J. Cell Set. 43, 253-267 (1980) 253Printed in Great Britain © Company of Biologists Limited 1080

THE POLYPEPTIDES OF RAT LIVER NUCLEAR

ENVELOPE

I. EXAMINATION OF NUCLEAR PORE COMPLEXPOLYPEPTIDES BY SOLID-STATE LACTOPEROXIDASELABELLING

JONATHAN C. W. RICHARDSON* AND ALUN H. MADDYUniversity of Edinburgh, Department of Zoology,West Mains Road, Edinburgh, EHg jjfT, Scotland

SUMMARY

Purified nuclei retaining a high degree of ultrastructural integrity were isolated by conven-tional centrifugation techniques. The cytoplasmic surface of these nuclei was iodinated usinglactoperoxidase immobilized onto giant Sepharose beads; thus the outer nuclear membraneand the cytoplasmic surface of nuclear pore complexes were selectively labelled. Pore complexesin association with a fibrous lamina were isolated from these nuclei by removal of the nucleo-plasm and extraction with Triton X-100. The chemical composition of the pore-laminafraction was 93-6 % protein, 6 % RNA, 0-4 % phospholipid. The labelling suggests that majorpolypeptides Ni (70000) and N2 (67000) and more than 10 other more minor polypeptides,ranging from 33000 to 200000 mol. wt, as being components of the nuclear pore complex.Polypeptide N3 (58000) is shown to be present only on the nucleoplasmic face of nuclearenvelopes, probably in the fibrous lamina.

INTRODUCTION

The nuclear envelope is a characteristic feature of the eukaryote cell. It comprises2 concentric membranes, pore complexes which punctuate the membranes and anunderlying fibrous lamina. The outer nuclear membrane is continuous with the innernuclear membrane at the level of the pore complex (Watson, 1955), bears ribosomes(Watson, 1955; Palade, 1955) and provides a surface to which structural elements ofthe cytoplasm may attach (for references see Franke & Scheer, 1974). The innernuclear membrane closely apposes the fibrous lamina (Fawcett, 1966; Aaronson &Blobel, 1975), which is contiguous with the pore complexes and represents theperipheral aspect of the nuclear matrix (Berezney & Coffey, 1977).

The nuclear pore complex is not a discrete structure (which is why a bulk methodfor its isolation has proved elusive) for it is connected both with the fibrous laminaand with the inner and outer nuclear membranes at their point of fusion. Its structurehas been studied in detail for more than a decade, and the main reason it has attractedso much interest is that it is widely believed to be the principle pathway by which

• Present address: Department of Physiology and Pharmacology, University of St Andrews,Bute Medical Buildings, St Andrews, Fife, Scotland.

254 3- C. W. Richardson and A. H. Maddy

nascent RNA is transported from the nucleus to the cytoplasm. Despite at least 2attempts (Aaronson & Blobel, 1975; Harris, 1977), the pure pore complex has not beenisolated from mammalian cells although a highly enriched fraction has been obtainedfrom Xenopus laevis oocytes (Krohne, Franke & Scheer, 19786). Analysis of thenuclear envelope's constituent polypeptides has extended little beyond the relativelytrivial establishment of its electrophoretic profile. There is a great deal of informationabout enzyme activities associated with nuclear envelope preparations (for refs seeFranke, 1974a, b; Harris, 1978), some of which may catalogue cross-contaminationfrom other membrane systems, but such information loses much of its value in theabsence of detail as to the location of components.

