the membranes.
In this report I have gathered recent data concerning the primary structure of some integral membrane proteins and show that one can divide them into two classes which can be distinguished on the basis of their amino acid sequences. One class can be arranged to cross the membrane only once. The membrane penetrating fragment is clearly hydrophobic and, in addition to other features, I show that the parameters used by Segrest and Feldmann [2] are useful to describe this property. A second class seems to involve a complex folding inside the membrane and the analysis of the amino acid sequences rather support the model of Engelman and Zaccai [3] which accounts for the intramembrane localization and the burial of charged residues.
326
J.-M. CIdment.
+-
HI3
NI31~LDNHT
HAl
KLESMG
HA2
KLSSMG
÷
Influenza virus haemagglutinins
HA3
CSNGSLQCR . . . . .
WfQILAIYATVAGSLSLAINMAGISFWM
CSNGSLQCRICI
+-
~
÷
VILWFSFC_~SC_.FLLLAI LVGLVFI WILWISFLISCFLLCVVLLGFIM~CA
@
ELKSGYKD
WILWISFAISCFLLCVVLLGFINA
CQR GNIRCNICI
.[9,10]
HA3
ELKSGYKD
WILWISFAISCFLLCVVLLCFIMNVA
CQ~ GNIRCNICI +
. [II, I~[
÷
÷+
TALSWWQK
El
ISGGLGAFAIGAILVLVVVTCIGL
RR
E[
AI SKTS
WS~t.FALFC_f._~SSLLI IGLMIF
Semi i k i virus
E2
QYYYGL
YPAATVSAVVOdSLLALISIFASCYMLV
Forest
[13]
ACS/~ATLSTRR +
E2
HPVYT I L A V A S A T V / k M M I
GVTVAVL
+
glycoprotein Rabies virus glycoprotein VSV
IASFFF
I IGL I
+
+
IGLFLVL RVG I H L C ql'+
"1-
[ 10]
I YT
4"
--
PriSE
GLFNKS
I:~/FTTLISTIMGPIVLLMILLFGI:~IL
TGT
41- +
FCTALLS 1TALALVCTLLYL
. [ 1 5]
. . . [ 16]
4"-+
NRFVQFVKDK . . .
+
÷----++
KYKSRF I RDEKKMP
[ 17] [ 1g]
t-4-4-
VHLLKG ++
LLLV I LLLVVCLPCLLQML
+
CGNRRKM I NN5 I ELPHG I ÷÷
RRLGRT
LLLVTFLAALLGICLMLFILI
[ 19]
÷
KRSRHF ÷
um
EEGFEN
L~I'TASTF I VLFLLSLFYSTTVTLF
Iporcine
VESNSS
WWTNWVIPAISALVVSLMY
[ 20] ÷
KVK
[ 21]
MFYTSEN
I
÷
[equine I Ibovine
. .
4-
LPNWGK YVLLSAGALTALMLI I F I M TCCRRVNRSEPTQHNLRGTGR
Adenovirus E3/16 Avian sarcoma virus glycoprotein Polyoma virus middle T antigen
Cytochrome b5
.
4-'11-'11"
4-
Irrrnunoglobulin
. .
I KLKHTKKKQ
4"
[ 13]
4-4--
CACKARREC
"IF
SS
WFSSWK
[ I g]
AARSKC • • • +
MLV
..[8]
@
HA3
Forest
virus
..[7]
+
CQR GNIRCNICI
Sindbisvirus
Sindbis
..[6]
+
+
+--
+
[5]
CVKNGNk~.CTICI
~-
ELKSGYKD --
Semi i k i virus
IYQILAIYSTVASSLVLLVSLGAIS~
KLSSGYKD --
RDNVSCSICL . . [ 0 ]
-
÷
HA3
VILLYYSTAASSLAVTLMIAIFIVYMVS
[22]
- -
VDSNSS
WWTNWVIPAISAVVVALNY
RIYTAED
[ 23],
IDSNPS
~I~/'I'NWLIPAISALFVALIY
MLYTSEN
[.24]
VDSNSS
W~'N~/IPAISALIV~
RLYMAI3D
[ 25]
t Irabbit
Histocompatibility antigens
H2d-3
PP F T[3S
H2k-b
PS
YMVIVAVL GV
L~IL
GAVVAFVM
~, RRRNTGCK+÷+ ÷ [ 26t. "1-
TVSN
MATVAVL
VV
LGA
AIVTGAVVAFVM
-I-+÷
41-
KMRRRATGGK
[ 27,']
~ RRRNTGGK +++ +
[ 28 ]
4-
H2d-I
PSS
TKTNT
V l IAVP VV
LGAVVIL GAVrV~u~VM
t-÷÷
HLAB 7
PSSQST
VPVGVAGAVAVVVILLGAVVI
A
A VM
CRRKSSGGK -It-++
HLAA-I
•
.
.
VLAVVV I
GAVV I
GAVVAAVM
+
[ 29 ]
"IF
CRRKS SGGK
[ 3 0.1
FIG. 1. - - C-terminal sequences o] 27 membrane proteins. + + C h a r g e d residu.es are indicated : K (lys), R (arg), D (asp) and E (giu). Other symbols ere : W (trp), F .(pl~e), I (il;e)~ Y (tyO, L ([eu), V (vM), M (m,eO, P' (~ro); A {ala), H ~h.ils)), T (~hr), G (gly), C fcys), S (ser), Q (gila), N (nsn). M e m b r a n e penetratk~g segmen~ts are written in bold-f~ced type a~ad underEr~ed, [References are indicated in parenflleses o n the righ¢ of We figure]. B I O C H I M I E , 1983, 65, n ° 6.
On the insertion o[ proteins into membranes. 1. PROTEINS WITH A SINGLE PEPTIDE PENETRATING THE MEMBRANE. T h i s p e p t i d e m a y be l o c a t e d at d i f f e r e n t p l a c e s a l o n g the s e q u e n c e of the p r o t e i n .
1.1. C-terminal peptide. A n u m b e r of a n i m a l virus g l y c o p r o t e i n s b e l o n g to this c a t e g o r y ( v e s i c u l a r s t o m a t i t i s , rabies, s e m liki forest, m o l o n e y l e u k e m i a , i n f l u e n z a , a v i a n
327
s a r c o m a and a d e n o viruses) as w e l l as p o l y o m a m i d d l e T a n t i g e n , c y t o c h r o m e b5, i m m u n o g l o b u lins m u - m a n d h i s t o c o m p a t i b i l i t y antigens. I n m o s t cases, t h e C - t e r m i n a l p e p t i d e has b e e n s h o w n to cross t h e m e m b r a n e by c o m p a r i n g the s e q u e n c e s of d e t e r g e n t - e x t r a c t e d p r o t e i n s to t h o s e of the s a m e p r o t e i n r e l e a s e d f r o m the m e m b r a n e by a m i l d p r o t e o l y s i s . T h e s e q u e n c e s of t h e s e p e p t i d e s h a v e b e e n listed in f i g u r e 1. N o s e q u e n c e
TABLE [,
Characteristics o/ C-terminal membrane penetrating segments. (a) A.A.
(b)
(c)
(d) A.A.
(e) H.I.
(f)
(g)
~h) +
(i)
225 ? 222 221 220 2'21 221
[4] [5] [6] [7] [8] [9, 10] [11, 12:
[31] [31] [31] [31] [31] [31] [31]
26 2'8 28 24 25 25 25
2.69 2.37 2.30 3'.02 3.36 3.28 3.28
OUT OUT OUT OUT OUT OUT OUT
11 12 12 13 12 12 12
1 1 1 2 2 2 2
1 l 1 2 2 2 2
1 0 0 0 0 0 0
4,38
[1311
[32]
24
2.21
OUT
2
2
2
0
440
[141
[141
22
2.89
OUT
11
2
2
0
422
[13]
[32]
28
2.18
OUT
32
3
3
0
424 600 505
[14] [15] [16]
[14] [33, 34] --
25 30 19
2.14 3.07 2.63
OUT OUT OUT
35 2'9 47
4 8 8
3 6 3
1 2 4
196
[17]
28
2.8.2
OUT
33
4
2
3
159 180 432 597
[18] [191 [20] [21]
[36] --[36, 37]
20 20 21
2.30 2.75 2.90
OUT OUT OUT
15 17 6
6 3 3
4 3 3
2 1 0
25
2.52
OUT
3
2
2
0
Oyfto~hrome b5 poroh~ 6quin~ bo~e rabbit
133 133 133 133
[22] [23] [24] [25]
[39] [39] [39.] [391
l0 19 19 1'9
2.84 2.89 3.11 3.03
OUT OUT OUT OUT
7 7 7 7
0 l 0 1
0 1 0 1
1 2 1 2
an~iger~ H2 a-z H2 k-b H2a-x HLAB7 HLAA1
340 346 341 337 340
[26] [271 [28] [29] [30]
[40] [4'0] [40] [40] [~0]
25 23 25 22 > 20
2.60 2.30 2.46 2.05 2.30
OUT OUT OUT IN OUT
9 10 9 9 9
5 5 5 4 4
5 5 5 4 4
0 0 0 0 0
Influenza virt~s tra.emag~lutiHn~s HB HA1 HA2 HA3 HA3 HA3 HA3 Slemali'ld Forest virus E1 Siaxl~bis virus E1 SlemlNi Forest viTus E2 Sirxl~bis virus E2 VS~V gly,oopro~i~n l~abies v~rt~s gllyc~protein MILV PRI5E Adel-~ovin~s E3,/16 ASV glyooprotein Polyoma virus m~ddIe T antige~ I mmunoglobu.liin ~xm
[381
.(a) size of the wl-~le protein (in amino acids). (b) references, to sequenoe d~ta. (c) references *o data about b e localization of the segmen,t inside the membrane. (d) ler~gth of the membrane penetrati~ng segment (in amino adds). (e) hydropl~obiei~ inc[ex. (f) position towards .the S.F. triangle. (g) size of the C=termina/ following portion (in amino acids). (11) number of posi@ce e'harges in that re.glow. (i) number of co,secretive positive eharges. (j) number of negative ekmrges.
BIOCHIMIE, 1983, 65, n ° 6.
(j) __
328
].-M. Ctdment.
homology exists between peptides from non related proteins. Furthermore, in variations of the same protein (influenza virus haemagglutinins, cytochrome b5 or histocompatibility antigens), a
I
0
3O
•
•
#
t~
o
20
,al o o o
/
\ +
!
1
2
0
IO I
3
Fro. 2. - - H y d r o p h o b i c i t y diagram o] m e m b r a n e penetrating segments. T h e ami,no acid len~tlh ~of eaoh o1 t h e memJbran,e peaae~rating s e g m e n t s is plbNed a g a i n s t i~s re speeti~re h y d r o phobicity i n d e x (H.L) whilzh is capitulated as foltowed :
to each amino a~idJcorre.~o~ad.s art hydrophobicify value (trp = + 6.5, phe = + 5, lie = + 5, tyr = + 4.5, ten = + 3.5, val = + 3, rrtet = + 2.5, pro = + 1.5, ala = + 1, la~ = + 1, ~hr = 4- 0.5, eys = 0, gly = 0, 8er = 0:.5, g1~, = - - l;.a~n = - - 1.5). H.t. is the mean value of its composing amino acids. Almost all of the ,po~nUs eorre~pondilng to m-~charged pols'perptide segments from sc~lubt.e proteir~s. ~al,1 i~sfide .the tri~a~ngle (taken from [2]). The points correspondk~g t~ ~ e membrane penotra~infi segments are figured : I C-termi~nal segmenCs. 0 N-termirml ~,¢~naemts. • ir~ternal s'egme~ts. [] inter rtal seg~erdSs from bacteriotrt~.in.
