Exp, Eye Res. (1977) 25,511-526

Structure of Isolated Bovine Rod Outer Segment Membranes

IW oukr wpmcnt membranes from fresh bovine retinas havr kn srparatetl into di tl’tw~~t liwtions by crntrifugation on discontinuous sucrose gradients hrtfkwt \vith either .I, I~\IIn the Tris-kmflixrd gradient one t’rai,tioii ‘fris a&atc or 100 mwsodium phosphate. Iwntsinetl predominantly free discs which had retained their original morphology. Intac,l :Ippearing ollter segments were found in all fractions of the phosphatt,-l)ufr~,(l gradirnt. (Gel electrophorrsis showed that in the disc t’rac~tion mow than M”,, of thtl total prott~ili it:lin can bc assigned to rhodopsin/opsin; in tIlta rod outer segment frac~tions. this trrlrllhw I> ;\llollt X5” 0’ t~:lwtron micrographs of ncgstively stsinetl disc mcmbratlcs fixetl u ith pllttnr;~ltl(~l1~-(~(~ ~~~gg!casl- that polar channels (about 1.5-T nnl wide) exist in the mcmbra~~~~. Tlw W~V~LI rlistanrr of the channels is approximately Ci nm. Freeze-t’roct~uwl rod outw segments I‘(‘\ cnl a rough fracturr face on the plasmic leaflet of both the disc and of the pl;wna mombrant~. I II t,he fractlw face of the inner leaflet of the disc memhranr? a fine granularity would I):% IV ~olvc~d (IO.~lW~ grains/~m--“). Freeze-dried and t,hrn shadowed t1isc.s carry some big part ic4w (20 nm itr diameter) protruding from the otherwise smooth external surfacc~. The rcs~~lts XI’, ~~c~nsistcwt uitik the assumption that, rhodopsin is a t.ransmemhr:rne yottGn.

512

W. KREliS

Ah-J)

2. Materials

H. IiUHX

and Methods

Fresh cattle eyes were obtained from a, local slaughter house. Irnme~liatel~+ wfter slauglltt~r the eyes were excised, wrapped in aluminiunl foil, and stored in the (lurk on ice for W\YMI hours until used. The cl&e&ion and the preparation of the rod outer segments MXS (~OIIV at (K! in dim red light or in the dark using a11 infraretl viewing device. The outer segnlellt< were broken off by agitating the ret.inas in 45’i, (w/v) sucrose solution. about 1 11111):‘~’ retina. The outer segment membranes were first floated in this solution bv centrifuqtioli (Papermaster, 19741, and then sedimented after three-fold dilution &h buf?‘er. ‘I’ll? sediment WM gent,lv homogenized in 0.77 M sucrose solution by hand in a, glass -Teflorr homogenizer and f&ther purified by centrifugation on a discontinuous densit!? gratlient collsisting, in a.scending order, of 1.14, 1.07, 1W and 0.92 M-SUCrOSe solut,ions. All thca solutions used were buffered either with 5 mLv-Tris-acetate (pH 7.4) cont,aining 1 n151i&Cl, (Papermaster and Dreyer, 1974), or with 100 mM-h’a-phosphate (pH 7.2) (‘o!jt,ililling 1 mlzr-MgCl,. In the Tris buffered system, the 450/o sucrose solution contained fi.5 n b11. KiaCl (Paperma.ster and Dreyer, 1974): th e other solutions containetl no N&l. The typtl of buffer WM not changed throughout’ each preps&ion. The yield of the combined fractions 1 and II from the gradients (Fig. 1) l!riLs normall! about 0.5 mg of rhodopsin (spectroscopically determined) per retina, and the rirtio (11 4?,0/4,,,, was 2.1-2.6. Sucrose concentr

[Ml ------=-o~77---Y lhite layer --A====z--

ROS ..:,:.~:.:,:.:.~:.:.:.:.:.:.:.:.:.:.:.

I

----0,92--

Free discs >:.>:.:.:.:.~:.:.:‘:‘:‘:‘:‘:‘:‘:’:’

ROS ‘.:.:.~:.:.:.:.:.:.:.:.:.~:,:,~:.:,:

II

---loo--Free discs ROS ;:j:::::::: :,:,>>;>>:.:.:.:.:.:.:.

ROS,RIS B

Ill

*--1.07-----c

ROS,RIS

Mitochondril RIS

m T

P

FIG.

