Proc. Natd Acad. Sci. USAVol. 79, pp. 3208-3212, May 1982Cell Biology

Lenticular intermediate-sized filaments: Biosynthesis andinteraction with plasma membrane

(lens cytoskeleton/vimentin/a-crystallin/cytoskeletal protein biosynthesis)

F. C. S. RAMAEKERS*t, I. DUNIAt, H. J. DODEMONT*, E. L. BENEDETTIt, AND H. BLOEMENDAL*§*Department of Biochemistry, University of Nijmegen, Geert Grooteplein Noord 21, 6525 EZ Nijmegen, The Netherlands; and *Institut de Biologie Moleculaire duCentre National de la Recherche Scientifique et de l'Universit6 Paris 7, Paris, France

Communicated by Severo Ochoa, February 19, 1982

ABSTRACT Electron microscopical features of the lens fiberplasma membrane-cytoskeleton complex are suggestive of an in-timate association between the intermediate-sized filaments (IF)and the lipid bilayer. Biochemical analysis of this complex revealsthe occurrence of an appreciable amount of vimentin as a proteinsubunit of lenticular IF. Additional evidence for association be-tween IF and membranes is provided by the observation thatnewly synthesized vimentin is associated with plasma membranesadded to a reticulocyte lysate programmed with lens polyribo-somes. Concomitantly a-crystallin polypeptide chains (aA2) arealso found associated with the plasma membrane together with ahitherto unidentified 47-kilodalton protein. Once associated withthe lipid bilayer, the vimentin polypeptide resists urea treatment,suggesting that it has become an integral constituent associatedwith part of the membrane.

Better insight into the function ofthe cytoskeleton in nonmusclecells requires elucidation of the nature of the link between cy-toskeletal motility proteins and the cell membranes (1).Whether or not protein subunits of the cytoskeleton are con-tained within the lipid membrane core as integral membranecomponents is still an open question.

Recently great interest has been focused on the intermediate-sized filaments (IF) (2), which, together with microtubules andactin filaments, constitute the intracellular matrix of eukaryoticcells. IF have been subdivided into five classes according to theembryonic origin ofthe cell type and the nature oftheir proteinsubunit (3).

IF have also been described in intact lens, where they con-tribute to the organization of the cytoskeleton of both the epi-thelial cells and the lens fibers (4-6). Moreover, we have dem-onstrated the presence of IF in cultured lens cells (6-8).Immunofluorescence studies and biochemical data indicatedthat the protein moiety of these IF is vimentin (6). Electronmicroscopical evidence has been provided for an end-on at-tachment of IF to the cytoplasmic site of the lens fiber plasmamembranes (9). The link ofIF with various types ofmembraneshas been postulated as a nucleation site for filament formation.These reports, however, are mainly confined to the interactionof prekeratin IF with desmosomes (10), of desmin IF with theintercalated disks in muscle (11), and vimentin IF with the nu-clear membrane (12).

In this paper we show that the lenticular IF are intimatelyassociated with the plasma membranes. Moreover, we dem-onstrate that vimentin newly synthesized in vitro becomes as-sociated with the lipid bilayer when purified lens fiber plasmamembranes are added to the translation system.

MATERIALS AND METHODS

Isolation of Lens Fiber Plasma Membranes. Fresh calf len-ses were decapsulated and homogenized by continuous stirringfor 2 hr in either bicarbonate buffer (1 mM NaHCO3/1 mMCaCl2) or TKM buffer (50 mM Tris-HCl, pH 7.4/25 mM KCV5mM MgCl2).

Plasma membranes were isolated by discontinuous sucrosegradient centrifugation as described earlier (13) in either bicar-bonate or TKM buffer. The plasma membrane fractions ob-tained at sucrose densities 1.20-1.22 g/cm3, 1.18-1.20 g/cm3,and 1.16-1.18 g/cm3 were washed three times in the respectivebuffers and sedimented in a Beckman Ti 50 rotor.