+
g_. s t e a r o t h e r m o p h i
ius
:
glu, lys, arg). This fragment is clearly hydrophobic as shown in table I. Segrest and Feldmann [2] have defined and hydrophobicity index for uncharged polypeptide fragments. This index is the mean hydrophobicity value of its constituting amino acids. These values, established according to Nozaki and Tanford [41], are reported in the legend of figure 2. They are related to the free energy of transfer of the amino acids from ethanol to water. By plotting the size of the uncharged segments of soluble proteins against their hydrophobicity, Segrest and Feldmann [2] observed that all the points are clustered within a triangular space (let us call it the << SF triangle >>). In contrast, all the points obtained from the values of table I, except one, are clearly situated outside on the right of the SF triangle. This means that the membrane penetrating segments are more hydrophobic than the soluble protein fragments of equivalent size. This result fully confirms those of Segrest and Feldmann who had only four examples of membrane penetrating fragments at the time of their publication [2]. A second feature shared by the C-terminal part of this type of membrane proteins is the presence of several positively charged residues at the border of the hydrophobic part which is placed at the cytoplasmic side of the membrane. The only exception to this rule is the cytochrome b5 for which a loop model of anchoring has been proposed [25 bis]. The presence of a proline residue in the middle of the hydrophobic part is suggestive of a possible turn in the structure. We should notice the peculiar case of carboxypeptidases from B. subtiIis and B. stearothermophitus which
+
-
t-
+
• • -KAWFVLSMP,.AVGGLFVDLWTSVAKTVKGWL C(:~--~
+
B.
subtilis
:
• . . ANWFVLTMRS I GGFFAG ['~FGS [ VDTVTGWL COOH
FIG. 3. - - C-terminal s e q u e n c e s o] t w o c a r b o x y p e p t M a s e s [7]. + + C h a r g e d .reskl,~es a r e ind[.cated : K (lys.), R (arg), D (asp) a n d E (ghl). Oilier s y m b o l s
ave: W (,ta~), F (Nae), I file), Y (tyr), L (I~u), V.(vat), /~ (met), P ~ro); A (ala), H ~his), T (thr), G (gly), C (cys), Q (gin), N (asn).
greater divergence seems to occur in the C-terminal part than in the rest of the molecule which is quite well conserved. One common feature of these C-terminal portions is the presence of a long segment (> 19 residues) deprived of charges (i.e., absence of asp, BIOCHtMIE,
1983, 65, n ° 6.
are hnked to the membrane by the 25 amino acids long C-terminal part [6J. This region shows very strong hydrophobic properties although this character is not suggested by the sequence since it includes charged amino acids (figure 3) [7]. The authors have suggested a model for attachment to the membrane : the C-terminal portion
On the insertion of proteins into membranes.
329
T A B L E II.
Characteristics o[ N-terminal membrane penetrating ~ragments (a)
A.A.
Influerma v~rus neuram~izi&ase D~pep~idyl peptklase I V Isomilt~se Rhodopsiaae (bovi~e) Cy.tochro,me P 450 LM2 PB b a Etx)xtidhs"drase (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)
(b)
(c)
(a)
A.A.
(e)
H.I.
(f)
(h)
(g)
+
(i)
__
(9
(k) S.S.
461
[51
[491
28
2.78
OUT
6
t
1
0
--
1200 1300
[42] [43[
[42] [50]
9 > 2'2
o 3.39
? OUT
0 9
0 0
0 0
0 1
? ?
350
[441
[44]
27
2.46
OUT
17
0
0
t
?
500 500 500 500 500,
[451 [4g] [47] [47] [48]
? ? ? ? 9
3.06
OUT OUT OUT OUT OUT
2 2 1 3 4
0 0 0 0 0
0 0 0 0 0
1 1 I 1 1
--
> 18 > 18 18 > 20 > 15
2~.94 3.00 2.55 3.30
estimation of the size of the whole protein (in a m i n o ands). references' ~o the sequence data. referet~c~s ~c) ~ t ~ ~ u ~ V.he ,~oc~t.iz~ation of ~he fragmen:t ins:fd.e ~he membrane. size o f the membrarm pe~etradn~g f r a g m e m (in a.mino acids). hydrophobioitSr i~d~x. pos.ition totvcards ~he S.F. ,riar~gle. size o f tabe N-terrMn.al preceedil~g portion. ~umber of tX~i~i~,'e otmrges fn tNat region. n u m b e r ~of oonse~l~ve posieive charges. n u m b e r of negative oharges. when known, absence, of sig~lal sequence (S.S.) (--).
Influenza virus neuraminidase Dipeptidyl peptidase
41-
~vINPNQK
LGFAL/kFI
1V
Isomaltase Rhodopsine (bovine)
PB
L...HQGSD
retinal V FGPI~IPAFF~TSAWCNPVITIMN
FSLLLLLAFLAGLLLLLF
b MLD
Epoxidhydrase
[[5 l
[42 I
ITLIVLFVIVFIIAIALIAVLA
~
SHS1Q • • •
...
AVNAFSGLE
LM2 Cytochrome P~SO
I ITIGSICLV.VGLISLILQIGNI I 5 I W I
XNoXPAV . , ,
+
[g3]
+
KQFR . . .
[#4]
.,.
[gS]
PSILLLLALLVGFLLLLV . . .
[g6.]
PT1LLLLALLVGFLLLLV
[471
RG . . ,
TGLLLVVILATLTVMLLLTL
t'vl~%E LVLA5LLGFVIY'~V$ . . .
...
[48] [g9 ]
FIG. 4. - - N-terminal sequences o[ 9 membrane proteins. + Ohargeod re~idtles are .ill~i,ca~ed : K (lys), R (arg), D (asp) a.t~d E (glu). O ~ e r symbols are : W (trp), F (phe), I (ile), Y (tq'r), L (leu), V (val), M (met), P fWo), A (al:a), H (laSs), T (thr), G (gly), C (cys), S fser), Q (gl.~), N (ash). M e m b r a n e penetrating segments are written in bold-faced type and underlined. [References are indicated fin parentheses, on the right e£ the figurel.
BtOCHtM1E, 1983, 6.5, n ° 6.
o ? ?
J.-M. Cldment.
330
s h o u l d a d o p t a p e r i o d i c a l structure ( a l p h a helix or b e t a sheet) which is half buried p a r a l l e l to the plane of the m e m b r a n e . C h a r g e d residues would be aligned on the e m e r g i n g side of the structure. Glycophorin (human e r y t h r o c y t e s )
÷÷
. . /• AAFDSLQASAT E YIGYAW/IdglVVVlVGATIGIKLFKKFTSKAS..[53,5~,55) ÷ ÷÷ ÷ ..
Z32
FDSLQASATEY|GYAW/~VVVIYGAAIGI KLFKKFTSKAS.. %
coat
proteins
÷&
..GERVQLAHHFSEPE I T L I I F G ~ G V l G T I L L I S T G I RRLIKKSPSD..[52]
fd/Ml3/fi Phages major
a n d the h y d r o p h o b i c i t y indexes of the u n c h a r g e d p a r t are r e p o r t e d in table II. T h e s e values are again high a n d the c o r r e s p o n d i n g points are situated on the right o[ the S F triangle (fig. 2).
÷÷
[56]
÷
Ill
..A~AFDSLTAQATE MSGYAW/kLVVLVVGATVGIKLFKKFVSRAS..
[57]
Ike
. . A T E / ~ S L TQAID LISQTWPVVTTVVVAGLVI RLFKKFSSKAV,.
[37]
÷
A gacteriorhodopsin
B
-
÷
_
. . EAQITGRPE WlWLALGTALMGLGTLYFLVKGMGV5 D _P(out) ÷÷ ( i n ) . . G YGLI.MSLYMrFAIAPVLTTIAYF KKAD ÷
E
[58]
÷
• • TSTR FV~FAI 5TAAMSYILYVLFFGFTS KAFSMR
FIG. 5. - - Amino acid sequences o~ internal membrane penetrating segments oJ 6 membrane proteins. Ch.arged residues are i~ndical~ed : K f~ys), R (arg), D (~sp) and -E (gllu). Ol~her sym°ools are : W (trp), F (phe), I (tie), Y (tyr), L (l.eu), V (val), M (me0, P (pro), A (ala), H ~ s ) , T (l~hr), G (g17¢), C (cys), S (ser), Q (glrt), N ('asn'). Membrane penetrating segments are written in bold-faced type and underlined. [References are inculcated in ,parenCheses on the righ't of ~he figure]. TABLE III.
Characteristics of internal membrane penetrating segments.
(a)
Gl'y,cophorin (humar~ ory~lwocytes) Ph~ges major c~at prot~i'aa~s Bacteriorhodopsin (a) (b) (c) (d) (e) (f) (g)
fd/M,l 3/fi ZI2 I~1 lke A B E
A.A.
(b)
131 50 5,0 51 53 2*8 248 248
[52] [53, 54, ,55] [56] [57] [57] [58] [~8] [~18]
(c)
(d)
A.A.
[59] 19 [60l 19 i60] 19 160] 19 [~] 19 [61, 62] 2,0 [61, 62] 23 161, I~] 22
(e)
H.I,
(f)
2.81 2.84 2.76 2.29 2.32 2.85 2.65 2.95
OUT OUT OUT OUT OUT OUT OUT OUT
(g) + 4 4 4 4 4
size of [he 'whole pr0~ei'n (in amino acids). re:fences ~o sequeaaceJd~m. references' to d~ta eon~ern.i,n,g t~e l,ocalizat/~31aof the fragmen,t i~aside fire anembr~n,e. ler~gth of the mea~brar~e per~elar~th~gsegment (in ami~no acids). hydroph,0biqeiltyindlex. position ~owards the S.F. ,triangle. number ,of conse~a~tive posi'tive charges i.n the C-~rmbrml following region.
1.2. N-terminal fragments. V a r i o u s p r o t e i n s a n c h o r e d by their N - t e r m i n a l segment are listed in figure 4. T h e i r characteristics BIOCH1MIE, 1983, 65, n ° 6.
These sequences are often c o n s i d e r e d as signal sequences [51] which are n o t processed : in such a perspective the anchorage of the p r o t e i n to the
33I
On the insertion of proteins into membranes.
(table 111) is again high (figure 2). The charge distribution on each side of the sequence is as'fmetrical : negative charges are on the N-terminaI and positive ones on the C-terminal side of the fragment (figure 5).
membrane resembles an abortive secretion- In two instances (cytochrome P-450 and neuraminidase), the primary translational product is identical to the final one. Bacillus licheni/ormis penicillinase is synthetized as a precursor. The N-terminal end is a somewhat peculiar signal sequence [51bisl which is necessary for the protein to anchor in the membrane in a transitory step which precedes the excretion.
1.4. Conclusion. The membrane penetrating segments responsible for anchoring proteins in the membrane are deprived of charged residues and are more hydrophobic than the uncharged segments of soluble proteins. This point was postulated by Segrest and Feldmann [2] and nearly all the examples I found fulfill this assumption. As stressed by Von Heijne [68], the specific amino acid sequence is relatively less important than the overall hydro-
The analogy between signal sequences and the N-terminal segments considered here is however limited, since the hydrophobic part of the signal sequences is usually preceded by positively charged residues whereas in penetrating fragments one can usuaIIy observe a negative charge on the Nterminal side of the hydrophobic segment.
