J. Jlol.

Biol. (1978) 123. 259-274

Specific Labelling

RIC:HAKD

Nrdical

of the Protein and Lipid on the Extracellular Surface of Purple Membrane

HENDERSOS.

,JANET R. ,JWSB AND SIYAN

Research ~‘ouwil Laborutory of’ Molecular Hills Road. Cambridge CB2 PQH. Euglmtl (Rweiwd

M:RTTOCK

Biology

22 December 1!177)

‘1‘11~ ferritirl/avidin/biotin labelling procedure of Heitzmanu Cy Ricllards (1974) lens been applied to purple mcambrane. l’wo biot,in reagents of differing specificity 11ave been used and the biotin then visualised in t,he electron microscope lzsinp avidiniferritin conjugate. \Vith biotin N-hydrox), succinimidr ester only tile protein molecules are labelled ; with biotin hydrazide, using periodate-oxidised

Inrmbranes,

only tile lipid molecules are labelled;

for both of t~hesereagents, t,he

distribution of biotin on each side of tlw membrane has been correlated with previous stru&ural shdies nsittg electron diffraction and freeze-fracture electron microscopy. The above experiments enable three drduct,ions t,o b(> Inadr. almost, certainly at an accessible lysine (1 ) Biotirr labelling of tlwt protein, residnt:, occurs only on the extracellular surface of the membrane. (2) Hiotin labelling of lipid, almost certainly plyrolipid, also occllrs only on the

cxxtjracellular surface of the membrane. (3) Tile bothom scc~py (Henderson trwmbrarw.

of the model of purple membratw obtained by electron micro& Unwin, 1975) corresponds to tile rxt~racellular surface of the

1. Introduction The primary aim of the experiments reported here was to label purple membrane from Halobacterium halobium specifically on one side, so that the top and bottom of t’he model obtained by electron microscopy (Henderson & Unwin, 1975) could be related to its sidedness in the cell. Earlier work ha,d shown that the t’wo sides of the purple membrane have substantial1.y different properties. In the first study of the structure of purple membrane. Blaurock & Stoeckenius (1971) freeze-fractured whole cells. They found that the cytoplasmic fracture face of the purple membrane patches displayed a clearly textured appearance, with a strong indication of t’he hexagonal lattice which they also found to be present by X-ray diffraction of a suspension of purple membranes in solution. By contrast, the extracellular fracture face was smooth and seldom displayed t,he hexagonal lattice structure. Most, of the mass of material in the membrane was found to stick to the cytoplasmic side. Later (Stoeckenius, 1976: Hwang t Stoeckenius. 1975)! the freeze-fracture pattern was interpreted to indicate t,hat the protein molecults stick more tightly to the cytoplasmic side of the membrane t’han to t’he extracellular side, and that it is t’he protein molecules. therefore. which give the cvtoplasmio fracture face its characteristic textured appearance (see also Fishei $ Stoeckenius, 1977). “59

260

R. HEKJ)ERROS,

.J. S. .JUH13

.4X1)

S. VVHYTO(‘K

More extensive S-ray analyses of the diffraction pattern of the purple membrane (Henderson, 1975; Blaurock. 1975) enabled the structure to be described more accurately. In particular. purple membrane was found to be a highly ordered crystal of space group P3, the thickness of the membrane corresponding to one unit cell and therefore one molecule. The protein molecules were oriented vectorially in the same direction across the membrane, and the two surfaces were crystnllographically distinct. The implication of this for bhe structure of t’he membrane is that t,he t,wo surfaces must, also be chemically distinct. However. the extent of t,he difference in t,he chemistry of the two surfaces remains to be det,ermined. The 7 .A resolution model of the purple membrane (Henderson & Unwin, 1975). sh owed that each protein molecule was composed of seven rod-shaped features, interpreted as x-helices. which were oriented roughly perpendicularly to the plane of the membrane. The packing of theso helices was different in detail at the two surfaces, because four of the seven helices were significantly tilted from being perpendicular to the plane of the membrane. These differences in structure between the two sides of purple membrane are those \vhich are of major concern to us here. In terms of the techniques, the freeze-fracture pictures published by Blaurock & Stoeckenius (1971) provide an unambiguous method for the identification of the two sides of the membrane in the cell. The electron diffraction pattern provides a link with the structural work. The ferritin/avidin/biotin labelling. which is described below, serves as a marker for one surface of the membrane which is compatible with both techniques and can be used to correlate the results obtained with the two methods. In addition, the specificity of the labelling itself provides a glimpse of the difference in surface chemistry of the two sides of purple membrane, which will undoubtedly be worked out in much greater detail in the future. Other differences in the properties of t,he two sides of purple membrane have been discovered which are less directl) interpretable. For example, there is a difference in the frequency of cracking when the membranes are dried with one or other of their two surfaces in contact with mica, or carbon (Kushwaha et al., 1976: Oesterhelt, 1976: Neugebauer 8-zZingsheim, 1978). a difference in the metal decoraGon pattern (Neugebauer & Zingsheim. 1978): and a difference in the surface charge on the two sides as revealed by ionic binding of ferritin at acid pH (Neugebauer et ~1.. 1978). These properties have been correlated with one another (Neugebauer et aZ.. 1978; Neugebauer & Zingsheim. 1978) and with the ferritin/avidin/biotin labelling that, is described here (Zingsheim rf al., 1978). The work described here has been presented in preliminary form (Henderson. 1978).