In the isolation procedure using TKM buffer, a membrane-containing layer is formed only at 1.20-1.22 g/cm3. Proteinconcentrations were determined by the Lowry method (14).De Novo Synthesis of Lens Proteins. Calf lens polyribo-

somes were isolated as described by Bloemendal et aL (15).Rabbit reticulocytes were prepared as described either by Ev-ans and Lingrel (16) or by Pelham and Jackson (17). A 30,000x g supernatant fraction of the lysed cells was used as the cell-free system, and incubations were performed at 30°C for 90 min(cf. ref. 18).The lysate was made mRNA dependent by preincubation

with micrococcal nuclease at 10 pg/ml in the presence of 1 mMCaCl2 for 15 min at 20°C. After the incubation 2mM EGTA wasadded to chelate the calcium ions. Polyribosomes were addedin a concentration of 0.4-1 mg/ml (determined spectrophoto-metrically and assuming an extinction coefficient of 13 ml/mgper cm at 260 nm).

Incubation and Reisolation of Lens Plasma Membranes.Ninety minutes after addition of lens polyribosomes the retic-ulocyte cell-free incubation mixture was centrifuged for 10 minat 10,000 X g and supplemented with purified lens plasmamembranes, isolated in either bicarbonate or TKM buffer. Insome experiments membranes isolated in bicarbonate that wereexhaustively washed with 6 M urea were used. The total mix-ture was incubated as indicated at 30°C with occasional stirring.When membranes isolated in TKM buffer were added to thereticulocyte cell-free system, the ionic conditions of this incu-bation were adjusted to the salt concentrations ofthe TKM buf-fer. Then the membranes were reisolated in bicarbonate orTKM buffer as described above or simply by centrifugation ina minicentrifuge. The membrane pellets were dissolved for gelelectrophoretic analysis.

Abbreviations: IF, intermediate filaments; kDal, kilodalton(s); IEF, iso-electric focusing.t Present address: Dept. ofExperimental Pathology, St. Radboud Hos-pital, Geert Grooteplein Zuid 24, 6525 GA Nijmegen, The Netherlands.

§ To whom reprint requests should be addressed.

3208

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. NatL Acad. Sci. USA 79 (1982) 3209

Polyacrylamide Gel Electrophoresis. Analysis of the poly-peptides was performed by NaDodSOJpolyacrylamide gelelectrophoresis according to Laemmli (19) with the modificationthat slab gels instead of gel rods were used.The gels were 12 cm long and contained either a NaDodSOJ

13% acrylamide gel or a 7-18% acrylamide gradient with 0.4%methylenebisacrylamide and 0.1% NaDodSO4. In this methoda stacking gel was applied. Staining and destaining were per-formed as described by Weber and Osborn (20).

Gels were processed for autoradiography. For the detectionofthe labeled proteins the procedure ofBonner and Laskey (21)was used in combination with the drying procedure describedby Berns and Bloemendal (22). Two-dimensional gel electro-phoresis was performed according to O'Farrell (23).

Electron Microscopy. Electron microscopic observations onthe isolated lens fiber plasma membrane cytoskeleton complexwere carried out on thin sections fixed in glutaraldehyde/os-mium tetroxide and embedded in Vestopal (24) or by the neg-ative staining technique. Freeze-fracture experiments were

performed on isolated fractions that were rapidly frozen in liq-uid Freon 22. The freeze-fracture replica was obtained in aBakzers BAF-301 apparatus. The unfixed specimens were frac-tured at - 150'C. The replica was made immediately after thefracture by evaporation of platinum and carbon, using an elec-tron gun (25). A Philips EM 400 microscope was used.

RESULTSElectron microscopical observation demonstrates a strildng dif-ference when the lens plasma membranes instead ofbeing iso-lated in sodium bicarbonate are fractionated under cytoskeletonstabilizing conditions CJKM buffer) (9). In the former methodthe membranes are practically devoid of filamentous structures.Conversely, when the cytoskeletal organization is preserved IFand actin filaments are clearly visualized in close associationwith the cytoplasmic site of the membrane (Fig. la).