÷
~
-
MYYLKNTNFT/MFGLFFFFYFFI~A YFPVFPIWLH~INHISKSDTGIIFA t
AISLFSLLFQPLFGLLSDKLGLRKY LLW[ITC~LVMFAPFFIFIFGPLLQ -
-~
÷÷
-
+
+
YNILVGSIVC.~IYLGVCFNAGAPAV EAFIEKVSRRSNFEFGRAR3AFGCVG WALCASIVGIMFTINNQFVFWLGSG CALILAVLLFFAKTDAPS$ATVANA VGANHSAFSLKLALELFRQPKLWFL 5LYVIGVSCTYDVFk.X~FANKFTSF FATGEQGTRVFGYVTTMGELLNASI MFFAPLIINRtC_,C_,KNALLLAGTIM$ VRIIGSSFATSALEVVIL~TLI~IFE VPFLLVGCF~YITSQFEVRFSATIY ÷
LVCFCFFKQLAMIFMSVLAGN~YE5 IGFQGAYLVLGLVALGFTLISVFTL Q ÷÷
-
SGPGPLSLLRRQVNEVAg[7
Fro. 6. - - Amino acid sequence of Lac Y protein. The amino ~cid ,se~tu,enc~e is ~aken fi'orn [56].
1.3. Internal [ragments. Two cases are presented : the erythrocyte glycophorin and the major capsid protein of phage M13 or related phages (figure 5). In both instances, the portion of the protein that spans the membrane is a 19 residue-long fragment. Its hydrophobicity B I O C H t M I E , 1983, 65, 11° 6,
phobicity of such segments. Indeed, no sequence homology does exist between unrelated proteins in these regions. Moreover, for related proteins (influenza virus haemaggtutinins or HLA and H2 antigens), the variations inside the hydrophobic segment seem more extensive than in the rest of the molecule.
332
J..M. Cldment.
covalently-linked fatty acids to the membrane. A similar case is the Thy-1 glycoprotein from the rat brain whose sequence is known [65]. It seems to be linked to some unidentified lipid elements at the C-terminal end. i have also discarded the erythrocyte band-3 protein which is an anion transporter. Its structure is partially elucidated [66] but its sequence remains unknown. We shall focus on 5 prokaryotic membrane proteins : the products of the genes lacY, ompA, ompF and lamB of E. coli and the Halobacterium halobium bacteriorhodopsin.
One constant particularity of membrane penetrating segments is the occurrence of positive charges at the C-terminal end which is in most cases on the cytoplasmic side of the membrane. These charged residues could possibly "interact with the polar heads of phospholipids. So that anchoring of these proteins may be due at the same time to the presence of the hydrophobic segment in the membrane and their interaction. 2. PROTEINS BURIED IN THE MEMBRANE. 2.1. Introduction.
2.2. E. coli lactose perrnease.
The polypeptide chains of this type of protein generally cross the membrane several times and are often responsible for the passage of ions or
The lacy gene has been sequenced 1167]. The protein has been extracted and its NH2 terminus
APKBNTWYTGAKLGWSQYHI3TGFI N NNGPTHENQLGAGAFGGYQVNPYVG ) -
-
41-
4"
--
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F~YDWLGRMPYKGS VENGAYKAQ GVQLTAKLGYPI TE~t3LDI YT~LGC/d
VWRADTKSNVYGKNHDTGVS P VFAG GVF:yA1TPE" I ATRLEYQWTNNI GI3A +
-
+
-
-
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+
-
HT IGTRPDNGMLSLGVSYRFGQGEA APVVAPAPAPAPEVQTKHFTLKSDV +
4-
-
-
-
+ -
-t-
-
LFNFNKATLKPEGQAALDQLYSQL5 NLDPKDGSVVVLGYTDRI GSDAYNQ - ÷ +
-
+
-÷
+
-
_
+
+
GLSERRAQSVVDYLISKGIPADKIS ARC/v~ESNPVTGNTCDNVKQRAALI -
. + +
-
-
+
+-
DCLAPDRRvE1EVKGIKDVVTQPQA325 @ Fro. 7. - - Amino acid sequence o/OmpA protein. The amino ~eid sequence iS ,taken from [79].
hydrophilic molecules through it. The existence of a hydrophilic pore traversing the membrane suggests that charged residues are expected to be found inside the hydrophobic part of the membrane. Only few examples of sequenccs of this type of proteins are known. I have discarded the case of E. coli lipoprotein [frl], since there is no proof that this protein is really inserted inside the outer membrane. More likely, it is attached through its BIOCHIMIE, 1983, 65, n ° 6.
sequenced in the particular case of a strain carrying a plasmid containing the lacY gene [68]. This strain overproduces the protein (15 per cent o~ the total inner membrane proteins) but the transport of beta-galactosides is normal. The NH,, termini of both the protein synthetized in vivo in that strain and the in vitro product are identical and fit with the predictions of the DNA sequence. This lead the authors [68] to state that lactose perrnease is not processed in vivo although the
333
On the insertion of proteins into membranes.
NH,2 terminus much resembles typical signal sequences. This point is not really clearly determined since it has not been proved that all of the permease molecules are active in the plasmid carrying strain. Since it is overproduced, it remains possible that a few molecules are actually processed - - and active - - while the bulk is unprocessed, uncorrectly positioned in the membrane, and devoid of activity.
other E. coli outer membrane proteins, OmpA does not form any pore in the membrane. Its activity for the penetration of amino acids through the membrane is quite controversial [75, 76]. Nevertheless, the protein crosses the membrane, since it is linked to the peptidoglycan [77] and it emerges at the surface, being accessible to CNBractivated dextran [78], as well as to the phages mentioned above.
The sequence of the protein is shown in figure 6. As can be seen, several uncharged fragments are hydrophobic (they are outside the SF triangle)
The sequence of the protein is known [79] (figure 7), as well as the nucleotide sequence of the gene [80]. It has been clearly demonstrated,
--
+ -
+
-
+
+
-
-
+
+
-
AE I YNKDGNKVDLYGKAVGLHYF S K GNGEN S YC__~NGI~MTYAR LG F KGE TQ
INSSLTGY~EYNFEGNNSEGADA QTGNKTRLAFAGLKYA.DVGSFDYGR ÷ . . . . + NYGVVYI3ALGYTI~LPGFGGgTAYS DDFFVGRVC~VATYRNSNFFGLVDG -+ + ÷
- + -
++
.
LNFAVQYLGKNERDTARRSNGDGVG --
++
-
+
--
-
.
.
+
+
GSISYEYEGFGIVGAYC_.,AADRTNLQ
--
EAQPLGGNKKAEQWATGLKYD~NNI +
.
-
+
+
YLAANYGETRNATPITNKFTNTSGF ÷
+
•
.
.
.
.
.
ANKTQDVLLVALYQFDFGLRPSIAT TKSKAKDVEGIGDVDLVNYFEVGAT
YYFN~r'~STYVDY[INQIDSDN~LG
VGSDDTVAVGIVYQF3a 0
FIG. 8. - - Amino acid sequence of OmpF protein. T1ae a.mino acid sequence .is .taken from [92].
but their position with respect to the membrane remains unknown. All these fragments are situated in the N-terminal half of the protein. No obvious model for the insertion of the lactose permease is at present available. 2.3, O m p A protein of E. coll. OmpA is abundantly present in the E. coli outer membrane. It is a plurifunctional protein : receptor for the phages Kz [69], TulI * [701 ; it is necessary for the activity of colicin L [71]; it plays a role in F-dependant conjugation [72, 73], and is also necessary for the functional integrity of the membrane [74]. In contrast to some BIOCHIMIE, 1983, GS, n ° 6.
in particular by the study of the expression of plasmids containing various portions of the o m p A gene, that all the known functions of the protein (including anchoring in the membrane) are carried out by the 193 first amino acids [81] (the mature protein is 325 amino acids long). It has also been shown that a mild protease treatment generates a N-terminal fragment which is 177 residues long and retains all the OmpA functions. However a 133 residues long fragment has lost all of the activities. Examination of the protein sequence does not reveal any particular hydrophobic segment. An uncharged peptide, 19 residues long (from amino 25
334
J.-M. Cldment.
Its sequence has been determined [92] and is indicated on figure 8. The most striking character is the great number of charges : the largest uncharged segment is 11 amino acids long and none is hydrophobic. The protein itself is however extremely hydrophobic. No information is available concerning its structure. Chen et al. [92] suggested that insertion in the membrane was mediated by association with other membrane proteins. This hypothesis has not yet received any experimental support.
acids 33 to 51) is rather polar. Once again, no model is available for the in situ structure of the OmpA protein. One must imagine a complex folding drawing charges inside the hydrophobic hydrocarbon bilayer. 2.4. O m p F protein of E. coil This <>E. coli outer membrane protein belongs to the <>family [.82, 83, 84] which creates hydrophilic channels across the membrane. This has been extensively studied both in vivo [84, 85], and in vitro [82, 83]. The pores, approximately 0.9 nm diameter [86, 87], specifically facilitate the diffusion of low molecular weight hydrosoluble components. The protein is active as a trimer [87]. It crosses the membrane, linked by
2.5. L a m B protein of E. coll. L a m b is an E. cofi outer membrane protein which is inducible by maltose [93]. It is a porin somewhat different from the others since it shows
VDFHGYARSGIGWTGSGGEQQCFQT TGAQSKYI~LGNECETYAEL~LC.-QEV WI~EGfKSFYF6TNVAYSVAQQNDWF- ATDPAFRE~VQGKNL|E~LPGSTI *÷ -
WAGKRFYC~.HDVr~IDFV~VO~SGP CmL~NI~VGFG~LSLAAT~SS~AG GSSSFASNNIYDYTNETANfVFDVR LAQMEINPGGTLELGV6YG~L~(13 ÷
-
+ -
-
+
+
-
÷
NYRLVDGAS~FTAEHTQSVLK GFNKFVVQYATDSMTSQGKGLSQGS -
- +
+
.-
GVAFDNEKFAYNI NNNGFgdLR1LDH GA [ $ 1 k ~ , D ~ V C / d Y Q D I NWI3NI3 NGTKWWTVGIRPMYKWTPIMSTVME IGYDNVESQRTGDKNK~YK[TLAQQ
"~AGD S IWSRPAi RVFATYAKWDEK VCGYI~YT~NAI3NNANFG~.AVPAI3FNG
GSFG~G6S~
F ~
I~ 4 Z
FIG. 9. - - A m i n o acid s e q u e n c e of L a m B protein.
The anairro ac2d Sexluen.c~is ta&en @om [ ~ ] .
an ionic bond to the peptidoglycan [88], and emerges at the surface where it serves as receptor for various bacteriophages : Tula [69, 89], TPI [90], and T2 [91]. BIOCtth'vllE,
1983, @5, n ° 6.
a marked specificity to facilitate the diffusion of maltodextrins and maltose through the outer membrane [94]. It is a transmembrane protein since, on the one hand it is bound to the peptidogly-
335
On the insertion of proteins into membranes.
2.6. Bacteriorhodopsin.
can [95] and interacts with a periplasmic protein, M a l e [96, 97], and on the other hand it is used by several bacteriophages 198, and M. Hofnung, personal communication] as receptor for adsorption at the surface of the bacteria.