(a) Preparation

2. Materials and Methods of membrane and synthesis of reagents

Purple membrane was prepared from 10 1 cultures of H. halobium itI as described b> Oesterbelt & Stoeckenius (1974). Biotirl &-hydroxy succinimide ester. biotin hydrazide, and avidin/ferritin conjugate were prepared as described by Heitzmann 8: Richards (1974). Radioactive [‘%]biotin (57 mCi/mmol ; Amersham Radiochemicals) xvas converted to [‘%]biotin N-hydroxy succinimide ester following the procedure of Wallace (1976). Briefly, freeze-dried [‘*C]biotin (50 &i/O.21 mg) was dissolved in 40 ~1 of dimothylformamide; 20 ~1 of A’-hydroxy succinimide (15 mg/ml in dimetllyl formamide) was added followed by 20 ~1 of dicyclohexylcarbodiimide (23 mg/ml in dimethyl formamide). The solution was incubated at 45°C for 24 11 with occasional mixing and then evaporated to dryness under reduced pressure. The r4(1-labelled ester was stored at 4”C, and used witlrout

I’1:KPLE

MEMBRASE

I’KOTEIS

.ASI)

f,IEfI)

L:~l3ELLISU

“li I

pllrificatiorl. [I% Jhiotin tiydrazidc was prepared accordiri g to tile fotlouing proceclurc~. To X ( 14C]biotirl (50 &i/O.21 mg) was added 10 ~1 of thionyt ctlforide and 20 ~1 of mct,llanot. 257; (v/v) solution of bydiazine tlydrate in mettlanol \vas made and 5 ~1 of this \vns addrcl slowly to the biotin acid chloride. The reactioll was cont.ilIrled ovrrl~ipht, at 20°C: a~ttl tlrca solution was the11 evaporated to dryness nndrr rettr~c~l t~~ss~~ro. Ttltx radioact iv0 hic)titl tlyctr;u.idc was used without purification.