We have therefore sought to develop a suitable labelling method to study theorientation of the polypeptides. Several methods have been developed for labellingthe cell surface (Hubbard & Cohn, 1976; Hynes, 1976) but none of these may beapplied immediately to the labelling of the nuclear surface. The great structuralcomplexity of the nuclear envelope and its permeability, even to macromolecules(Bonner, 1975a, b; De Robertis, Longthorne & Gurdon, 1978; Paine, Moore &Horowitz, 1975), set problems which are without parallel in a membrane-labellingstudy. Simple chemical probes and soluble enzyme labelling methods are suspectsince they might gain access both to the cisternal surfaces of the 2 membranes and tothe nucleoplasm (via the pore complexes). We have overcome this problem byimmobilizing the labelling enzyme lactoperoxidase onto giant Sepharose beads. Asiodination catalysed by lactoperoxidase takes place at the enzyme surface via anindiffusible activated I~ the iodinating sites are thus confined to beads several timeslarger than the nuclei. The beads are too large to pass through the pore complex orinto the perinuclear cisternum of isolated nuclei and are restricted to the nuclearsurface where their lactoperoxidase may catalyse the radio-iodination of pore complexand outer nuclear membrane polypeptides. In conjunction with a procedure forisolating pore complexes in association with the fibrous lamina, such labelling hasenabled examination of pore complex and fibrous lamina polypeptides. It has alsopermitted the comparison of outer nuclear membrane and rough endoplasmic reticulumpolypeptides of the same membrane plane, which is to be reported in a subsequentpaper.

MATERIALS AND METHODS

Preparation of nuclear envelopes

Nuclei from rat livers were isolated by a dense sucrose procedure similar to that of Kay,Fraser & Johnston (1972).

Nuclear envelopes were prepared by a double DNase digestion procedure modified from.Kay et al. (1972).

1st DNase digestion (pH 8-5)

A pellet of nuclei derived from. 20 g of rat liver was resuspended by the addition of a fewdrops of glycerol and vortexing. To the suspension were added, with vigorous vortexing, 75 mlH,O followed by 375 fi\ DNase 1 (100 fig/ml H2O; Sigma type DNEP) and 30 ml of a solution

Polypeptides of rat liver nuclear envelope. I 255

of 10% sucrose, 10 mM Tris.HCl, o-i mM MgCla, and o-i mM PMSF, pH 8 5 . The mixturewas incubated at 22 °C for 15 min with vortexing every 5 min. After 15 min the digestion wasslowed by the addition of 40 ml ice-cold distilled water and the suspension centrifuged at40000 g mMX for 15 min at 4 °C yielding a supernatant and pellet (DNAt pell).

2nd DNase digestion (pH 7-5)

The pellet was resuspended, using a syringe and fine-gauge needle, into 75 ml of a solutionof 10 % sucrose, 10 mM Tris.HCl, 0 1 mM MgCls and o-i mM PMSF, pH 7-5. To this suspen-sion 375 fi\ DNase (100 fig/ml) were added. After incubation for 20 min at 22 °C the digestionwas slowed by the addition of 9 ml ice cold distilled water and the suspension centrifuged for10 min at 4 °C at 20000 g „ . in a 10 x 10 titanium rotor (MSE rotor 43114-128), yielding asupernatant and a pellet (DNAa pell) of nuclear envelopes.

Isolation of pore-complex lamina fraction (Modified from Dwyer & Blobel, 1976)

Triton X-100 wash of nuclear envelopes: The DNase-digested pellet (DNA,pell) wasthoroughly resuspended into 2-5 ml of ice-cold solution of 10% sucrose, 0 1 n w MgClj,10 mM Tris.HCl, pH 7 5 to which 025 ml 20 % (v/v) Triton X-100 (British Drug HousesScintillation grade) was added with vortex mixing. Incubation of the mixture on ice for 10 minfollowed by centrifugation for 10 min at 4 CC at 200000 g ,T. in the 10x10 titanium rotoryielded a supernatant and a pellet of crude pore laminae.

High salt extraction

The resulting pellet was gently, but thoroughly resuspended into 0-2 ml 10% sucrose,o-i mM MgCla, 10 mM Tris.HCl pH 7-5. Homogeneous resuspension was essential. (If thepellet was resuspended directly into the high salt medium, it tended to clump and the prepara-tion remained contaminated with nucleoplasm). To this suspension were added 25 ml 10%sucrose, 2-0 M NaCl, 0 1 mM MgCl2, 100 mM Tris.HCl pH 7 5 . Incubation of the mixture for10 min on ice, followed by centrifugation as above yielded a pellet of purified pore complex-lamina fraction.