It constitutes roughly the only proteic element of the ~ purple membrane >> of Hatobacterium halobium. It is constituted of 248 amino acids with a retinal molecule covalently bound to iysine 216 [100J. The protein has been sequenced 1158, 61, 62] as well as the structural gene [101, 10~.] (figure 10). The protein is synthesized as a precursor. The structure of the i3 residues long amino-terminal extension is different from all the known prokaryotic and eukaryotic signaI sequences. The C-terminal asp which precedes the stop codon is absent from the mature protein.
The gene has been sequenced [99] revealing the protein sequence (figure 9). As in the case of OrnpA or OmpF, the charges are dispersed throughout the molecule. An hypothesis has been suggested [99] concerning the charge distribmion which is periodical. This model was based on a relatively high alpha-helix content in the secondary structure predictions for LamB. Recent phy-
. . . . . . . . .
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. . . . . . . .
MLELLPTAVEGVS QAQ{ TG~PEWIW3-ALGTAL/~LGTLYFLVKC~'vSGVSD
-
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-
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--
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-
PDAKKFYA 1 TTI_ VPA l AFTMYL SMLLGYGLTMV P FGGEQ'qP I TWARY/~Iq~LFTT P L L LLDLALL VDA
- - - -
. . . . . .
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.
.
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DQGT I LALVC_dkIX] IMI QTGLVGALTKVY SYRFVVA!/AI STAAMLT I LYVLFFGFT SKAES
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--
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MRPE V/kSTFKVLRN VT VVLWNAYPV~OIgLI GS EGAG I VPI.N I ETLLFMVLIDV,SAKVGFGL I LLRSR
AIFGF.,AEAPEPSAGE~:3AAATS D2~ $
1"to. 10. --Amino acid sequence o] Bacteriorhodopsin, "1-/~ ~m.~o acid ~equen~e is taken from [92l. T h e two. a r r o ~ s ind,kzete ~t~he Pelrtide bonds cleaved dlard0ag proce.ssiag o f the pvoteila. T,tte 7 m e m b r a n e pene~ratirtg segmezts (A to G) and Che charged amino acids internal to tthese segmen,ts (acec~d~g ,to [911) are in bold-faced type and overIdned.
sicaI studies (J.M. Neuhaus and J. Rosenbush, personal communication) reveal that actuMl7 little i~ any of the protein is helical. Once more, the structure of the protein in the membrane remains speculative. However it will be possible to define portions of the molecule which are responsible for various functions, particularly the regions accessible from the outside of the cell since mutations affecting some of the functions are being sequenced (J.M. C16ment et al., work in progress). BIOCHIMIE, 1983, 65, n ~ 6,
The bacteriorhodopsin acts as a light driven proton pump. Models for the tertiary structure have been proposed. They are based on radiocrystaUography studies of the protein in situ and on its accessibility to proteases. In all the models, 7 alphahelical fragments cross the membrane perpendicularly. The majority of,the charges are external to the lipid bilayer [61, 103, 104]. Three alpha-
336
J.-M2 C l d m e n t .
helical fragments are uncharged and analogous to the m e m b r a n e penetrating segments described in § 13 (figure 5). Nonetheless, 9 charges are buried inside the m e m b r a n e and the authors built a model of interactions between opposite charges belonging either to the same alpha-helical fragment or to distinct ones. N e u t r o n diffusion studies [3] suggest that the charges are indeed located inside the channel formed by the 7 alphahelices while the h y d r o p h o b i c residues are left on the external face which is in contact with the phospholipidic h y d r o c a r b o n chains. E n g e l m a n and Zaccai [3] imagine that bacteriorhodopsin is an <~ inside out >> protein, in opposition to soluble proteins where the core is h y d r o p h o b i c while the external surface contains the charged residues free to interact with the polar solvent [105]. This conformation would allow the fulfilment of two requirements of bacteriorhodopsin : anchoring and carrying protons across the membrane. 3. CONCLUSION. I have emphasized two modes of insertion of integral proteins in membrane. 1) Integration by a single peptide. This peptide is longer and m o r e h y d r o p h o b i c than uncharged peptides of soluble proteins and is limited by several positive charges on the C-terminal side. 2) Integration by a complex folding inside the phospholipid bilayer. The <~ inside-out >> model of the bacteriorhodopsin m a y partially or totally apply to the 5 integral m e m b r a n e proteins I focussed on. It favours the absence of contact between charged residues and the h y d r o p h o b i c h y d r o c a r b o n chains of the phospholipids. The surface of the protein is deprived of charges, and this m a y explain the extreme h y d r o p h o b i c character of some of these proteins which nonetheless m a y contain a high proportion of charged residues. In addition, this model takes into account the function of this type fo proteins, which is often to create a channel through the m e m brane. Acknowledgements. I thank M. Hofnung and S. Wain-I-Iobson ]or their useful comments on the manuscript. This work was supported by grants from the D G R S T and C N R S (C.P. 960002), the N A T O (Grant N ° 1297), the Fondation pour la Recherche Mddicale and the Association pour la Recherche sur Ie Cancer.
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5.9. Fur~hmayer, H., 13alas'dy, R. E., Tomi~a, M . . ~ Marehes.~, V. T. (1978). Arch. Biochem. Biophys., 185, 21-29. 60. Ohkawa, I. a Webster, R. E. (1981) J. Biol. C h e m , 256, 9954-9958. 61. Ovehinr~ikvf, Y. A., Abdaxhiev, N. G., F,eigina, M. Y., Kiselev, A. V. e Loba~ev, N. A. (t979) F.E.B.S. Lett., ll~0, 21,9-224. 62. WaI,ker, J. E., Car~e, A. F. a Schmi~t, H. W. (1979) Nature, 278, 6313-654. 63. Vort Heijne, G. (19,81') Eur. J. Biochem., 120, 275278. 64. Braun, V., Roterilag, H., Ohms, J. P. e Hagenmayer, H. ,(t'976) Eur. J. Biochem., 70, 601-610. 65. Campbel'l, D. J., 13agn,on, J., Rekt, K. B. M. Wil|ian~s, A. F. (198I) Biochem. J., 195, 15-30. 66. Williams, D. J., Jenkins, R. E. e Tanner, M. J. A. (1979) Biochem. J., 181, 477-493. 67. BucJael, D., Gronnenborg, B. e Mall er-Hidl, B. (19'80) Nature, 283, 541-545. 68. Ebring, R., B e2cre~h~r, K., WNgt~t, J. K. ~ Overat-'h, P. (1980) Nature, 283, 537-540. 69. Da~la, D. B., Arde~, B. ~ Hen~Nr~g, U. (1977) 1. Bacteriot., 131, 82.1-8~9.. 70. Van Alphwn, L., ~avel~es, L. -~ Lug~eaaberg, B. (1.977) F.E.B.S. Lett., 75, 2'85-290. 71. ChM, T. a Fo~ldJs, J. (1974) J. Mol. Biol., 85, 465zb74. 72. Schwei~er, M. a Henni~g, U. (I977) J. Bacteriol., 129, 165~1-t652. 73. Sl~urray, R. A., Hancock, R. E. W . . a Reeves, P. (1974) J. Bacteriot., 119, 726-73'5. 74. Seabag, I., Sc,hvcartz, H., I-I~ra~a, Y. ~, Hennin..g, U. (19'78) J. Bacteriol., I~6, 280-2~5. 75. M,a~niag, P. A., Pugsle~¢, A. P. ~ Reeves, P. (1977) J. Mot. Biol., 11~, 185-300. 76. Lugtenbexg, V., Var~ Box~l, R., Ve.vt~oe:f, C. ~ Van Alphen, W. (1978)F.E.B.S. Lett., 96, 99-105. 77. En~etrma~n, R., Keam~r, C. e Henm)'n.g, U. (1978) F.E.B.S. Left., 86, 21-24. 78. Kamio., Y..~ N~kzA,'tto,, H. (1977) Biochim. Biophys. A eta, 464, 5.89,-601. 79. Cben, R., Sc~hmidmayer, W., Chen-Sch.meisser, U. a ~on~hag, U. ,(1980) Prec. Natl. Acad. Sci. USA, 77, 4592-4596. 80. Beck, E. • Brenner, E. (1980) Nuct. Acids Res., 8, 3,011-3024. 81. Bremer, E., COle, S. T., Hir~de~n,azh, I. a Henning, U. (1982) Eur. J. Bioehem., 122, 223-231. 82. N~a~zae, T. (10,70) Bioehem. Biophys. Res. Commun., 71, 8'77-88& 83. Nakae, T. (1976) Y. Biol. Chem., 251, 2176-2178. 84. N~kaido, H., Ia~key, M. a Rosenbe~g, E. (1980) J. Supramolec. structure, 13, 305-3'13. 85. Bay.ohm',P., Nfik~,N,o, H. a V
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J.-M.
93. Rartdal[-Hazdbauer, L. L. ~ Schwartz, M. (1973) 1. Bacteriol., 116, 14r3.6-1446. 94. Ferenci, T., Schwentorat, M., Ulrich, S. ~ Vilmart, J. (1'980) J. Bacteriol., 14J2, 5,21-526. 9'5. G~b:ay, J. e Y~s,tm,aka, K. (1'9~8~1),Eur. J. Biochem., 104, 13-18. 96. Wa,nder~a,an, C., Sdh~v~z, M. ~ Feren~ci,, T. (1979) J. Bacteriol., 140, 1-13. 97. Ferenci, T. ~ Boos, W. (1980) J. Supramolec. Structure, 18, 101~106. 98. Schwartz, M. (1980) in : ~ Virus Receptors (Receptors and Recognition, Series B)~, vol. 17. (L. L. Rand~al,1 ~ L. Pl~ilipson Editors) Chapman and Ha~, London, 60-94. 99. C16ment, J. M. a Hofnung, M. (1981) Cell, 27, 507514.
BIOCHIM1E, 1983, 65, n ° 6.
Cldment.
100. Bayley, H., Hu~ng, K. S., P~affl~akrlsl~r~n, R., R•ss, A. H., Ta:kag 'ak~, Y. ~ Kl~oraa~a, H. G. ,(1981) Proc. Natl. Acad. Sci. USA, 78, 22~25-2229. 101. Dtmn, R., M~c Coy, J., Simsek, M., M~aj~md,ar, A., Chang, S. H., P~ajb~ndhary, U. L. ~ I~h~rana, H. G. (19'8'1) Proc. Natl. Acad. Sci. USA, 78, 67446?48. 102. Chang, S. H., M,~j,tmadar, A., I)unn, R., Makabe, O., Rajbaaad'.hary, U. L., Kh~ovana, H. G., O~htsuka, E., Tanaka, T., Tan~yama, O. ~ IIk~h~ra, H. (19,81) P,roc. Natl. Acad. Sci. USA, 78, 3398~-3402. 103. H~nderson, D. M..a Ur~i',n, P. N. T. (1'975) Nature, 25'7, 28-32. 104. Er~g~lman, D. M., Hemtorson, R., M~c Lacht~an, A. D. ~ VCall'aee, B. A. (1'~8~0) Proc. Natl. Acad. Sci. USA, 77, 21)23-2027. 105. Ja~in, J. (1979) Nature, 277, 491-492.
On the insertion of proteins into membranes. (Refu le 20-4-1983, acceptd le 27-4-1983).