1. Schematic presentation of the distribution of bovine rod outer segments on discontinuous gradients. T: gradient buffered w&h 5 mxr-Tris acetate, I’: gradient buffered with l(lO miss phosphate buffer. ROS: large rod outer segment fragments (morphologically intact), RIS: rod inncl segments. The Roman numerals are the numbers of the fractions referred to in the text. The actual densities of the sucrose solutions are higher in 100 mwphosphatc buffer than in 5 m.nr-Tris; therefore, corresponding membrane fractions are collected at different sucrose concentration on the two gradients. SUCTOIB

Gel rlectrophoresis. This was performed according to Fairbanks (1971) on 58q;, polvacrylamide gels containing 1% sodium dodecyl sulfate. The gels were stained with Coomaskic Brilliant Blue R-250 (Fairbanks, 1971), destained with 1113; acetic acid until the hac,kground was clear (3-4 days), and scanned at 580 nm.

ISOLATED

ROD

OCTER

SEGMEXT

MEMBRAXEK

513

to a concentrati~~t~ l‘ltrathirb sectioniny. Suspensions were fixed by addin, 0 b&taraldehvde of c)+‘>& After 10 min at VC, samples were filtered via suction through millipore filters (0.1 pm). Phosphate buffer (100 HIM, pH 7.4) was used to wash the filters gently even whet1 Tris buffer had been used throughout the preparation in order to prevent the rot1 0ute1 segment membranes from dissolving during the subsequent treatment with 0.30, (1” b ith 100 mM-phosphate buffer) (Falk and Fatt, 1969). After dehydration with ethanol, thta millipore filt,:rs were dissolved in propylene oxide. The remaining fixed material WY enlbedded in epon. Ultrathin sections were stained with urnnyl acetate and lead citrate. Se@c*e stai&g. Phosphotungstic acid (.VC), ammonium molybdate (2’55) or uratlyl acetate (0+0 discs fixed with glutaraldehyde (05:;) and unfixed discs mere both stained at IYM)III t,eniperature. Eieeze-fructu,in,ll.Rod outer segment suspensions were fixed with 0.50/b glutaraldehyclc~. incubated in 2Wh glycerol and frozen in liquid Freon 22 (about -120°C). The samples were freeze fractured in a Balzer freeze-etch unit and shadowed with platinum and carbon from an ancrle of 45”. Frrrze-h&y cmd subsequentshadow&y. Aliquot’s of the disc suspensions were t&l with glutaraldehyde (050/) and filtered through millipore filters (0.1 pm). The filters wert’ \\nshed with 10 mM-l‘ris acetate (pH 7.2) and frozen in liquid nitrogen. The filters wert’ freeze dried in the evaporator and shadowed with platinum and carbon at a stage tet~~per>lt.ure of -:)(I% >mrl a. vacuum of less than 5 ;< 1~7 Torr. The angle of the shatlo\s \v;I~ 20’. The procedure3 usually were performed in the dark until the membranes were stiGllc,ll or frozen. Home s;Lmples’ were illuminated with bright white light (fluorescent bulb) f111 111 tlliti at 0°C prior to the fixation.

,ire;ts

were measured on The particles in t1elrsit.p of t,he particles was of 1 cm2 by placing a square phJlilIleter.

micrographs (magnification: 82 ()C)(j-fold) using :L “Hnff” the measured areas were counted. From these values the calculated. Densely packed particles were count,ecI in arr;li lattice over the print.

3. Results

Figure 1 shows schematically the results af t’he densit,?- gradient centrifugation. In the Tris-buffered gradient a fraction of predominantly free discs is enriched at t,hc interface between 0.92 RI and 1.00 >I-sucrotie (Fig. 2). Somet,imes several discs are attached at their edges (“loops”) to vesicles which are presumably derived from t,llcs plasma menlbrane (Fig. 3). Fraction II of the Tris-buffered gradient usually ;tlw contains free discs mixed wit#h larger fragments of outer segmentr in which st’aclw of tlisw are surrounded hy the plasma membrane. Fraction III, containing mostI> sue Ii large outer segment fragments. is contaminated by inner segments and frw iuitochondria (Fig. 4). A layer of white mcmbraneous material was sometimes Jtut not always -visible at the interface of 0.7i/O-93 Ji sucrose, and both the Tris-butK>rtbcl and the phosphate-buffered gradients contained a pellet at their bottom. The yivl(l of free discs on the Tris-buffered gradient varied between t#he difierent preparatlolls, but normally. frac%ion I c0nsiste.d pretlominantly of free discs. On the phosphwtc*buffered gradient, the outer segments arc also separated int)o three fractions which, in contrast to the T&gradient fractions, all show the same n~orphologv, namely large

TSOLATED

Fit. 4. Fraction of inner segments

ROD

III of the Tris-buffered (18) and mitochondria

FIG+. 5. Fraction I of the phosphate-buffered outrr wgmentx is pwsrrved. C‘. “contracted”

OUTER

SEGMEKT

MEJIBRAKES

gradient. The outer segments are relatively (m) are present,. Bar = 1 pm. ( 15 750). gradient. rod outer

.i I .i

intact.