Lactoperoxidase labelling

Iodination conditions. Iodination conditions for the complete system per millilitre of finalsolution: nuclei from 0-333 S liver, 1 fimol glucose, 33 fig lactoperoxidase coupled onto Sepha-rose in the ratio of 1-33 mg LPO per ml of settled beads (see below), 0-7 fig glucose oxidase(Sigma, Type V), 33 fid N a m l (Amersham Radiochemicals) in 10% sucrose, o-oooi %butylated hydroxytoluene (from a stock of 0 5 % in ethanol), 20 / J M K 1 I J I , I mM glucose,10 mM Tris.HCl, pH 7 2 . Incubation was 12 min at 23 CC in a test tube rotating end-over-endat 4 rev/min. The reaction was stopped by the addition of an equal volume of ice-cold stopperbuffer (10% sucrose, 0 0 0 0 1 % butylated hydroxytoluene (Welton & Aust, 1972) 20 fiM 3-amino, 1,2,4-triazole (Harris, 1978), 20 fiM sodium sulphite, 10 mM Tris.HCl, pH 72) . Themixture was filtered through 80-fim mesh nylon gauze to remove the Sepharose beads, under-layed with 1 vol. of 20 % sucrose, 10 mM /?-mercaptoethanol in stopper buffer, and centrifugedto pellet the nuclei (1000 g for 10 min at 4 °C in a 6 x 100-ml swing-out rotor). The supernatantwas discarded, and the nuclei were washed twice in 2 vol. 10% sucrose in stopper buffer bypelleting at 700 g for 5 min in the same rotor at 4 °C.

Immobilization of lactoperoxidase. One gramme of CNBr-activated Sepharose 6MB (Phar-macia Fine Chemicals) was swollen in a beaker and washed for 15 min on a glass fibre filterwith 1 mM HC1 (200 ml). Lactoperoxidase (Sigma, lyophilized powder) dissolved in o-1 M sodiumphosphate buffer (pH 72), was mixed with the gel in a test tube, and the mixture rotated end-over-end at 4 rev/min overnight at 4 °C. Unbound material was washed away with 200 mlphosphate buffer (coupling efficiency was always greater than 999 %), and any remainingCNBr groups were reacted with 1 M glycine for 2 h at room temperature. Three washingcycles were used to remove any possible non-covalently adsorbed protein (none was everdetected), each cycle consisting of a wash in 0 2 M sodium phosphate buffer (pH 7-2) followed

256 J. C. W. Richardson and A. H. Maddy

by a wash in 1 M glycine. Lastly, the beads were washed with 200 ml 10 % sucrose, 1 mM MgClj,0-2 mM NaHCO3 (pH 7-4) and stored for up to 4 h prior to use.

Radioactive counting. TCA-precipitated samples on glass fibre disks were counted in aNuclear Enterprises gamma counter (efficiency about 75 %).

Assay methods

Protein was assayed by the method of Lowry, Rosebrough, Farr& Randall (1951) with bovineserum albumin as standard.

DNA was measured by Giles and Myers modification (Giles & Myers, 1965) of the methodof Burton (1956), with deoxyadenosine monophosphate as standard.

RNA was assayed by a modification of the orcinol method (Richardson, 1979).Phospholipid phosphorous was determined according to Chen, Toribara & Warner (1956)

on lipid samples extracted according to Bligh & Dyer (1959) and evaporated to dryness.Succinate dehydrogenase activity of freeze-thawed and briefly sonicated samples was assayed

by the reduction of phenazine methosulphate (Singer, 1975).