Unitd de Programmation Moldculaire et Toxicologie Gdndtique ( C N R S L A 271, I N S E R M U 163). lnstitut Pasteur, 28, rue du Docteur-Roux, 75015 Paris.
R6sum6.
Summary.
Les donndes rdcentes concernant la structure primaire et l'interaction entre les protdines et les membranes suggkrent l'existence d e deux classes de protdines intdgraIes de membrane. Dans la premiere Classe, hi Chatne polypeptidique traverse une seule lois la membrane. Le segment transmembranaire est particuli~rement hydrophobe et est limitd du c6t~ C-terminal par plusieurs charges positives. Dans la seconde classe, la protdine est replide de fafon complexe au sein de la membrane et la seule connaissance de sa structure primaire ne permet pas de pr~dire son mode d'interaction avec la membrane.
Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins. In the first class, the polypeptide chain crosses the membrane only once. The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border. In the second class, the protein is Jolded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.
Mots-el6s :
Key-words :
Mots-el6s : prot~ines int~grales de membrane / polypeptides hydrophobes / interactions prot~ines-membrane.
Key-words : integral membrane proteins / hydrophobie polypeptides / protein-membrane interactions.
Jean-Marie CLI~MENT.
Introduction.
Membrane proteins are usually divided into two classes [1] : Peripheral membrane proteins are not deeply associated with the phospholipid bilayer and can be released from the membrane using mild treatments such as increasing the ionic strength. Integral membrane proteins cross the phospholipid bilayer at least once and can be released only through disruption of the membrane (use of detergents) or mild proteolytic treatment. Integral membrane proteins are generally very hydrophobic and are therefore difficult to handle. This probably explains why, until recently tittle was known which could explain their localization. However technical advances in the miniaturization of protein sequencing and the development of gene cloning and sequencing have given some insight into the nature of the attachment of protein to the membranes.
In this report I have gathered recent data concerning the primary structure of some integral membrane proteins and show that one can divide them into two classes which can be distinguished on the basis of their amino acid sequences. One class can be arranged to cross the membrane only once. The membrane penetrating fragment is clearly hydrophobic and, in addition to other features, I show that the parameters used by Segrest and Feldmann [2] are useful to describe this property. A second class seems to involve a complex folding inside the membrane and the analysis of the amino acid sequences rather support the model of Engelman and Zaccai [3] which accounts for the intramembrane localization and the burial of charged residues.
326
J.-M. CIdment.
+-
HI3
NI31~LDNHT
HAl
KLESMG
HA2
KLSSMG
÷
Influenza virus haemagglutinins
HA3
CSNGSLQCR . . . . .
WfQILAIYATVAGSLSLAINMAGISFWM
CSNGSLQCRICI
+-
~
÷
VILWFSFC_~SC_.FLLLAI LVGLVFI WILWISFLISCFLLCVVLLGFIM~CA
@
ELKSGYKD
WILWISFAISCFLLCVVLLGFINA
CQR GNIRCNICI
.[9,10]
HA3
ELKSGYKD
WILWISFAISCFLLCVVLLCFIMNVA
CQ~ GNIRCNICI +
. [II, I~[
÷
÷+
TALSWWQK
El
ISGGLGAFAIGAILVLVVVTCIGL
RR
E[
AI SKTS
WS~t.FALFC_f._~SSLLI IGLMIF
Semi i k i virus
E2
QYYYGL
YPAATVSAVVOdSLLALISIFASCYMLV
Forest
[13]
ACS/~ATLSTRR +
E2
HPVYT I L A V A S A T V / k M M I
GVTVAVL
+
glycoprotein Rabies virus glycoprotein VSV
IASFFF
I IGL I
+
+
IGLFLVL RVG I H L C ql'+
"1-
[ 10]
I YT
4"
--
PriSE
GLFNKS
I:~/FTTLISTIMGPIVLLMILLFGI:~IL
TGT
41- +
FCTALLS 1TALALVCTLLYL
. [ 1 5]
. . . [ 16]
4"-+
NRFVQFVKDK . . .
+
÷----++
KYKSRF I RDEKKMP
[ 17] [ 1g]
t-4-4-
VHLLKG ++
LLLV I LLLVVCLPCLLQML
+
CGNRRKM I NN5 I ELPHG I ÷÷
RRLGRT
LLLVTFLAALLGICLMLFILI
[ 19]
÷
KRSRHF ÷
um
EEGFEN
L~I'TASTF I VLFLLSLFYSTTVTLF
Iporcine
VESNSS
WWTNWVIPAISALVVSLMY
[ 20] ÷
KVK
[ 21]
MFYTSEN
I
÷
[equine I Ibovine
. .
4-
LPNWGK YVLLSAGALTALMLI I F I M TCCRRVNRSEPTQHNLRGTGR
Adenovirus E3/16 Avian sarcoma virus glycoprotein Polyoma virus middle T antigen
Cytochrome b5
.
4-'11-'11"
4-
Irrrnunoglobulin
. .
I KLKHTKKKQ
4"
[ 13]
4-4--
CACKARREC
"IF
SS
WFSSWK
[ I g]
AARSKC • • • +
MLV
..[8]
@
HA3
Forest
virus
..[7]
+
CQR GNIRCNICI
Sindbisvirus
Sindbis
..[6]
+
+
+--
+
[5]
CVKNGNk~.CTICI
~-
ELKSGYKD --
Semi i k i virus
IYQILAIYSTVASSLVLLVSLGAIS~
KLSSGYKD --
RDNVSCSICL . . [ 0 ]
-
÷
HA3
VILLYYSTAASSLAVTLMIAIFIVYMVS
[22]
- -
VDSNSS
WWTNWVIPAISAVVVALNY
RIYTAED
[ 23],
IDSNPS
~I~/'I'NWLIPAISALFVALIY
MLYTSEN
[.24]
VDSNSS
W~'N~/IPAISALIV~
RLYMAI3D
[ 25]
t Irabbit
Histocompatibility antigens
H2d-3
PP F T[3S
H2k-b
PS
YMVIVAVL GV
L~IL
GAVVAFVM
~, RRRNTGCK+÷+ ÷ [ 26t. "1-
TVSN
MATVAVL
VV
LGA
AIVTGAVVAFVM
-I-+÷
41-
KMRRRATGGK
[ 27,']
~ RRRNTGGK +++ +
[ 28 ]
4-
H2d-I
PSS
TKTNT
V l IAVP VV
LGAVVIL GAVrV~u~VM
t-÷÷
HLAB 7
PSSQST
VPVGVAGAVAVVVILLGAVVI
A
A VM
CRRKSSGGK -It-++
HLAA-I
•
.
.
VLAVVV I
GAVV I
GAVVAAVM
+
[ 29 ]
"IF
CRRKS SGGK
[ 3 0.1
FIG. 1. - - C-terminal sequences o] 27 membrane proteins. + + C h a r g e d residu.es are indicated : K (lys), R (arg), D (asp) and E (giu). Other symbols ere : W (trp), F .(pl~e), I (il;e)~ Y (tyO, L ([eu), V (vM), M (m,eO, P' (~ro); A {ala), H ~h.ils)), T (~hr), G (gly), C fcys), S (ser), Q (gila), N (nsn). M e m b r a n e penetratk~g segmen~ts are written in bold-f~ced type a~ad underEr~ed, [References are indicated in parenflleses o n the righ¢ of We figure]. B I O C H I M I E , 1983, 65, n ° 6.
On the insertion o[ proteins into membranes. 1. PROTEINS WITH A SINGLE PEPTIDE PENETRATING THE MEMBRANE. T h i s p e p t i d e m a y be l o c a t e d at d i f f e r e n t p l a c e s a l o n g the s e q u e n c e of the p r o t e i n .
1.1. C-terminal peptide. A n u m b e r of a n i m a l virus g l y c o p r o t e i n s b e l o n g to this c a t e g o r y ( v e s i c u l a r s t o m a t i t i s , rabies, s e m liki forest, m o l o n e y l e u k e m i a , i n f l u e n z a , a v i a n
327
s a r c o m a and a d e n o viruses) as w e l l as p o l y o m a m i d d l e T a n t i g e n , c y t o c h r o m e b5, i m m u n o g l o b u lins m u - m a n d h i s t o c o m p a t i b i l i t y antigens. I n m o s t cases, t h e C - t e r m i n a l p e p t i d e has b e e n s h o w n to cross t h e m e m b r a n e by c o m p a r i n g the s e q u e n c e s of d e t e r g e n t - e x t r a c t e d p r o t e i n s to t h o s e of the s a m e p r o t e i n r e l e a s e d f r o m the m e m b r a n e by a m i l d p r o t e o l y s i s . T h e s e q u e n c e s of t h e s e p e p t i d e s h a v e b e e n listed in f i g u r e 1. N o s e q u e n c e
TABLE [,
Characteristics o/ C-terminal membrane penetrating segments. (a) A.A.
(b)
(c)
(d) A.A.
(e) H.I.
(f)
(g)
~h) +
(i)
225 ? 222 221 220 2'21 221
[4] [5] [6] [7] [8] [9, 10] [11, 12:
[31] [31] [31] [31] [31] [31] [31]
26 2'8 28 24 25 25 25
2.69 2.37 2.30 3'.02 3.36 3.28 3.28
OUT OUT OUT OUT OUT OUT OUT
11 12 12 13 12 12 12
1 1 1 2 2 2 2
1 l 1 2 2 2 2
1 0 0 0 0 0 0
4,38
[1311
[32]
24
2.21
OUT
2
2
2
0
440
[141
[141
22
2.89
OUT
11
2
2
0
422
[13]
[32]
28
2.18
OUT
32
3
3
0
424 600 505
[14] [15] [16]
[14] [33, 34] --
25 30 19
2.14 3.07 2.63
OUT OUT OUT
35 2'9 47
4 8 8
3 6 3
1 2 4
196
[17]
28
2.8.2
OUT
33
4
2
3
159 180 432 597
[18] [191 [20] [21]
[36] --[36, 37]
20 20 21
2.30 2.75 2.90
OUT OUT OUT
15 17 6
6 3 3
4 3 3
2 1 0
25
2.52
OUT
3
2
2
0
Oyfto~hrome b5 poroh~ 6quin~ bo~e rabbit
133 133 133 133
[22] [23] [24] [25]
[39] [39] [39.] [391
l0 19 19 1'9
2.84 2.89 3.11 3.03
OUT OUT OUT OUT
7 7 7 7
0 l 0 1
0 1 0 1
1 2 1 2
an~iger~ H2 a-z H2 k-b H2a-x HLAB7 HLAA1
340 346 341 337 340
[26] [271 [28] [29] [30]
[40] [4'0] [40] [40] [~0]
25 23 25 22 > 20
2.60 2.30 2.46 2.05 2.30
OUT OUT OUT IN OUT
9 10 9 9 9
5 5 5 4 4
5 5 5 4 4
0 0 0 0 0
Influenza virt~s tra.emag~lutiHn~s HB HA1 HA2 HA3 HA3 HA3 HA3 Slemali'ld Forest virus E1 Siaxl~bis virus E1 SlemlNi Forest viTus E2 Sirxl~bis virus E2 VS~V gly,oopro~i~n l~abies v~rt~s gllyc~protein MILV PRI5E Adel-~ovin~s E3,/16 ASV glyooprotein Polyoma virus m~ddIe T antige~ I mmunoglobu.liin ~xm
[381
.(a) size of the wl-~le protein (in amino acids). (b) references, to sequenoe d~ta. (c) references *o data about b e localization of the segmen,t inside the membrane. (d) ler~gth of the membrane penetrati~ng segment (in amino adds). (e) hydropl~obiei~ inc[ex. (f) position towards .the S.F. triangle. (g) size of the C=termina/ following portion (in amino acids). (11) number of posi@ce e'harges in that re.glow. (i) number of co,secretive positive eharges. (j) number of negative ekmrges.