F;(~.~~IIICIIIS

The original arrangement of the discs within segment. Bar =- 2 pm. ( ‘, 4590).

t IN,

Electrophoretic analysis on MDS~~~l:olyacr!-lallliclc g‘t’I,q show that in fraction I to III, rhodopsin (plus opsin) is the major protein component comprising at- Iwht x.3”,, of the total protein. In Fig. 6(a) and (b). scansof a typical rod outer segmwlt fractiotl

i

FIG. 6. Electrophoretic separation of proteins in difl’erent rod outer segment fractions on SDSpolyacrylamide gels, stained with Coomassie Brilliant Blue and scanned at 580 nm. (a) Outer segments (fraction II of a phosphate-buffered gradient): (b) free discs (fraction I of a Tris-buffered gradient); (c) disc membranes after washing with 5 mwEDTA/5 m>rITris. 411 three fractions contain the same amount of rhodopsin (5pg). The rhodopsin peak is fully shown only in scan (cJ which was ran at a sensitivity four times lower than the scans (cl), (b) and (a). The numbers at the bottom indicate approximate molecular weights in thousands corresponding t,o different positions on the gels.

and of a typical disc fraction are shown. Most of the minor proteins present in whole outer segments are missing or present only in smaller amounts in the disc fraction. Essentially onlp two Coonlassie-stainedcomponents are found in the disc membranes, namely rhodopsin/opein and a high molecular weight (about 200 000 or higher) COIHponent. The area under this high molecular weight peak is only 2:/,, and t.he area under the rhodopsin peak is about 97-989!; of t.he total area of stained material in the scans.The small peaks seenin the scansat molecular weights higher than rhotiop~in represent real stained bsnds on t,he gel:: and not just background noise. However, these proteins are present only in minute amount,s as compared to rhodopsin ‘l’hc~~ are partially extracted if the discs are washedwith 5 mnf-Tris/5 WV-EDTA [Fig. 6(c)]. Repeated washing of whole rod outer segments by low ionic strength buffers led to lossof most,of the minor proteins previously present and resulted in the samesimple protein pattern as seenin Fig. 6(c). Most of these soluble “minor” proteins could 1)~ washedout with isot,onic Ringer’s solution after the outer segmentshad been mechnnitally broken hy repeated freezing and thawing and this suggestst,hat they are cytoplasniic soluble proteins.

ISOLATED

The contracted

ROD

OUTER

SEGRIEKT

MEMBKASES

517

rod outer segments(Figs 7 and 8)

In all fractions and in all preparations, regardlessof the buffers used, a significant number of heavily stained particles are present (Fig-s 2, 4). These are generally about :! 10 pm long and l-2 pm thick and at higher magnification appear to be rod outer segments, in a highly contracted or condensed state (Figs 7, 8). Even in unstainetl

FIG. 7. “Contracted” rod outer segment derived from a phosphate-buffered gradient, fixed with glut :,I alclehyde and with osmium tetroside. L: lumen of the discs, I: space between the discs. Bar y 0.1 ~~~~). ( 14x 500). FII:. ~~nwws

8. ‘Y’ontrwted” indicate

loops

rod outer segment as in Fig. 7 but additionally of the discs. Bar = 0.1 pm. ( /: 148 500).

stained

with

katl

eit,ratv.

‘1’11~~

w&ions fixed only wit,h 0~0, (Fig. 7) electron densematerial is seenbetween t!he light profiler of the disc membranes. In stained sections the loop-like edgesof the disc:: a,r(b .~eenclearly (Fig. 8). ThevY are normally filled with dense material and the lumen of cliw in unstained sections is marked by the 3-5 nm thick electron-dense line with ;I club-shaped ending, 9 nm in diameter. The disc membrane is vLible only as a liw allout~3 nm thick alt8houghin stained sections, at the edgesof the t1iFc.sthe mem1)ran(b of the loops is about 6 nm thick. The repeat distance of the discs (centre to ccntre) i-: I?--15 mn. The distance between two adjacent discs (i.e. the thickness of the RIXJ nlatclrial between the light lines) is about 6 nm. After washing bhe preparations with sucrose-freebuffer, the number of the contracted organelles is greatly reducwl. Thc~! clisappear also after mild sonication and hvpot’onic shock a3 well a after trratmellt n-it11low concent,rations of digitonin. Se!g&w stnining (Figs 9-11). The three negative staining agents (Isedgave \-irtwtll!* itlentical results. In unfixed membranes. disc-like sbructures could not lw f’ountl after ntxg;lti\Te &a.ining; the membranes were transformwl iltt c) t,ubular antI \wic~ul;~.~