TII(~ procedure of Heitzmanti & Klctlards (Igil) uxs followed. Hricfly. I 1111of ii solrltioll of 20 ~ng of the raster/ml of dimettlyl formarnide \vas lnixcrt \vittl 0 ml of a sltspctAon of pllrpte Inernhrar~c~ (1 mg/ml) in O-f lr-NaHCO, (pH X.5) for I II at 20°C’. Ttw rncrnbrunf5 WX’TPttI(LIl washed 3 times by ccntrifugatioll and res~q~ensio~r III 0.1 Jr-KaHCO:, (pH 8.5). iLlId left o\-ernight in ttle same bllf&r before carI:\-itlp ant a fitA \\-a~11 ill distillctt Lvatcr. Tlris sribstsclllent alkaline treatment is important 111rc%rnovilq lul\\ar,trtl I)iot,in \vllicll I~as c~)\~ptctl to hydroxyt groups on ttlr membrane (see latc>r.). lieactiori witlr tllc, “(‘-laf~~~ll~~tl clstcr MXS carried o\lt, by adding 100 ~1 of a srlsprarlsiotl of pllrplc 1ilcmhratlc (IO mg/ml) to I(1 ,*I of the 14(‘-fabelled-ester in dimettlvl formamid(L (0.8 ~ng rst,cbr: 50 &i). followed h> ttlc \\trsttirrg procedure described abovr. Sificc t.hc rc>sults slIo\*-cd t,tlc illcorporatioll of 0111y ahollt 0.2 biotic ,grol1ps/protein molrcrde. ttir rctactioll \vas somc%imcrs rt~pt~atctt if ~norc~ stoichiometric incorporatiotr \vas reqllircd. Ho\vv\.w. i~on(b of ttlo csxpcriltlc,tlts rcportc~ct tlr-ptlosptlatr, pH 7.3). washed twiccx by c,(,ntrifileat,iol~ and resuspension it1 PBS, ar1c1 finally reacted \vith 10 rnbr-biotin tlydrazidtx in PBS for 2 h at 20°C. Ttle procctl~u~ \vas ttie same wt~cthcr t.tic uulabelted or 14C-tahelt~~d I\vdrazide wxs used, except ttrxt t.tl(s cotlccnt,ratiotl of pllrptc mcmbranc~ wax dift‘errrlt in the 2 cases: 1 m&n1 for tllcx rcactiotl with thus llnlabelted and 20 mg/mt Lvittl tllr 14~‘-lahrlled hvdrazide. Similar 01 slightly IOWY incorpornt~ion of biotirl was obtained usi,lg I)ir)tin tIydrazidbra~l(~sltclet,s, \vcrc then rc\vtbtted \vitlI OII(~ drop of PRS. This \~as hiot,ted off and rct)lacrd t)y a drop of a\-idil>,‘fcrritirl sofnt~ion (-0.5 mp/ml) cofltaining I y,, oratbllmi~r in I’M. ‘I’llis \vits aflo\ved to react for 30 s witlr t,llr biotin groups on tllo mernbrallrs. ‘l’l~c grid wa.s the11 blotted off and \vasllctt with 3 successive drops of PBS and. finall~~. I drop of distilled \vat,rlr or. for c~lf~ctrotl diffraction, 1 drop of 1”; g111cosc. 0.1 O0 ovalbnmitl. Ttl(% last tlrop was blotted off and tlrfs grid alto\ved to dry.

(f) Sotlinwr

tlotlecyl

s~~l~~l~ate,‘~~~lya.crylrrrn~tlr gel electropl~orrsis

and 0. I”{, sotliurn Polyacrylamido (2096) slab gels containing O~O(ifi”,, I,isuol?.laitritlc, dodecyl sulphato were prepared and rittt ttsing tltr discotltinrtous brtffixr system of Lactmnli (1970). Samples, wittt tltc addition of .i mg sodium dodrcyl sttlpttate/rng rtwrnbratle. IVP~C~ lteated in a boiling waterbatlt for 3 min before applicatiott in tluplicate to the gel. AftfIr c~lectroplioresis the gels were ciit. Ottc half LL‘BSfixed itt 1A”;, mr~tttattol~!~“~ acetic acitl. awtir acitl and dcstaittctl itI q5”,, stained it1 0.1 S& Coomassie blue it2 4:it+b rtwtltanol/R”, metltanol/70/ acetic acid. Tlic other suctiott was dried tttrdcr twlttced pressure (Honttc~r i!! Laskey, 1974) and atttoradioRrra,phet1 at room temperatrtw. A wparattb gel, rtt n rtntlcr identical conditions to tltc above, was stainc>cl for carboll)-dratc. tisitjg ttw trwtltotl of Zarltarius et a/. (19G9).

3. Results (:L) Riotiwlahelling The

first

N-hydroxy

observations

succinimide

qf th,e protein

were made with purple membrane labelled with biotin ester. The membranes were applied to the electron microscope

PURPLE

MEMBRAXE

PROTEIN

Awl)