BIOCHIMIE, 1983, 65, n ° 6.
(j) __
328
].-M. Ctdment.
homology exists between peptides from non related proteins. Furthermore, in variations of the same protein (influenza virus haemagglutinins, cytochrome b5 or histocompatibility antigens), a
I
0
3O
•
•
#
t~
o
20
,al o o o
/
\ +
!
1
2
0
IO I
3
Fro. 2. - - H y d r o p h o b i c i t y diagram o] m e m b r a n e penetrating segments. T h e ami,no acid len~tlh ~of eaoh o1 t h e memJbran,e peaae~rating s e g m e n t s is plbNed a g a i n s t i~s re speeti~re h y d r o phobicity i n d e x (H.L) whilzh is capitulated as foltowed :
to each amino a~idJcorre.~o~ad.s art hydrophobicify value (trp = + 6.5, phe = + 5, lie = + 5, tyr = + 4.5, ten = + 3.5, val = + 3, rrtet = + 2.5, pro = + 1.5, ala = + 1, la~ = + 1, ~hr = 4- 0.5, eys = 0, gly = 0, 8er = 0:.5, g1~, = - - l;.a~n = - - 1.5). H.t. is the mean value of its composing amino acids. Almost all of the ,po~nUs eorre~pondilng to m-~charged pols'perptide segments from sc~lubt.e proteir~s. ~al,1 i~sfide .the tri~a~ngle (taken from [2]). The points correspondk~g t~ ~ e membrane penotra~infi segments are figured : I C-termi~nal segmenCs. 0 N-termirml ~,¢~naemts. • ir~ternal s'egme~ts. [] inter rtal seg~erdSs from bacteriotrt~.in.
+
g_. s t e a r o t h e r m o p h i
ius
:
glu, lys, arg). This fragment is clearly hydrophobic as shown in table I. Segrest and Feldmann [2] have defined and hydrophobicity index for uncharged polypeptide fragments. This index is the mean hydrophobicity value of its constituting amino acids. These values, established according to Nozaki and Tanford [41], are reported in the legend of figure 2. They are related to the free energy of transfer of the amino acids from ethanol to water. By plotting the size of the uncharged segments of soluble proteins against their hydrophobicity, Segrest and Feldmann [2] observed that all the points are clustered within a triangular space (let us call it the << SF triangle >>). In contrast, all the points obtained from the values of table I, except one, are clearly situated outside on the right of the SF triangle. This means that the membrane penetrating segments are more hydrophobic than the soluble protein fragments of equivalent size. This result fully confirms those of Segrest and Feldmann who had only four examples of membrane penetrating fragments at the time of their publication [2]. A second feature shared by the C-terminal part of this type of membrane proteins is the presence of several positively charged residues at the border of the hydrophobic part which is placed at the cytoplasmic side of the membrane. The only exception to this rule is the cytochrome b5 for which a loop model of anchoring has been proposed [25 bis]. The presence of a proline residue in the middle of the hydrophobic part is suggestive of a possible turn in the structure. We should notice the peculiar case of carboxypeptidases from B. subtiIis and B. stearothermophitus which
+
-
t-
+
• • -KAWFVLSMP,.AVGGLFVDLWTSVAKTVKGWL C(:~--~
+
B.
subtilis
:
• . . ANWFVLTMRS I GGFFAG ['~FGS [ VDTVTGWL COOH
FIG. 3. - - C-terminal s e q u e n c e s o] t w o c a r b o x y p e p t M a s e s [7]. + + C h a r g e d .reskl,~es a r e ind[.cated : K (lys.), R (arg), D (asp) a n d E (ghl). Oilier s y m b o l s
ave: W (,ta~), F (Nae), I file), Y (tyr), L (I~u), V.(vat), /~ (met), P ~ro); A (ala), H ~his), T (thr), G (gly), C (cys), Q (gin), N (asn).
greater divergence seems to occur in the C-terminal part than in the rest of the molecule which is quite well conserved. One common feature of these C-terminal portions is the presence of a long segment (> 19 residues) deprived of charges (i.e., absence of asp, BIOCHtMIE,
1983, 65, n ° 6.
are hnked to the membrane by the 25 amino acids long C-terminal part [6J. This region shows very strong hydrophobic properties although this character is not suggested by the sequence since it includes charged amino acids (figure 3) [7]. The authors have suggested a model for attachment to the membrane : the C-terminal portion
On the insertion of proteins into membranes.
329
T A B L E II.
Characteristics o[ N-terminal membrane penetrating ~ragments (a)
A.A.
Influerma v~rus neuram~izi&ase D~pep~idyl peptklase I V Isomilt~se Rhodopsiaae (bovi~e) Cy.tochro,me P 450 LM2 PB b a Etx)xtidhs"drase (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)
(b)
(c)
(a)
A.A.
(e)
H.I.
(f)
(h)
(g)
+
(i)
__
(9
(k) S.S.
461
[51
[491
28
2.78
OUT
6
t
1
0
--
1200 1300
[42] [43[
[42] [50]
9 > 2'2
o 3.39
? OUT
0 9
0 0
0 0
0 1
? ?
350
[441
[44]
27
2.46
OUT
17
0
0
t
?
500 500 500 500 500,
[451 [4g] [47] [47] [48]
? ? ? ? 9
3.06
OUT OUT OUT OUT OUT
2 2 1 3 4
0 0 0 0 0
0 0 0 0 0
1 1 I 1 1
--
> 18 > 18 18 > 20 > 15
2~.94 3.00 2.55 3.30
estimation of the size of the whole protein (in a m i n o ands). references' ~o the sequence data. referet~c~s ~c) ~ t ~ ~ u ~ V.he ,~oc~t.iz~ation of ~he fragmen:t ins:fd.e ~he membrane. size o f the membrarm pe~etradn~g f r a g m e m (in a.mino acids). hydrophobioitSr i~d~x. pos.ition totvcards ~he S.F. ,riar~gle. size o f tabe N-terrMn.al preceedil~g portion. ~umber of tX~i~i~,'e otmrges fn tNat region. n u m b e r ~of oonse~l~ve posieive charges. n u m b e r of negative oharges. when known, absence, of sig~lal sequence (S.S.) (--).
Influenza virus neuraminidase Dipeptidyl peptidase
41-
~vINPNQK
LGFAL/kFI
1V
Isomaltase Rhodopsine (bovine)
PB
L...HQGSD
retinal V FGPI~IPAFF~TSAWCNPVITIMN
FSLLLLLAFLAGLLLLLF
b MLD
Epoxidhydrase
[[5 l
[42 I
ITLIVLFVIVFIIAIALIAVLA
~
SHS1Q • • •
...
AVNAFSGLE
LM2 Cytochrome P~SO
I ITIGSICLV.VGLISLILQIGNI I 5 I W I
XNoXPAV . , ,
+
[g3]
+
KQFR . . .
[#4]
.,.
[gS]
PSILLLLALLVGFLLLLV . . .
[g6.]
PT1LLLLALLVGFLLLLV
[471
RG . . ,
TGLLLVVILATLTVMLLLTL
t'vl~%E LVLA5LLGFVIY'~V$ . . .
...
[48] [g9 ]
FIG. 4. - - N-terminal sequences o[ 9 membrane proteins. + Ohargeod re~idtles are .ill~i,ca~ed : K (lys), R (arg), D (asp) a.t~d E (glu). O ~ e r symbols are : W (trp), F (phe), I (ile), Y (tq'r), L (leu), V (val), M (met), P fWo), A (al:a), H (laSs), T (thr), G (gly), C (cys), S fser), Q (gl.~), N (ash). M e m b r a n e penetrating segments are written in bold-faced type and underlined. [References are indicated fin parentheses, on the right e£ the figurel.
BtOCHtM1E, 1983, 6.5, n ° 6.
o ? ?
J.-M. Cldment.
330
s h o u l d a d o p t a p e r i o d i c a l structure ( a l p h a helix or b e t a sheet) which is half buried p a r a l l e l to the plane of the m e m b r a n e . C h a r g e d residues would be aligned on the e m e r g i n g side of the structure. Glycophorin (human e r y t h r o c y t e s )
÷÷
. . /• AAFDSLQASAT E YIGYAW/IdglVVVlVGATIGIKLFKKFTSKAS..[53,5~,55) ÷ ÷÷ ÷ ..
Z32
FDSLQASATEY|GYAW/~VVVIYGAAIGI KLFKKFTSKAS.. %
coat
proteins
÷&
..GERVQLAHHFSEPE I T L I I F G ~ G V l G T I L L I S T G I RRLIKKSPSD..[52]
fd/Ml3/fi Phages major
a n d the h y d r o p h o b i c i t y indexes of the u n c h a r g e d p a r t are r e p o r t e d in table II. T h e s e values are again high a n d the c o r r e s p o n d i n g points are situated on the right o[ the S F triangle (fig. 2).
÷÷
[56]
÷
Ill
..A~AFDSLTAQATE MSGYAW/kLVVLVVGATVGIKLFKKFVSRAS..
[57]
Ike
. . A T E / ~ S L TQAID LISQTWPVVTTVVVAGLVI RLFKKFSSKAV,.
[37]
÷
A gacteriorhodopsin
B
-
÷
_
. . EAQITGRPE WlWLALGTALMGLGTLYFLVKGMGV5 D _P(out) ÷÷ ( i n ) . . G YGLI.MSLYMrFAIAPVLTTIAYF KKAD ÷
E
[58]
÷
• • TSTR FV~FAI 5TAAMSYILYVLFFGFTS KAFSMR
FIG. 5. - - Amino acid sequences o~ internal membrane penetrating segments oJ 6 membrane proteins. Ch.arged residues are i~ndical~ed : K f~ys), R (arg), D (~sp) and -E (gllu). Ol~her sym°ools are : W (trp), F (phe), I (tie), Y (tyr), L (l.eu), V (val), M (me0, P (pro), A (ala), H ~ s ) , T (l~hr), G (g17¢), C (cys), S (ser), Q (glrt), N ('asn'). Membrane penetrating segments are written in bold-faced type and underlined. [References are inculcated in ,parenCheses on the righ't of ~he figure]. TABLE III.
Characteristics of internal membrane penetrating segments.
(a)
Gl'y,cophorin (humar~ ory~lwocytes) Ph~ges major c~at prot~i'aa~s Bacteriorhodopsin (a) (b) (c) (d) (e) (f) (g)
fd/M,l 3/fi ZI2 I~1 lke A B E
A.A.
(b)
131 50 5,0 51 53 2*8 248 248
[52] [53, 54, ,55] [56] [57] [57] [58] [~8] [~18]
(c)
(d)
A.A.