Poly aery lamide gel electrophoresis

Samples were prepared for electrophoresis by precipitation in 2 vol. ethanol at — 20 °C for16 h in order to decrease the presence of detergent and salts. The alcohol precipitate waspelleted and resuspended into 5 vol. of a solution containing 3 % w/v SDS, 5 % v/v 2-mercap-toethanol, 20% v/v glycerol, 1 mM EDTA and 62-5 mM Tris.HCl (pH 6-8). The sampleswere then incubated for 10 min at 70 °C and for 5 min at 100 °C. Particulate material remainingafter this time was removed by centrifugation at 3000 g mtI for 5 min.

Analytical SDS /'polyacry lamide — gel electrophoresis was carried out in the buffersystem of Laemmli (1970) in vertical slab gels (15 x 15 x 02 cm) cast between glass plates,comprising a i-5-cm stacking gel (375 % w/v acrylamide, o-i%w/v N.AP-methyleneiu-acrylamide) and a 13-5-0x1 resolving gel (16 % w/v acrylamide, 0-09 % w/v A .̂A '̂-methylene-ftiracrylamide). The gels were polymerized by addition of ammonium persulphate and N,N,N',-iV-tetramethylethylenediamine. After electrophoresis gels were fixed and stained accordingto Fairbanks, Steck & Wallach (1971).

Gels were dried onto thick filter paper under vacuum at 90 °C. Dry gels were either exposeddirectly to X-ray film (Kodak X-Omat H film) or the film was first flash exposed, backed withan intensifying screen (Ilford Fast Tungstate) and closely apposed to the gel in a cassette at— 70 °C for approx 3 days. E. coli /?-galactosidase (130000), bovine serum albumin (68000),chick brain tubulin (55000), rabbit muscle actin (46000) and lactate dehydrogenase (35000),bovine /?-lactoglobulin (17500) and heart cytochrome C (12500) were used as standards formol. wt determinations.

Lipid chromatography

Lipid extracts were run in 2 dimensions (Zwaal & Roelofsen, 1976) on 8 x 8 cm TLC plates(Polygram SIL NHR, Camlab). The distribution of radioactivity was determined by auto-radiography.

Electron microscopy

Samples were fixed with glutaraldehyde and osmium (Dwyer & Blobel, 1976), embedded inAraldite and sectioned using a diamond knife. Sections were stained with uranyl acetate(Watson, 1958) and lead citrate (Reynolds, 1963).

Electron micrographs were taken with a Philips EM 300 operating at 80 kV. Kodak ' Estar'sheets were used in the camera.

Polypeptides of rat liver nuclear envelope. I 257

Morphometry

Morphometric determinations of nuclear integrity and membranous contamination werecarried out on electron micrographs taken at magnifications between x 3000 and x 5000(Richardson, 1979)- Negatives were displayed on a microfilm reader and examined at magni-fications between 6-5 and 17-5 diameters. Length measurements were made at 6-5 diametersusing a ' Map Measure' and converted to microns original membrane.

Nuclear membranes were classified as membrane profiles which were associated with nuclearchromatin in at least one site, and which contained pore complexes (Franke et al. 1976). Thecircumference of very small vesicles was approximated to 3 times the longest axis.

RESULTS AND DISCUSSION

Preparation of nuclei

Nuclei were prepared at a yield greater than 75 % (estimated by the recovery ofDNA) by chemically mild means. Electron micrographs of purified nuclei showedlittle cytoplasmic membrane contamination and the nuclei were rounded and showedonly minor disruption of the nuclear envelope. Succinate dehydrogenase activity of

Table 1. Morphometric measurements

Membrane profile lengths, /«ni * v Nuclear

Nuclear preparation nuclear other mem/total, %

1 682 24 972 678 41 943 849 72 924 IO94 83 93

Values are expressed in microns of membrane profile. Nuclear membranes were classedas aJl membranes profiles which were associated with nuclear chromatin in at least one site andwhich contained pore complexes (Franke et al. 1976).

the preparation was typically 0-5 units (/miol dichloroindophenol reduced per minper mg of protein) i.e. less than 0-5 % of the activity of purified mitochondria. Theproportion of membrane profiles which in thin section were clearly nuclear membraneprofiles was determined by morphometric means (see Table 1); on average, this was94% - a value close to that of Franke et al. (1976).