LTPID

LABELLING

263

grid and subsequently react,ed with the avidin/ferritin conjugate so that only the surfaw of the membranes not in contact with t,ha carbon film was accessible. When this was done with ester-labelled membranes prepared as described in Materials and Methods, section (In), the picture shown in Figure 1 was obtained. Half of the membranes were heavily labelled wit’h avidin/ferritin and t,he obher half almost completely unlabelled. Presumably the two types of labelling are simpl,v due to membranes having either of their two surfaces in contract’ w&h t)hr carbon film. The ratio of Iabelling on t,he two sides was greater than 30: 1. Occasionally a folded membrane could ltr seen which showed clearly the difference in Iabelling on the two sides of t)he same nwmbranc (Fig. 1). Whw 20 of thesr ester-labelled membranes were subj&ed to c,lrctron ditiraction, the wsults shown in Figure 2 and in Table 1 were obt’ained. All the membranes with tht lahrlled side t,owards the &ct,ron source had ‘left-ha.nded’ diffraction patterns (\\,heu \-iewed from t.he eleckon source), and all those with the unlabelled side towards the ralcctrou source had ‘right’-handed’ diffract,ion patterns. The handedness is defined in Txbk 1. In previous electron diffraction of purple membrane, the ‘right-handed’ pattern is that shown in Figure 2 and used to product Plate Vi(b) of Vnwin & Henderson (1975). The ‘right’-handed’ pattern also (*orresponds t,o the view from t’hc top of Figures 3 and 4 of Henderson $ Unwin (1975). Thus the biotin incorporated using the succinimide ester must be on the bottom of the 7 .k resolution model of purple membra,ne (Henderson & Unwin, 1975). Two further experiments suggest that biotin N-hpdroxy succinimide est’er labels to that shown in Figure 1. at lysine residues of the protein. ,A similar pxperitncnt TABLE

Ofelectrou

HamMness

Electron pattern from

diffraction viewed above

Avidin/ferritin on top surface

reaction (biotin

0

Right-handed

1

d~ffrnction patterns qf ar,irli)t/~~rritilr!hiolin purylr ttlemhra7le present side up)

ester-labelled

No reaction (biotin side down)

17

1;

IL

c Left-handed

3

Ii c

k

(1) Thr definition of the handedness of the electron diffraction pattern is based on the assignmrnt of the index (4,:s) t,o the strongest reflection in the pattern. It is considerably stronger than t,he (3,4) and Y~~YCX as a convenient reference point. (2) The distribution of 3 labelled to 17 unlabelled membranes does not indicate any preferential orientation on t,hcx grid, thv distributmn was approx. 50:50 in practice. Tt r&her indicates a conscious dwision to concentrate on unlabelled membranes in t,his experiment, because of the por;sibillt,y that a labelled membranr llot in propw contact, with t,he carbon film could bind ferritin on it,s unde,mid+~ and t,hemby give t.hl, appearance of a mernhranc the othrr way up (sep Tabk 2, whew thw ciifiioalty is more obvious). 10

:I

26G

R. HENDERSON,

J. S. JUBB

AND

S. WHYTOCK

except that the ester-labelled membranes were quickly washed in distilled water 6) instead of in buffer (pH 8.5). showed that membranes could be labelled (PH with avidinlferritin on both surfaces under these conditions, although not t’o the same degree. The initial labelling on both sides seems to occur more easily in 4 M-salt as, for example, in the labelling of purple membranes in whole cells. If these membrane are t)hen left for a few hours in 0.1 M-sodium bicarbonate buffer at pH 85 (or several days at lower pH) the avidin/ferritin Inbelling pattern reverts t’o that shown in Figure 1. Some of the biotin linkages are therefore labile and are hydrolysed off at, mildly alkaline pH values. This is confirmed when the 14C-labclled t:st.er is used to follow the extent of biotinylation. About twice as much biotin is covalently bound initially, than remains after the final wash. This behaviour would be expected for the reaction of an activated ester such as this with the membrane. Presumably, reaction occurs initially with both amino groups (such as the c-amino group of lpsine) to form amides, and hydroxyl groups (such as in t,yrosine or lipid head groups) to form esters, but any ester linkages formed would be expected eventually to hydrolgse. Since there et ~1.. l975), and the residue at the are no nitrogen-containing lipids (Kushwaha N-terminus of the polypeptide is pyrolidone carboxylic acid (Walker, unpublished experiments). the remaining biotinyl residues are probably amide linked by reaction with the E-NH, of lysine. An autoradiogram of sodium dodecyl sulphate/polyacrylamide gel of ] 14C]biotin N-hydroxy succinimidc ester-labelled membrane shows labelling of only the protein and not the lipid (Fig. 3). The conclusion from ester labelling and electron diffraction is that there is probably one or more accessible lysine residue located on the bottom of the 7 Ak resolution model of purple membrane. (b) Biotin-lahelling