[59] 19 [60l 19 i60] 19 160] 19 [~] 19 [61, 62] 2,0 [61, 62] 23 161, I~] 22
(e)
H.I,
(f)
2.81 2.84 2.76 2.29 2.32 2.85 2.65 2.95
OUT OUT OUT OUT OUT OUT OUT OUT
(g) + 4 4 4 4 4
size of [he 'whole pr0~ei'n (in amino acids). re:fences ~o sequeaaceJd~m. references' to d~ta eon~ern.i,n,g t~e l,ocalizat/~31aof the fragmen,t i~aside fire anembr~n,e. ler~gth of the mea~brar~e per~elar~th~gsegment (in ami~no acids). hydroph,0biqeiltyindlex. position ~owards the S.F. ,triangle. number ,of conse~a~tive posi'tive charges i.n the C-~rmbrml following region.
1.2. N-terminal fragments. V a r i o u s p r o t e i n s a n c h o r e d by their N - t e r m i n a l segment are listed in figure 4. T h e i r characteristics BIOCH1MIE, 1983, 65, n ° 6.
These sequences are often c o n s i d e r e d as signal sequences [51] which are n o t processed : in such a perspective the anchorage of the p r o t e i n to the
33I
On the insertion of proteins into membranes.
(table 111) is again high (figure 2). The charge distribution on each side of the sequence is as'fmetrical : negative charges are on the N-terminaI and positive ones on the C-terminal side of the fragment (figure 5).
membrane resembles an abortive secretion- In two instances (cytochrome P-450 and neuraminidase), the primary translational product is identical to the final one. Bacillus licheni/ormis penicillinase is synthetized as a precursor. The N-terminal end is a somewhat peculiar signal sequence [51bisl which is necessary for the protein to anchor in the membrane in a transitory step which precedes the excretion.
1.4. Conclusion. The membrane penetrating segments responsible for anchoring proteins in the membrane are deprived of charged residues and are more hydrophobic than the uncharged segments of soluble proteins. This point was postulated by Segrest and Feldmann [2] and nearly all the examples I found fulfill this assumption. As stressed by Von Heijne [68], the specific amino acid sequence is relatively less important than the overall hydro-
The analogy between signal sequences and the N-terminal segments considered here is however limited, since the hydrophobic part of the signal sequences is usually preceded by positively charged residues whereas in penetrating fragments one can usuaIIy observe a negative charge on the Nterminal side of the hydrophobic segment.
÷
~
-
MYYLKNTNFT/MFGLFFFFYFFI~A YFPVFPIWLH~INHISKSDTGIIFA t
AISLFSLLFQPLFGLLSDKLGLRKY LLW[ITC~LVMFAPFFIFIFGPLLQ -
-~
÷÷
-
+
+
YNILVGSIVC.~IYLGVCFNAGAPAV EAFIEKVSRRSNFEFGRAR3AFGCVG WALCASIVGIMFTINNQFVFWLGSG CALILAVLLFFAKTDAPS$ATVANA VGANHSAFSLKLALELFRQPKLWFL 5LYVIGVSCTYDVFk.X~FANKFTSF FATGEQGTRVFGYVTTMGELLNASI MFFAPLIINRtC_,C_,KNALLLAGTIM$ VRIIGSSFATSALEVVIL~TLI~IFE VPFLLVGCF~YITSQFEVRFSATIY ÷
LVCFCFFKQLAMIFMSVLAGN~YE5 IGFQGAYLVLGLVALGFTLISVFTL Q ÷÷
-
SGPGPLSLLRRQVNEVAg[7
Fro. 6. - - Amino acid sequence of Lac Y protein. The amino ~cid ,se~tu,enc~e is ~aken fi'orn [56].
1.3. Internal [ragments. Two cases are presented : the erythrocyte glycophorin and the major capsid protein of phage M13 or related phages (figure 5). In both instances, the portion of the protein that spans the membrane is a 19 residue-long fragment. Its hydrophobicity B I O C H t M I E , 1983, 65, 11° 6,
phobicity of such segments. Indeed, no sequence homology does exist between unrelated proteins in these regions. Moreover, for related proteins (influenza virus haemaggtutinins or HLA and H2 antigens), the variations inside the hydrophobic segment seem more extensive than in the rest of the molecule.
332
J..M. Cldment.
covalently-linked fatty acids to the membrane. A similar case is the Thy-1 glycoprotein from the rat brain whose sequence is known [65]. It seems to be linked to some unidentified lipid elements at the C-terminal end. i have also discarded the erythrocyte band-3 protein which is an anion transporter. Its structure is partially elucidated [66] but its sequence remains unknown. We shall focus on 5 prokaryotic membrane proteins : the products of the genes lacY, ompA, ompF and lamB of E. coli and the Halobacterium halobium bacteriorhodopsin.
One constant particularity of membrane penetrating segments is the occurrence of positive charges at the C-terminal end which is in most cases on the cytoplasmic side of the membrane. These charged residues could possibly "interact with the polar heads of phospholipids. So that anchoring of these proteins may be due at the same time to the presence of the hydrophobic segment in the membrane and their interaction. 2. PROTEINS BURIED IN THE MEMBRANE. 2.1. Introduction.
2.2. E. coli lactose perrnease.
The polypeptide chains of this type of protein generally cross the membrane several times and are often responsible for the passage of ions or
The lacy gene has been sequenced 1167]. The protein has been extracted and its NH2 terminus
APKBNTWYTGAKLGWSQYHI3TGFI N NNGPTHENQLGAGAFGGYQVNPYVG ) -
-
41-
4"
--
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VWRADTKSNVYGKNHDTGVS P VFAG GVF:yA1TPE" I ATRLEYQWTNNI GI3A +
-
+
-
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HT IGTRPDNGMLSLGVSYRFGQGEA APVVAPAPAPAPEVQTKHFTLKSDV +
4-
-
-
-
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-t-
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LFNFNKATLKPEGQAALDQLYSQL5 NLDPKDGSVVVLGYTDRI GSDAYNQ - ÷ +
-
+
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+
-
_
+
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GLSERRAQSVVDYLISKGIPADKIS ARC/v~ESNPVTGNTCDNVKQRAALI -
. + +
-
-
+
+-
DCLAPDRRvE1EVKGIKDVVTQPQA325 @ Fro. 7. - - Amino acid sequence o/OmpA protein. The amino ~eid sequence iS ,taken from [79].
hydrophilic molecules through it. The existence of a hydrophilic pore traversing the membrane suggests that charged residues are expected to be found inside the hydrophobic part of the membrane. Only few examples of sequenccs of this type of proteins are known. I have discarded the case of E. coli lipoprotein [frl], since there is no proof that this protein is really inserted inside the outer membrane. More likely, it is attached through its BIOCHIMIE, 1983, 65, n ° 6.
sequenced in the particular case of a strain carrying a plasmid containing the lacY gene [68]. This strain overproduces the protein (15 per cent o~ the total inner membrane proteins) but the transport of beta-galactosides is normal. The NH,, termini of both the protein synthetized in vivo in that strain and the in vitro product are identical and fit with the predictions of the DNA sequence. This lead the authors [68] to state that lactose perrnease is not processed in vivo although the
333
On the insertion of proteins into membranes.
NH,2 terminus much resembles typical signal sequences. This point is not really clearly determined since it has not been proved that all of the permease molecules are active in the plasmid carrying strain. Since it is overproduced, it remains possible that a few molecules are actually processed - - and active - - while the bulk is unprocessed, uncorrectly positioned in the membrane, and devoid of activity.
other E. coli outer membrane proteins, OmpA does not form any pore in the membrane. Its activity for the penetration of amino acids through the membrane is quite controversial [75, 76]. Nevertheless, the protein crosses the membrane, since it is linked to the peptidoglycan [77] and it emerges at the surface, being accessible to CNBractivated dextran [78], as well as to the phages mentioned above.
The sequence of the protein is shown in figure 6. As can be seen, several uncharged fragments are hydrophobic (they are outside the SF triangle)
The sequence of the protein is known [79] (figure 7), as well as the nucleotide sequence of the gene [80]. It has been clearly demonstrated,
--
+ -
+
-
+
+
-
-
+
+
-
AE I YNKDGNKVDLYGKAVGLHYF S K GNGEN S YC__~NGI~MTYAR LG F KGE TQ
INSSLTGY~EYNFEGNNSEGADA QTGNKTRLAFAGLKYA.DVGSFDYGR ÷ . . . . + NYGVVYI3ALGYTI~LPGFGGgTAYS DDFFVGRVC~VATYRNSNFFGLVDG -+ + ÷
- + -
++
.
LNFAVQYLGKNERDTARRSNGDGVG --
++
-
+
--
-
.
.
+
+
GSISYEYEGFGIVGAYC_.,AADRTNLQ
--
EAQPLGGNKKAEQWATGLKYD~NNI +
.
-
+
+
YLAANYGETRNATPITNKFTNTSGF ÷
+
•
.
.
.
.
.
ANKTQDVLLVALYQFDFGLRPSIAT TKSKAKDVEGIGDVDLVNYFEVGAT
YYFN~r'~STYVDY[INQIDSDN~LG
VGSDDTVAVGIVYQF3a 0
FIG. 8. - - Amino acid sequence of OmpF protein. T1ae a.mino acid sequence .is .taken from [92].
but their position with respect to the membrane remains unknown. All these fragments are situated in the N-terminal half of the protein. No obvious model for the insertion of the lactose permease is at present available. 2.3, O m p A protein of E. coll. OmpA is abundantly present in the E. coli outer membrane. It is a plurifunctional protein : receptor for the phages Kz [69], TulI * [701 ; it is necessary for the activity of colicin L [71]; it plays a role in F-dependant conjugation [72, 73], and is also necessary for the functional integrity of the membrane [74]. In contrast to some BIOCHIMIE, 1983, GS, n ° 6.
in particular by the study of the expression of plasmids containing various portions of the o m p A gene, that all the known functions of the protein (including anchoring in the membrane) are carried out by the 193 first amino acids [81] (the mature protein is 325 amino acids long). It has also been shown that a mild protease treatment generates a N-terminal fragment which is 177 residues long and retains all the OmpA functions. However a 133 residues long fragment has lost all of the activities. Examination of the protein sequence does not reveal any particular hydrophobic segment. An uncharged peptide, 19 residues long (from amino 25
334
J.-M. Cldment.
Its sequence has been determined [92] and is indicated on figure 8. The most striking character is the great number of charges : the largest uncharged segment is 11 amino acids long and none is hydrophobic. The protein itself is however extremely hydrophobic. No information is available concerning its structure. Chen et al. [92] suggested that insertion in the membrane was mediated by association with other membrane proteins. This hypothesis has not yet received any experimental support.
acids 33 to 51) is rather polar. Once again, no model is available for the in situ structure of the OmpA protein. One must imagine a complex folding drawing charges inside the hydrophobic hydrocarbon bilayer. 2.4. O m p F protein of E. coil This <
2.5. L a m B protein of E. coll. L a m b is an E. cofi outer membrane protein which is inducible by maltose [93]. It is a porin somewhat different from the others since it shows
VDFHGYARSGIGWTGSGGEQQCFQT TGAQSKYI~LGNECETYAEL~LC.-QEV WI~EGfKSFYF6TNVAYSVAQQNDWF- ATDPAFRE~VQGKNL|E~LPGSTI *÷ -
WAGKRFYC~.HDVr~IDFV~VO~SGP CmL~NI~VGFG~LSLAAT~SS~AG GSSSFASNNIYDYTNETANfVFDVR LAQMEINPGGTLELGV6YG~L~(13 ÷
-
+ -
-
+
+
-
÷
NYRLVDGAS~FTAEHTQSVLK GFNKFVVQYATDSMTSQGKGLSQGS -
- +
+
.-
GVAFDNEKFAYNI NNNGFgdLR1LDH GA [ $ 1 k ~ , D ~ V C / d Y Q D I NWI3NI3 NGTKWWTVGIRPMYKWTPIMSTVME IGYDNVESQRTGDKNK~YK[TLAQQ
"~AGD S IWSRPAi RVFATYAKWDEK VCGYI~YT~NAI3NNANFG~.AVPAI3FNG
GSFG~G6S~
F ~
I~ 4 Z
FIG. 9. - - A m i n o acid s e q u e n c e of L a m B protein.