In Fig. lb a high-resolution electron micrograph of isolatedIF is shown. From this picture and at higher magnification in

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FIG. 1. (a) Transmission elec-tron microscopic observation of theisolated lens fiber plasma mem-brane-yskeleton complex. IF (10nrm) are running in different direc-tions and have end-on attachmentsto membrane profiles (arrows). (bandc) Isolated IF negatively stainedby 1% uranyl acetate. Note the sub-unit organization of the filaments,probably consisting of four subfila-ments (arrows). (d) Freeze-fracturereplica of the isolated lens fiberplasma membrane-cytoskeletoncomplex. The arrows point to rod-like structures that apparently havean end-on association at the pro-toplasmic fracture face (PF), stud-ded by intramembranous particlesof heterogeneous size. Rodlikestructures are also visible far fromthe membranes (arrowheads). Theaverage diameter of the rodlike en-tities is about 12 nrm.

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3210 Cell Biology: Ramaekers et aL

Fig. Ic the subunit organization of these structures becomesclearly visible, showing that each IF is built up probably byfour subfilaments.The intimate association between IF and membrane leaflets

is further demonstrated by freeze-fracture aspects of the lensfiber plasma membrane cytoskeleton complex. In freeze frac-ture replicas rodlike structures, most probably correspondingto longitudinal fractures of IF, appear mainly associated to theprotoplasmic membrane fracture face (PF), where they areclosely packed with intramembranous particles (Fig. ld). Therods are continuous with filaments found in the cytoplasmicspace where the fracture aspects of the cytoskeleton structureare visible.NaDodSO4 gel electrophoretic analysis of the cytoskeleton

membrane complex shows a very strong band migrating in themolecular weight region of vimentin (Fig. 2). Less heavilystained bands represent the a-crystallin subunits, actin, and a

Sucrose density gradient

1.14

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FIG. 2. Final step of the purification of lens fiber plasma mem-branes in sucrose density gradients. In one case the plasma membraneswere isolated in 1 mM NaHCO3 at low temperature. Under these con-ditions electron microscopic observation showed that the membranefractions gathered at the interface between 1.16 and 1.14 g/cm3 werealmost completely devoid of filamentous structures. Some remnantsof the cytoskeleton were found in the plasma membrane fraction gath-ered at the interface between 1.18 and 1.20 g/cm3. The protein profileof the pool of membrane fractions isolated in NaHCO3 indeed con-tained a minor component comigrating with actin and practically novimentin. The major polypeptides were the intrinsic membrane com-ponents MP26 and MP34, and a-crystallin polypeptides (aB and aA).MP34 is represented by a doublet. When cytoskeleton-stabilizing con-ditions (TKM) were used during isolation and the whole procedure wascarried out at 15°C, the NaDodSO4 gel electrophoretic pattern of theplasma membrane-cytoskeleton complex was characterized by a pre-dominant band in the 57-kDal region, comigrating with vimentin.Another protein band comigrates with actin.

47-kilodalton (kDal) polypeptide. The lens fiber membrane pro-teins MP26 and MP34 (26) are masked by the predominanceof the cytoskeletal polypeptides. Only after removal of the cy-toskeleton by bicarbonate washings and subsequent reisolationby flotation of the membranes do the MP26 and a doublet inthe 34-kDal region become clearly visible on the electropher-ogram (Fig. 2).The cytoskeletal polypeptides were further characterized by

two-dimensional gel electrophoresis (Fig. 3). It can be seen thatvimentin is the most abundant component and that the 47-kDalpolypeptide differs from actin in its isoelectric point. The 100-kDal polypeptide coincides with a-actinin.