of the lipid

Membrane sugars oxidised with periodate give rise to free aldehyde groups. The aldehyde can then be reacted with biotin hydrazide to form a hydrazone (Heitzmann $ Richards, 1974). In the case of purple membrane, very little if any glycosylation of the protein is found (Kushwaha et al.. 1975) (see also Fig. 3) so t’he biotin hydrazide would be expected to react primarily wit’h the triglycosyl diether and glycolipid sulphate that together form 30% of t,he total lipids (Kushwaha et ul., 1975). Some reaction with phosphatidyl glycerol would also be expected, since periodate oxidizes tin-dials in general, but this is present in much smaller quantity. Autoradiography of a sodium dodecyl sulphate/polyacrylamide gel of ] 14C ]biotin hydrazide-labelled membrane indeed shows that, the label runs with the lipid (Fig. 3). Further work will be necessary to demonstrate unequivocally bhat it is the glycolipids that are labelled. However, it is difficult to see how any h~JdiIlg could occur in phosphatidyl glycerophosphate, the predominant. (SY’,) lipid in purple membrane (Kushwaha et al., 1975,1976). Application of the avidin/ferritin in the same way to biotin Iiydrazide-labelled purple membrane gives the result shown in Figure 4. The same pattern of asymmetric labelling occurs as with the succinimide ester. A control experiment, with no periodate oxidation gave completely unlabelled membranes. Table 2 shows t,ht: correlation of the electron diffraction pattern with the sidedness of biotic hydrazide est,er. The labelling. The results are similar to those shown for the succinimide labelling is found on the bottom of the model, The Iiydrazide and ester incorporat’ion. very probably into the glyoolipid headgroups and lysine residues of the protein, respectively, are therefore on bhe same side of the membrane.

a

b

C

d

l’lrRk’I,l+: it*)

Riofit,

MEMBRANE

N-hydroxy

I’HOTEIS

succinintide

.\SIJ

P.&r-lnldlirt~t

1.1 I’ll)

I,:IHEI,I,IS(:

L’(i!l

q[ ~11s clrd cdl ~~welop~s

i%oth this section of Results and section (d). IJ?lO\\ ~ are used t,o det~ermint~ \\.ht+ h(br it, is t8hr extracellular or intracellular side of the purple memhranc that is labt~llctl with t)iotin N-hydroxy succinimide ester. First. whole cells. freshly grown and suspended in basal salts (Oesterhelt & Stoeckeniu*. 1974) containing 0.1 &r-sodium bicarbonate (pH 8.5), were lahelled with the cstcbr as described in Materia,ls and M&hods. Purple membrane was subsequently isolattsd from these cells by the normal procedure (Oesterhelt & Stoeckenius, 1974). applied to an elect,ron microscope grid. and thta nvidinlferritin reacted as for t’he rxperinwnt shown in Figure 1. The same distribution of roughly 50 : 50 labelled and unlal~t~llt~cl mcmhranrs n-as obtained. Tht, result cwuld br obtained wit’h purple membrane (Bither from rst’er-labelled cells or 1974). Electron diffraction frtml wll envelopes (E-BS. Oestrrhelt’ & Stowkt~trius, sho\\wl (6 diffraction patt,erns : 1 right-handed from an unlal~t~llcd memhraw. 5 Itxtt Ilil tldtbtl from laholled membranw) complet’e wnsisttwry with t,htb rwAs of ‘I’al~lt~ I Thus put@ menil)rane can he lahelled in \\~holt~ wlls or in wll cwwlopt~s in t IJ~L ~~rtwnw of 1 M-salt in exactly thr a same \vay as in a -;usprwbion of puritit~d nwm1~ra nts in 0. I >I-sodium bicarbonate buffer. tn the next experiment,. the idea was to makr uw of’ the cell \\,a11 which is on thtb outsidt, of tha cell to protect the outside surface of thv wll mrrnhrane flwm acwssihility to the avidin. To do this. ester-lab&d cell t~nvelopcs. consisting of the wll mcmtw\~ne and the cell wall. were freeze-thalved thwr tirnw in the presetlcc of avidin (6 mgiml) to react all a,ccwsiblc b&in groups hoth inside and outsides the cell rnvt~lopt~ \\,it,h avidin and tht~rcforv nlakts tllt~ln unavailal)lt~ to il11J’ slit~st~t~ut~nt ;Ividill:ft,l.rit’iII watd iorl. A\ftclr t hck hczt~-t~ha\\~ tWiltIll~~Ilt. illI t’st*tw of’ IJiot iii \\‘iis :lddctl to satur;ltt~