Iodination of nuclei

It was important to establish that the iodination procedure did not strip away largeportions of the outer nuclear membrane and that the conditions of iodination weresuch as to provide a high specific activity. Morphometric determinations of theproportion of outer nuclear membrane covering the nuclear surface were made beforeand after the iodination reaction (Table 2). Approximately 89 % of the nuclear surfacewas covered with outer nuclear membranes prior to iodination, and this value wasnot significantly different in nuclei recovered from the iodination reaction. After

258 J. C. W. Richardson and A. H. Maddy

iodination, the nuclei were still largely rounded, showed intact pore-complexes, andretained their ribosom.es on the outer nuclear membrane (Fig. 1). The outer nuclearmembrane occasionally showed an increased tendency toward a bleb separation fromthe inner nuclear membrane; a phenomenon not generally seen prior to iodination(although see Kartenbeck, Jarasch & Franke, 1973). This was not seen in all prepara-tions and the outer nuclear membrane frequently remained closely apposed to theinner nuclear membrane after iodination.

The nuclei were iodinated to a level of io6 cmp/mg protein. Deletion of either theperoxide-generating system or lactoperoxidase resulted in less than 3-5% of theiodide incorporation achieved under standard iodinating conditions. The inclusion ofcarrier iodide in the reaction was essential both in order to obtain a satisfactory levelof radioiodination and to ensure that the ratio of lactoperoxidase-dependent to

Table 2. Morphometric determination of the extent to wJiich the innernuclear membrane is covered by outer nuclear membrane in purified nucle i

before and after the iodintaion reaction

Preparationof

nuclei

Profile lengths, /ttn,measured before

iodination" , ONM/%

Nuclear Outer Nuclearsurface membrane surface

Membrane profile lengths,fim, measured after

iodination

Nuclearsurface

Outermembrane

ONM/%nuclearsurface

366353427

323375

8692

5854007H

510365608

87

85Values are expressed in microns of membrane profile seen in thin section. Nuclei were

labelled for 12 min at room temperature under standard conditions (see Materials and Methods).

lactoperoxidase-independent labelling was high. In the absence of carrier iodide, theratio was about 2:1, whereas if carrier iodide was included at a concentration of20 /IM this ratio could be raised to greater than 25:1 with a more than 50-fold stimula-tion of 126I incorporation (cf. Hubbard & Cohn, 1976).

If the temperature of the reaction was raised from 6 ° to 23 °C, the level of iodina-tion increased 7-fold to an incorporation efficiency of greater than 10% into trich-loroacetic acid-precipitable material. Thus all iodinations were conducted at this lattertemperature.

Endogenous generation of hydrogen peroxide from glucose within the reactionmedium led to significantly greatly incorporation efficiencies than did the singleaddition of peroxide to 8/tM. The use of glucose oxidase to generate peroxide atlow levels seemed preferable to the addition of concentrated peroxide which wouldcreate spatial and temporal gradients of peroxide and can result in lipid peroxidation andloss of enzyme activity (Welton & Aust, 1972). As the pattern of iodination was identicalfor both methods glucose oxidase does not itself contribute to the iodination pattern.

Polypeptides of rat liver nuclear envelope. I 259

mmm

Fig. i. Survey electron micrograph of iodinated nuclei ( x 6700). The nuclei are roundedand show a high degree of integrity. The outer nuclear membrane frequently showsa bleb separation from the inner nuclear membrane (arrow), a phenomenon not gene-rally seen prior to iodination. One of the features of iodinated nuclei is that, becausethe membranes show a wider separation than in un-iodinated nuclei, separate mor-phometric determinations of the 2 nuclear membranes are much easier to perform.Inset: Higher-power micrograph ( x 27500) showing detail of the outer nuclear mem-brane of an iodinated nucleus. Pore complexes in transverse section are clearlyvisible (arrows). Ribosomes can be seen all over the surface of the outer nuclearmembrane.