Because the morphological evidence was suggestive for anintimate association between plasma membranes and IF, weexamined the fate of newly synthesized vimentin in the pres-ence ofpurified lenticular plasma membranes in vitro. For thispurpose lens fiber polysomes were translated in a reticulocytelysate. Two-dimensional gel electrophoresis showed that thepolyribosomes direct the synthesis not only of crystallins butalso ofcytoskeletal polypeptides. Among the latter polypeptidesvimentin is again predominant (Fig. 4a). Thereafter the follow-ing recombinant experiments-were carried out. The lens poly-some/reticulocyte lysate mixture was supplemented, upon 90min ofincubation, with purified plasma membranes from whichthe cytoskeleton was detached by bicarbonate washings. In-cubation was continued for 1 hr and the membranes were re-isolated by flotation. Two-dimensional electrophoretic analysisyielded the pattern shown in Fig. 4b. Obviously, only a fewcomponents have been selected from the protein populationsynthesized de novo, namely the major a-crystallin chain A2 andthe IF constituent vimentin. In addition the yet-unidentified47-kDal protein also becomes associated with the plasmamembranes.When urea-extracted membranes were used in the recon-

stitution experiments it could be shown that these membranesalso are capable of selectively sorting out newly synthesizedtranslation products (Fig. 5). Of the products of translationshown in lane b, vimentin preferentially interacts with themembranes (Fig. 5, lane c). Also actin, MP47, aA, and IBla aresorted out, but although other ,B-crystallins were synthesizedde novo in appreciable amounts, they were not found to be as-sociated with the membrane. This latter result indicates that

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FIG. 3. Two-dimensional gel electrophoretic analysis of the iso-lated plasma membrane-cytoskeleton complex. Gel electrophoresiswas carried out by isoelectric focusing (IEF) in 9 M urea followed byelectrophoresis in 0.1% NaDodSO4 (13% polyacrylamide slab gel). Onemajor component comigrates with vimentin (V). Minor components areactin (A) and aA2-crystallin. The 47- and 100-kDal polypeptides arealso indicated.

Proc. Nad Acad. Sci. USA 79 (1982)

-aB

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Proc. Natd Acad. Sci. USA 79 (1982) 3211

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FIG. 4. (a) Two-dimensional gel electrophoretic pattern of theproducts obtained by translation of free lens fiber polyribosomes. Notethatamong these products both vimentin (V) and actin (A) are present.T, tubulin. (b) Lens fiber plasma membranes isolated in bicarbonateand thoroughly washed in the same medium were incubated for 1 hrwith the products of translation of the lens polyribosomes in a retic-ulocyte cell-free system. After reisolation, even after repeated washingin bicarbonate buffer, the membranes selectively retain vimentin, aA2,and MP47 but not actin and other polypeptides visualized in a.

there is, at least for this class ofproteins, no exchange betweenpreexisting and newly synthesized polypeptides but a selectiveassociation. The gel pattern (Fig. 5) also shows that the newlysynthesized vimentin becomes attached to the lipid bilayer andresists further urea extraction (Fig. 5, lane d). Crystallins, on

the other hand, are partially solubilized by this treatment.An interesting feature ofthis association ofnewly synthesized

protein is shown in Fig. 6. This gel pattern shows the time de-pendence of the association process for the individual transla-tion products. Whereas there is little or no increase of the vi-mentin and the MP34 doublets after 10-min incubation, thereis a clear increase in the sorting out of aA, aB, and /Bia poly-peptides with increasing, time of incubation. Unlike vimentin,actin association becomes detectable only after 60 min of in-cubation; thereafter it remains virtually constant. The same

holds true for a high molecular weight component that coincideswith a-actinin.