Electron

diffraction pattern

Right

handed

Avidin/ferritin on top surface

reaction (biotin 2t

present side up)

No reactmn (blotin side down) 6

PURPLE

MEMBRrZSE

PROTETS

AND

LIPIT)

LARF,T,T,TNG

271

t,he remaining avidin binding sites. the cell envelopes nerr \\ashrd several times in basal s&s and purple membrane isolated in tht, usual wa.y with find washings in distilktl water and purification on a sucrose gradient (Oest,ctrhclt & St,oeckenius. 1974). A control experiment with purified purple membrane was suhjcckd t)o the identical procedure t,o ensure that the ester-labelling and the subsequent avidin-biotin reaction survived t#he high salt and distilled mater treatment. Final examination of the purpk tnemhratw isolated from ester-labelled, nvidin-freeL,cb-t’hawed. whole cells showed the normal 50:50 distribution of labelling on the elect)ron microscope grid and the santt’ correlat)ion with the electron diffraction pattern (5 diffraction patterns : 3 left-handed from labelled membranes, 2 right-handed from unlabelled membranes). The cottt~rol exprrimmt with purified purple membrane showed no labelling. confirming that avidin can react under the conditions used. That thr cells really \\‘ere lysed so that t,ltcb avidirt would have access t.o the evtoplasmic side trf the membrane was confirmed Lye teleas~ of soluble (cytoplasmic) protein and DNA (highly viscous and removrd by 1)R’a~) in the initial freeze-thaw treatment, and incorporation trf ‘*CLlabelled inulin unpublished observations) into some of t’ttra in heparate experiments (Henderson, vehicks. which subsequent’lg reseal. The freeze-thawing procedure has itself bctbn widely used and forms part of the normal procedure for preparation of cell membt*attt~ (0rstrrhelt & St,oeckenius, 1971). Thr conclusion is that the est~er~labelled side of the membrane in cells which are rrpeatcdly freeze-thawed appears to be inaccessible to avidin. a macromolecule with n tnol~*oular weight of 60,000 (Grerin & Toms. 1970). The natural deduction is tttat~ this protrctivc side of t,hc tnemljrane is the extracrllular sidta and tlrat the cell wXl1 forms l)arrjt~t. to macromolecules. HOWWW. t.hr t!xp~ritttc~ttt. is t>ssettt ially a negatives one and it is conceivable that t.iterth is also some’ kind of i~a.t~rieron t.itc cytoplasm ic: surfib(:t~ of t II(n ~11 membrane which is not seen by elt~ctroti ttiir:rosc:op,v of t bin stactions of ~11 envelopes prepared under essentially the same conditions as used here (Sto~ckt~nius & Houen. 1967). The result’ is t)herefore consistcnt \vit)lt t’hc\ clst’er-labrllinp being on t hc* c~xtracellular surface of purpl’ membrane but tnorcb direct tlvidcnce is rt~quirtd. i%

Two kinds of freeze-fracture experiment wtar(’ pcrformcd. In the first. avidin/ ftlrril in/biotin ester-labelled ~nt~mbranes. prcparrd in solution so thilt all metnbranc‘s WPW labelled with ferrit,in on r)nt* side. were applied t’o an r~lrctron microscope grid. wssl~c~d. and then freeze-frnct.urtxd and slightl~r etc+tl \vltil(i on the carbon tilm. ‘l’ht resrrtts of’ this we sliown in E‘igure 5(a.). ‘i%vf~ s;iitHr tttetttiwittt(5 \\.itiictut fwemf’ract~urr are shown simply shadoH.(~d in Figure 5(l)) for (*c)tttpat.incltt. It is clear that tlttb f+itin molecules are on t)hc sitit* opposit,r that, \vhiclr in frt~ezt,-ft,actur~, rtxveals t,ltcL normal hf~xagonal lattice of protein molrrules ant1 which cotttainh most of the mass of m;ttcrial in the membratte (tlrc cvt~oplustttio side*) ((Gf. Fisltc~r & Stocy:krnius. 1977).