518

\s. I\I',El:s

ANI)

H. KiiHN

TSOLATED

ROD

OC’TER

SEGMEST

MEMBR.ASES

519

structures (Fig. 9) which were often fused together. After glutaraldchyde fixation c)f the suspended free discs, the structure of the membranes was stabilized t,o sustain the stress of the negative staining procedure (Fig. 10. 11). The rim of the discs was frequently broken away or opened and the double layer of membranes was then apparent. Fig. 10 shows such a disc which is doubled over at one edge. At the line ot the fold the negatively stained membranes can be seen in cross section (Fig. 11). Light subunits. 3-4 nm in diameter, can be discerned separated from each ot,her l)!dark channels 1.5-Y nm wide. The center-to-center spacing of the particles is approsimatclv 6 nm and the thickness of the membranes is 5-6 nm. Ko substructure ot’hcl than a random granularity can be seen in the plane of the membrane. No differenccb were observed between illuminated and unilluminated disc?. Globular subunits (4. null tlialuetcr) as seen in micrographs of Blasie, Worthington and Dewey (1969) occasiotrally were seen but only in tubular extensions of unfixed membranes. E’wcze fractwinq (Fiqs 12-15). Th e nomenclature proposed by Branton et, al. (I 975) is used to designate the fracture faces of the membranes. Thus, the t~sopI;~stuic~ leaflet of the split membranes is the outer part of the cytoplasmic membrane of thri rod outer segments or it is the inner half of the disc membrane. The fracture face t)t this leaflet is designated as EF. The ot,her half of the membrane is the plasmic leaflet. t,hc fract’ure face of which is designated as PF (Fig. 12). The exoplasmic half of the>

,

/

f/,‘I ,, “i

/’ /

FM:. 12. Schematic presentation of the fracture plamx qtopla~mic leatlrt of the split membrane: EF. fracture brane: I’M, plasma membrane; CYT, cytoplasm.

in a rod outw sezmrnt. face of the csoplasmic

j

i

PF. fructurv Iwflct: I)M.

fiwr r~t‘t ht. ~lisr ~~~III-

mcluhrane fixed in the dark was usually relatively smoot’h on its fracture face with sonle solitarv particles scattered over it (Figs 13-15). These particles appearecl tcb l)th nlore sparsely distributed in illuminated rod outer segments. This wws t.tle otrl! possit,le effect of light we found. However, due to the large variation of the uun~h~t OF particles per pm 2 between the different preparations (Tal~lt I) of unillunlin:~tr~tI uI(lml)ranes the significance of this finding is doubtful. FIG:. 9. Discs, unfixed, and negatively stained with phosphotungstic ttrcir original shape illustrating the highly fluid &ate of their mcmbranrs.

acid. The discs have chan& Bar = 0.2 pm. ( : 55 01~~).

FIG. 10. A disc fixed with glutaraldehyde and negatively stain& with phosphotungstic acid. ‘l’hc membrane now is stiffened and t,he original shape of the disc is preser\-ed. The rim (R) of the disc IS hrolwn and the double layer of membranes is evident. The upper right edge of the disc is f’ol~lcd ovw. lh

:~ 0.2 pm.

( .55

000).

I:Ic:. 11. Hiphw magnification of the upper right edge of the disc shown iiltll arlrl at the broken rim the membrane can be seen in cross stvti granularity (ahout 20 000 particles ~nl-~) in the frxcturc face of thca inner ltalf of’ tltcl disc8 mettthrane (Fig. 15). The fracture face of the plasntic leaflet of lwtlt tttetttl)rittt(i< \V;LS tlmselv covcred with part’icles to give a rough surface. In the plasma Illenll)ri~tl(~ it) oftw cotttaiiied round areas. approxiniat,ely 100 ntn in diameter. free of Icwticl~~< (Fig. I-L). Such areas were never present in the plastic fracture faw of tlw tliw metttl~twtc.

l~‘rc~c,n~~-/l~ri~rldiscs (Fig. 26). The surface of the discs displavecl of :tl)ottt fO 000 grains per pm2 which cannot he discerned

a VCI’V fine gr;tt~u tarit \front the l)~t(‘li~t7~lltt(l

(a)

(b)

C’ytoplasma (1) particks (2) solitary

membrane in PFt particles

in EFI

Disc membrane (1) part,icles in PFT (2) grains in EF$ (3) solitary particles in EFt unilluminated illuminated (4) big particles on the cptoplasmic surface