DISCUSSIONOur present reconstitution experiments provide evidence foran intimate association of newly synthesized motility protein,

a b c d

FIG. 5. Reconstruction experiment using lens plasma membranes,thoroughly washed with urea. Lane a, stained pattern of a NaDodSO4gel electrophoretic analysis of the membrane fraction used in this ex-periment. Note that besides MP26 virtually no protein bands are pres-ent. Lane b, autoradiograph of a NaDodSO4 gel electrophoretic anal-ysis of products newly synthesized in a reticulocyte cell-free systemunder the direction of lens polyribosomes (jS indicates ,-crystallin sub-units, aA2 and aB2 are the two subunits of a-crystallin). Lane c, au-toradiograph of a NaDodSO4 gel electrophoretic analysis of radioac-tive polypeptides that interact with the plasma membrane fractionshown in lane a when incubated with the products shown in lane b for3 hr. V, vimentin; A, actin. Lane d, like lane c, but the reisolated mem-branes were washed with urea before gel electrophoretic analysis.

in particular vimentin, with the membrane. The same holdstrue for MP34, which is an integral urea-insoluble plasma mem-brane protein found in both lens epithelium and fiber cells.These proteins vanish after protease treatment (27). However,the fact that they are not affected by urea favors the assumptionthat vimentin and MP34 interact strongly with the lipid bilayer,although the inserted sequence of the molecule may be rathersmall. Thus, sensitivity to proteolytic enzymes may not nec-

essarily mean that newly synthesized polypeptides do not be-come associated with the membrane as an integral constituent.A still unresolved problem concerns the mechanism of in-

teraction. There is as yet no evidence that the proteins sortedout from the bulk of translation products contain a "topogenic"amino acid sequence (28) that would favor integration with thehydrophobic core of the membrane. At least for a-crystallinchains whose complete primary structure is known (29), asso-ciation with the membrane does not seem to require a specificsignal sequence. We are aware that in other membrane systemsthe assembly of protein components synthesized de novo mayfollow a different design principle. In one model, membraneprotein assembly may occur even when the translation is com-pleted at a distance from the membrane target (30). In the othermodel, close proximity of the lipid bilayer is necessary shortlyafter initiation ofthe protein (31). At least the lenticular proteinsvimentin and MP34 follow the first model, because they rapidly

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3212 Cell Biology: Ramaekers etaP

M.

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10 30 60 90 120 180

FIG. 6. Time dependence of invitro insertion of newly synthesizedlens polypeptides into the plasma membrane. Times (min) are indi-cated under each lane. Labels as in other figures.

associate with the lipid bilayer after translation is completed.Itds tempting to speculate that the difference in association

rates of the various translation products reflects the sequenceand specificity of their interaction with the plasma membrane.It is, however, not readily apparent whether association of a

particular polypeptidewith membranes depends upon the pres-ence ofother protein factors-e.g., the 47-kDal component thatpresumably also occurs in'other cell types (32). aA2-Crystallinmay also represent such a factor. Obviously vimentin interactsvery rapidly -with the plasma membrane, and the amount ofassociated subunits does not increase with prolonged incuba-tion time. For other proteins e.g., aA-, aB-, and (3Bla-crystallin-such an increase is clearly demonstrated (compareFig. 6). The fact that urea treatment cannot completely removenewly synthesized proteins, such as vimentin and MP34, re-

flects their strong interaction with the plasma membrane.Moreover, we cannot rule -out that remnants of vimentin or

other cytoskeletal constituents may act as nucleation sites forfurther vimentin assembly at the plasma membrane level.

As for interaction ofother components with the plasma mem-branes, the A2 subunit ofa-crystallin deserves special attention.Evidence is accumulating that this polypeptide attaches to het-erologous and homologous membranes (18, 33). The followingmechanism seems to be operative in the lens. Initially newlysynthesized aA2 subunits interact with the plasma membraneand thereafter the aB2 chains join the complex to form the a-

crystallin high molecular weight aggregate. This model is con-

sistent with a proposal for the architecture ofnative a-crystallin(34) and studies on its biosynthesis in heterologous cell-free sys-tems or oocytes (35), where it was found that a core ofaA2 chainsis required before aB2 chains can copolymerize to form the aaggregate.

This work was carried out under the auspices of the NetherlandsFoundation for Chemical Research (SON) and with financial aid fromthe Netherlands Organization for the Advancement of Pure Research(ZWO). E.L.B. was a recipient of a visitor fellowship from the latterorganization.

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