“7”

R,. HESDERSON,

.J. S. .JUBB

,4Sl)

R. WHYTOC’K

J’l’RPJ,li:

RZEMBRANE

PROTEJS

ANI)

LIPII)

L.~HISLLTNC:

273

Apparently, when purple memhrane is freeze-fractured on a flat’ surface, the characteristic pattern seen on the cgtoplasmic face (PF) of cells is modified and no longer has t)he mosaic appearance seen, for example, in Figure (i(a) and (b). However, the appearance of particles and t,hr t,hick structure remaining after fracture, showing most of the mass of the membrane on this side. remains quite! characteristic (Stoeckenius, personal communication). In the second freeze-fracture experiment (Fig. H(a) and (I))) w similar fracture and c+ch proccdurc as abovr is applied but’ to ;Ividitl/fi’l’ritill~~~i[)tin c+:r-labelled membra,ncr in a prllct. I’urplc mcmbranc, having such a high prottbin content. is difficult. to frtrtmt-frxt.urv

and wcn

swtr

awas

as WIT sho\\m

in Fipuw

(;(:I) and (1)) owur

onI>

occa~sionally. Much more frqucnt is a cross-f’racturti pattern \\.h(>re the texture of tht: int,(srnal cleavagt: of the mcml)rano is not r~vt&tt. St~vert~hclcss. it, is clear from t”igurtl G(l)) t,hat t,he fprrit,in motr~c~ules are l~untl to tjhts side of thcb membrane which must, tsa removed in freeze,-fractt~lr,(: to rcvcbal the VCI’J. chi~l.ilot,t,ristit.. tjextBurcd patttlrrr of the cytopla.smic fracture fac:~~.Thus both fr(~t,xt,-fr;-~~tut.t: c~xpf~riments show t,ht: fi,~ritill:avidiri/l,ic,tin lat)t:l on tht: c~xtract~llutat sido of thcb tn(Lrnt)ranc. in il~tWtt?l(!tlt~ \vit,h thtb al)ovc inaccessibility of’ tjhc: t)iotin to avidin in I’rc~c*xt~-t hawutl. hi&in cstcrlabelkd oclls dut: to the prescnct’ of tho cell watt.

4. Discussion ‘1’11t~(~spt~ritnt~nts reported tl(brti provirtc a highly sp~itic markrr for the extrac~~llular surface of the purple membrane. Both t)iotin X-hytlrox>succinimide e&t (at pH 8.5) and biotjin h.ydrazidc! (after oxidation of thtb memhranc: component~s w&h perioda,te) are convenient to use and with the atta‘. the observation of’ rr:act,icm onl,v with the c~xtracellular surface direct I,v indicates that certain part’s of the protr:itl and lipid ate yuite asyrnmctrically distrihutcd. Lt. seems vary likely that an a,ccessil)tr l.vsinrb side-chain of thcl protein (possit)ly tnot’c ttiatl one) is the group labelled by biotin N-hydroxy succinimidc ester. and that t,ht: gI,vc~olipid sugars arc rrspolLsit&: for t,tle react,ion wit,h hiotin hydrazidc:. IMlr amino ;\(%I se(luenc~ anal,vsis a.nd scparxtion of the lahelled lipids will be nerdad l)eforc it will bc possible to say in d&ail \\-hich amino acid and which of t,he lipids are lat~trlled. Xt the moment, it is simply of interest that a part. of t7he protein in purple membrane can bc labelled on the si& of the membrane opposit)e to the side it, sticsks to in freeze-fract’ure. Similarly. but more significantly, it seems clear t’hat t#he glyuolipitl forms a large part of the t~xtracrllular half of the lipid bilayer of purple tnernhrane. Since the glycolipid

R. HENDEBSON,

274

J. S. JUBB

ANI)

S. WHYTOCB

sulphate and the triglycosyl diether together form 3094 of the lipid in purple membrane (Kushwaha et al., 1975). this means that most of the lipid in the outer leaflet is glycolipid. Another lipid, phosphatidyglycerophosphate (Kushwaha et al., 1975), forms 52:$, of total lipids, so presumably most of this should form the inner leaflet. This finding of glycolipids outside, and acidic phospholipids inside is consist,ent with the results of other experiments in animal cells and bacteria (Rothman & Lenard, 1977; Bretscher. 1973). However. it is the first time that glycolipids have been shown to exist on the outside of a bacterial cell membrane. The extent of the chemical asymmetry of both protein and lipid is not a, surprise, considering the vectorial nature of t,he proton pumping activity that, is the function of the protein molecules in purple membrane (Oesterhelt & Stoeckenius. 1973; Lazier of nl., 1976). It should be expected that many more chemical differences will be found between the two surfaces of purple membrane in addition to those already document’ed. We are grateful

to Dr Don

Engelman for drawing our attention to the usefulness Ann Wallace for supplying an introductory sample ester.