The anairro ac2d Sexluen.c~is ta&en @om [ ~ ] .
an ionic bond to the peptidoglycan [88], and emerges at the surface where it serves as receptor for various bacteriophages : Tula [69, 89], TPI [90], and T2 [91]. BIOCtth'vllE,
1983, @5, n ° 6.
a marked specificity to facilitate the diffusion of maltodextrins and maltose through the outer membrane [94]. It is a transmembrane protein since, on the one hand it is bound to the peptidogly-
335
On the insertion of proteins into membranes.
2.6. Bacteriorhodopsin.
can [95] and interacts with a periplasmic protein, M a l e [96, 97], and on the other hand it is used by several bacteriophages 198, and M. Hofnung, personal communication] as receptor for adsorption at the surface of the bacteria.
It constitutes roughly the only proteic element of the ~ purple membrane >> of Hatobacterium halobium. It is constituted of 248 amino acids with a retinal molecule covalently bound to iysine 216 [100J. The protein has been sequenced 1158, 61, 62] as well as the structural gene [101, 10~.] (figure 10). The protein is synthesized as a precursor. The structure of the i3 residues long amino-terminal extension is different from all the known prokaryotic and eukaryotic signaI sequences. The C-terminal asp which precedes the stop codon is absent from the mature protein.
The gene has been sequenced [99] revealing the protein sequence (figure 9). As in the case of OrnpA or OmpF, the charges are dispersed throughout the molecule. An hypothesis has been suggested [99] concerning the charge distribmion which is periodical. This model was based on a relatively high alpha-helix content in the secondary structure predictions for LamB. Recent phy-
. . . . . . . . .
A
. . . . . . . .
MLELLPTAVEGVS QAQ{ TG~PEWIW3-ALGTAL/~LGTLYFLVKC~'vSGVSD
-
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-
at
--
-
-
PDAKKFYA 1 TTI_ VPA l AFTMYL SMLLGYGLTMV P FGGEQ'qP I TWARY/~Iq~LFTT P L L LLDLALL VDA
- - - -
. . . . . .
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.
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.
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.
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.
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. . . . . . . . .
DQGT I LALVC_dkIX] IMI QTGLVGALTKVY SYRFVVA!/AI STAAMLT I LYVLFFGFT SKAES
. . . . . . . . . . . 4"
--
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.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
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MRPE V/kSTFKVLRN VT VVLWNAYPV~OIgLI GS EGAG I VPI.N I ETLLFMVLIDV,SAKVGFGL I LLRSR
AIFGF.,AEAPEPSAGE~:3AAATS D2~ $
1"to. 10. --Amino acid sequence o] Bacteriorhodopsin, "1-/~ ~m.~o acid ~equen~e is taken from [92l. T h e two. a r r o ~ s ind,kzete ~t~he Pelrtide bonds cleaved dlard0ag proce.ssiag o f the pvoteila. T,tte 7 m e m b r a n e pene~ratirtg segmezts (A to G) and Che charged amino acids internal to tthese segmen,ts (acec~d~g ,to [911) are in bold-faced type and overIdned.
sicaI studies (J.M. Neuhaus and J. Rosenbush, personal communication) reveal that actuMl7 little i~ any of the protein is helical. Once more, the structure of the protein in the membrane remains speculative. However it will be possible to define portions of the molecule which are responsible for various functions, particularly the regions accessible from the outside of the cell since mutations affecting some of the functions are being sequenced (J.M. C16ment et al., work in progress). BIOCHIMIE, 1983, 65, n ~ 6,
The bacteriorhodopsin acts as a light driven proton pump. Models for the tertiary structure have been proposed. They are based on radiocrystaUography studies of the protein in situ and on its accessibility to proteases. In all the models, 7 alphahelical fragments cross the membrane perpendicularly. The majority of,the charges are external to the lipid bilayer [61, 103, 104]. Three alpha-
336
J.-M2 C l d m e n t .
helical fragments are uncharged and analogous to the m e m b r a n e penetrating segments described in § 13 (figure 5). Nonetheless, 9 charges are buried inside the m e m b r a n e and the authors built a model of interactions between opposite charges belonging either to the same alpha-helical fragment or to distinct ones. N e u t r o n diffusion studies [3] suggest that the charges are indeed located inside the channel formed by the 7 alphahelices while the h y d r o p h o b i c residues are left on the external face which is in contact with the phospholipidic h y d r o c a r b o n chains. E n g e l m a n and Zaccai [3] imagine that bacteriorhodopsin is an <~ inside out >> protein, in opposition to soluble proteins where the core is h y d r o p h o b i c while the external surface contains the charged residues free to interact with the polar solvent [105]. This conformation would allow the fulfilment of two requirements of bacteriorhodopsin : anchoring and carrying protons across the membrane. 3. CONCLUSION. I have emphasized two modes of insertion of integral proteins in membrane. 1) Integration by a single peptide. This peptide is longer and m o r e h y d r o p h o b i c than uncharged peptides of soluble proteins and is limited by several positive charges on the C-terminal side. 2) Integration by a complex folding inside the phospholipid bilayer. The <~ inside-out >> model of the bacteriorhodopsin m a y partially or totally apply to the 5 integral m e m b r a n e proteins I focussed on. It favours the absence of contact between charged residues and the h y d r o p h o b i c h y d r o c a r b o n chains of the phospholipids. The surface of the protein is deprived of charges, and this m a y explain the extreme h y d r o p h o b i c character of some of these proteins which nonetheless m a y contain a high proportion of charged residues. In addition, this model takes into account the function of this type fo proteins, which is often to create a channel through the m e m brane. Acknowledgements. I thank M. Hofnung and S. Wain-I-Iobson ]or their useful comments on the manuscript. This work was supported by grants from the D G R S T and C N R S (C.P. 960002), the N A T O (Grant N ° 1297), the Fondation pour la Recherche Mddicale and the Association pour la Recherche sur Ie Cancer.
RI3FERENCF_N. 1. S~nger, S. J. (1974) Annu. Rev. Biochem., 43, 80583~3. B I O C H I M I E , 1983, 65, n ° 6.
2. Segrest, J. P. & Feldma~n, R. J. (19'74) J. Mol. Biol., 87, 853-858. 3. t3ngelrnann~ D. lVL & Zaoea4, G2 (I9'80) Proc. Natl. Acad. Sci. USA, 77, 5894-5~898. 4. Kry~ll, M., Eliott, R. M., Benz J;r, E. W. Young, J. F. & Palese, P. (1'982) Proc. Natl. Acad. Sci. USA, 79, 4800~4804. 5. F~elds, S., Wi~.ter, G. & Brovcnl'ee, G. G. (1981) Nature, 290, 213-217. 6. Weiner, A. M., Plait, T. ~ Weber, K. (1972) J. Biol. Chem., 256, 3242-3251. 7. VCaxman, D. J. e Strom~nger, J. L. (1'9'81) J. Biol. Chem., 266, 2067-2077. 8. F,a,ng, R., M~L~a-Jl~u,W., tffuyl,eb~oek, D., De~ros, R. ,& Fiefs., W. (19.8q')Cell, 25, 3,1'5-3~3. 9. Verh,oyen, M~,. Fang, R., M;i~n-J!ou, W., Devos, R., Hu3debroeck, D., Sama~, E. & lZ~ers, W. (1981) Nature, 286, 771-776. 10. Both, G. W. ,~ Slei'gh, M. J. (19'80) Nucl. Acids Res., 8, 2J56,1-2575. 11. Mirl-Jou, W., Verhoyen, M., D~evos, R., Saman, E., Fang, R., HuyltebrOek, D., Fiers, W., Threllfall, G., Barber, C., Carey, N..& Ermtage, S. (1980) Cell, 19, 6.8~3-696. 12. Both, G. W. & S~ei~h, M. J..(1981) J. virol., 39, 6636'72. 13. Garoff, H., Fri,s~hauf, A. M., Sin~ons, K., Lehrach, H..& Del'itrs, H. (19'80) Nature, 288, 236-241. 14. Rice, C. M..& Strauss, J. H. ,(198q)Proc. Natl. Acad. Sci. USA, 78, 2062-2066. 15. Rose, J. K., VCeloh, W. J., Sefton, B. M., Esch, F. S. & Ling, N. C. (lt980) Proc. Natl. Acad. Sci. USA, 77, 3884-388'8. 16. AnilL[oaais, A., H.urmer, W. H. ~ Curtis, P. J. (1981) Nature, 294, 275-278. 18. Persso,n, H., J,ansson, M. & Pl~li.pson, L. (19~0) J. Mol. Biol., 137, 3r75-394. 19'. Czerni,lofski, A. P., Levi.ns,on, A. D., Varmus, H. E., Bishop, J. M., Tisher, E. & Gooc[man, H. E. (1980) Nature, 287, 198-203. 20. Soeda, E., Arrand, J. R., Smolar, N., WMsh, J. E. & Griffin, B. E. (19'80) Nature, 283, 445-463. 21. Rogers, J., Earl'y, P., Carter, C., Calame, K., Bond, H., /-/o~1, L. & Wall, R. (1980) Cell, 20, 303~312. 22. Ozols, J. & Gerard, C. (1977) Proc. Natl. Acad. Sci. USA, 74, 3725-3729. 23. Ozol's, J. & ~erard, C. (19'77) I. Biol. Chem., 252, 8549-8'553. 24. Fl~mmin.g, P. J., Dai,tey, H. A., Coreora, n, D. & Strittmat*er, P. (1978) J. Biol. Chem., 253, 53685372. 25. Takag~ki, Y., Gerber, G. E., Nihei, K. e Kho~ana, G. (1980) J. Biol. Chem., 255, 1536-1541. 25 bis. Dailey, H. A. e Strittmatter, P. (1980) J. Biol. chem., 256, 1677-1680. 26. Br6g6g~re, F., Abastado, J. P., Kvist, S., Rask, L., Lalarme, J. L., Gnroff, H., Cami; B., W~nan, K., Larh'arnrrrar, D., PeterSo~n, P. A., Gachel~n, G., Kour~Nky, P. ~ Dobberstein, B. (1981) Nature, 292, 78-8q. 27. Coligan, J. E., Kin6t, T. J., Ueh,ara, H., Martinko, J. & N~ther~son, S. G. (I'9'81) Nature, 291, 35-39. 28. KviSt, S., Br,6g6g~re, F., R~sk, L., Carol, B., Garolt, H., D.ar~el, F., Win~an, K., Larl~amm,ar, D., Abastado, J. P., Gachel,in, G., Petersoaa, P. A., Do.bberstein, B. & Kour~lsk2¢, P. (1981) Proc. Natl. Acad. Sci. USA, 78, 2772-~776. 29. Orr, H. T., Lc~pez de Castro, J. A., Perham, P., Ploe/~h, H. L. & Strom~ger, J. L. (1979) Proc. Natl. Acad. Sci. USA, 76, 4395-4399.
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