.5’,50( * 16UO) 500( lrwGo0)

6BOO( * 1300) 19 YOO( *lSuo)

IOU

I4 111

161) are causctl 1)~.rhotlopsin. Jan and Revel (1974) a 1so argue t)hat, these particles are relattvl t,o that ~I~(WIICY’ of rhodopsin within the disc meml,rane. F%ochemical evidence exist’s t,ltilt t~ltotlolJ4tt in intact discs is accessible from the cytoplasmic phase for proteinasvs (S;t;tri. I97 1 ; Trayhurn: Mandel and Virmaus, 197d) anal concanavalin A (Hteincnlan ant1 Str!,cst,. 1973). This might suggest that the particles in the fracbure fact of t’hc ~!+(tl)lilhtl Iit. leafld of the split membrane are the rhodopsin molecules. Howcvcxr t,hc size iltlt1 t Irr deiisity of the particles in the c~~t~CJ$LStllic leaflet of the discs dcviatcs frottr k-ttou tt data of the size and of the concentration of rhodopsin in the IllPlll~Jl.m6?. Ifro~l~ N-ra,~ (Blasie, Dewey. Blaurock and Wort,hingt,on. 1965) and I~iocltrnrical (Ihettrrvt. I973) data the average repeat distance of rhodopsin should be i mtl. Fronl t,ltis folio\\,\ ;I postulaktl density of rhodopsin of about, 20 000 pm ?. The particles of the cytoplwstttiv fracture face have a density of 5000-6000 pn+. an average repeat, tlistatlcv c~fal,c,llt 13 nni, ant1 a size of some 10 nm in diameter and thus inclivitld Ilarticlrv vannot correspond to single rhodopsin moleculrs. The fracture face of the inner half membrane of the discs ( = EF) displays a clistitxt. granularit,y (Fig. 15). The fine grains in this fracture face have st,rikingly the satttv density and awrage repeat distance which is postulated for a ltl(JlidayW of' rhocioltsiti in the membrane (about 20 000 pm m2.Table I). Is rhodopsin t’hen sitttat’e(1 itt t h(A luminal half of the disc membrane? Jan and Revel (1974) found that specific antibodies to rhotlopsin bind to both surfaces of the membrane and they favor t’hc il Itsa that the rhodopsin molecule spansthe membrane. Neutron diffract ion csperiments OII isolated frog rod outer segmenhsshow that most of rhodopsin is 1)urietl in the hvtlrophobic core of the membrane (Saihil, (%ahre a,nd Worcester. 1976). The foll&ing interpretation of our freeze-fracture results may be proposed: The elonga,tetlrhodopsin moleculesare pulled to the outer half during the cleavage of the nxmtxanes and thrcvt or four neighhouring rhodopsins then combine to form one big particle ou the surfac(x of the fracture face. The spots where the rhodopsin moleculeshad been anchored ill the inner half of the membrane are seen as fine grains. The basic similarity between the fracture faces of the clisc and the cytoplasmic membrane suggestsa similar molecular architecture for both. The (JLikr half of the cytoplasmic membrane corresponds ult~rastructurally to the inner lea!det of the disc membrane. Although rhodopsin apparently is accessible at the cytoplasmic surface (Jf the discs (Saari, 1974; Steineman and Stryer, 1973; Trayhurn et al.: 1974) no granularit! other than the background of the platinum shadow (60 000 grains,‘pnlm2)co~&l IX seenon the surface of the freeze-dried discs (Fig. 16). This suggeststhat the external part of rhodopsin is relatively smoothly integrated into the surface of the t&c. The nature of the big particles on the disc-surface is obscure. ACKNOWLEDGMENTS Part of this work wassupported by SFB 160 of the DeutscheForschungsgemeinschaft. We thank Miss S. Bader, Miss M. Kuhnen und Mrs G. Theisenfor technical assistance,

ISOLATED

ROD

OCTER

SEGSIEST

MEJIBKASES

525

and Mr A. Harnacher for the weekly collection uf eyes frotn the slaughterhouse. One of us (K. I(.) thanks Dr Walther Stoeikenius for t,he privilege of working in his laboratory, Cardiovascular Research Institute of the University of California in San Francisco. His inspiring suggestions are gratefully acknowledged. Jly thanks further due to the people who shared space and equipment with me in the “surge unit” of the CVRI. EspecialI!, helpful have been *Town Ventura and 1stva.n Mare. REFERIEXCES Hlasir,

,J. K., Dewey. M. M., Bl aurock. A. E. and 1Vorthington.