these reagents, and to Dr Bonnie biot,in

N-hydroxy

snccinimido

of of

.l’ote udder? 1:n proof: ‘1’h(l S~IIIC assignmr~tlt of c~xt~raccllular surface t,o t,lla bottom of tllo 111odtl1 I~as bccxrr madc irttl~:pc,rrtfclltly by a tliffcrctlt, mcxt)lod (Huy~vurd, C;mt~o, Glaeser R: Fisller, manuscript in preparation). REFERENCES Blaurock, A. E. (1975). J. ,Viol. Uio!. 93, 139-158. Blaurock, A. & Stoeckenius, W. (1971). Nature New Riol. 233, 152-155. Bonner, W. M. & Laskey, R. A. (1974). Eur. J. Biochem. 46, 83-88. Branton, D. (1966). Proc. Nat. Acad. I%~., I:.S.A. 55, 1048-1056. Rretscher, M. S. (1973). Scl:erLce, 181, 622-629. Collins, T. It., Bartholomew, J. C. & Calvin, M. (1975). J. Cell. Biol. 67, 904-911. Fisher, K. A. & Stoeckonius, W. (1977). Science, 197, 72-74. Green, N. M. & Tams, E. ,J. (1970). Biochem. .I. 118, 67.-70. Heitzmann, H. & Richards, F. M. (1974). i’roc. IVat. Acad. Sci., U.S.A. 71, 3537-3541. .Henderson, R. (1975). J. Mol. Biol. 93, 123-138. J., Henderson, R. (1978). 31st Annu. Meeting Sot. Gen. Physiol. (Cone, R. A. & Dowling, eds), Raven Press, New York, in t,he press. Henderson, R. & Unwin, P. N. T. (1975). Nature (London), 257, 28-32. Hwang, S.-B. & Stoeckenius, W. (1977). ,I. Membr. Biol. 33, 325-350. Kl&waha, S. C., Kates, M. & Mnrt,in, W. (.:. (1975). Can. J. Riochem. 53, 284-292. Kushwaha, S. C., Kat,es, M. & Stoeckcnius, W. (1976). Biochim. Biophys. Acta, 426, 703-710. Laemmli, U. K. (I 970). I2Tat/&re (Lo?&n), 227, 680-685. K. A., Hwang, S.-H. & Stoeckenius, W. Lazier, R. H., Niederberger, W., Bogomolni. (1976). Biochim. Biophys. Acta, 440, 545.-556. Neugebauer, D. C., Oesterhelt, D. & Zingsheim, H. P. (1978). J. Mol. Biol. 123, 127-137. Neugebauer, D. C. & Zingsheim, H. P. (1978). ,J. Mol. Biol. 123, 115-125. Oesterhelt, D. (1976). Progr. Mol. Subcell. Riol. 4, 133-166. oesterhelt, D. & Stoeckenius, W. (1973). l’roc. Nat. Acad. Sci., U.S.A. 70, 2853-2857. Oesterhelt, D. & Stoeckenius, W. (1974). Methods Enzymol. 31, 667-678. Rothman, J. E. & Lenard, J. (1977). Science, 195, 743-753. 234, 6, 38-46. Stoeckenius, W. (1976). Sci. Amer. Stoeckenius, W. & Rowen, R. (1967). J. Cell. Biol. 34, 365-393. Unwin, P. N. T. & Henderson, R. (1975). .I. iMo/. Riol. 94, 425-440. Wallace, B. A. (1976). Ph.D. thesis, Yslr Ulliversity. Zacharius, R. M., Zell, T. E., Morrison, .J. H. & \?loodlock, .I. .J. (1969). Anal. Biochem. 30, 148-152. Zingsheim, H. P., Henderson, R. $ Neugebauer, D. C. (1978). J. Mol. Biol. 123, 275-278