The time course of the iodination reaction was studied over a period of more than20 min. Longer reaction times gave greater incorporation efficiencies and a greaterlactoperoxidase-dependent/lactoperoxidase-independent incorporation ratio but under'standardized conditions a reaction time of 12 min was found sufficient to allowadequate iodination at an acceptable lactoperoxidase-dependent/lactoperoxidase-independent ratio (~ 30:1) and was consistent with the need to ensure minimal damage

26o J. C. W. Richardson and A. H. Maddy

to the nuclei. Sepharose 6-MB beads have the advantage of being easily and simplyremoved from the nuclei after iodination.

If iodination of nuclei were to occur by a non-enzymic route via oxidation of iodideto the highly reactive iodine which permeates cells, then iodination of the lipids wouldbe expected (Hubbard & Cohn, 1976). It was thus essential to establish that iodinationdid not occur by such a reactive diffusible moeity, which would abolish the specificity

Fig. 2. Electron micrograph of the nuclear pore complex-lamina fraction in thinsection ( x 64500). Several pore complexes are seen in tangential section, connected bya fine, but irregular, fibrous meshwork. The internal structure of the pore complexesis, in general, rather diffuse but central elements and annular subunits are evident insome pore complexes (ringed).Inset: nuclear pore-lamina fraction in thin section (x 69000). Pore complexes areclearly identifiable in transverse section. The interconnecting lamina (arrows), whenseen in transverse section, appears as a thin and closely compacted layer. Dwyer &Blobel's study (1976) indicated that the lamina extends over the entire submembranousnuclear surface in a shell-like fashion.

of labelling. In 3 separate experiments less than 10% of the counts associated withwashed labelled nuclei could be extracted with organic solvents by the Bligh & Dyer(1959) procedure. When this extract was chromatographed in 2 dimensions on thinlayer plates, no significant portion of the radioactivity co-migrated with the 3 majornuclear phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidyl-ethanolamine) which, together, account for approximately 93 % of nuclear envelope

Nuc NE PLF

Polypeptidts of rat liver nuclear envelope. I

Nuc NE PLF

261

« - •• 4

N1 (70000)N2 (67000)N3 (58000)

lii

mol. wt

*- 200000^-160000—118000^97000^88000V 70 000

x 67 000^51000^47000

^38000—36000—33000

17000

-15000

Fig. 3. Sodium dodecylsulphate electropherogram of reduced polypeptides from nuclei(Nuc), nuclear envelopes (NE) and the pore complex-lamina fraction (PLF). Left-hand slots, Coomassie stained. Right-hand slots, fluorographs. Histone bands are indi-cated by white dots. The pore complex-lamina fraction exhibits a very complexpolypeptide pattern consisting, primarily, of polypeptides of greater than 46000 mol.wt. The pattern is dominated by 3 bands, Ni, N2 and N3 (Richardson & Maddy, 1979)only 2 of which (Ni and N2) are labelled by lactoperoxidase beads. More than 12labelled polypeptides are associated with the pore complex-lamina fracion, rangingfrom 33000 to 200000 mol. wt. These are the pore complex polypeptides. The in-creased density of the fluorograph of this fraction reflects its greater specific activity.

phospholipid (Kleinig, 1970). Greater than 95% of the radioactivity ran just behindand within the 2 solvent fronts, and probably represented unbound iodide.

Pore lamina fraction

The pore lamina appears as an extensive meshwork of densely staining pore com-plexes connected by fine fibrillar threads (Fig. 2). Some pore complexes retain aninternal structure comprising a central granule and centripetal elements but suchdetail is usually difficult to discern. Thus although nuclear pore complexes seated on

262 J. C. W. Richardson and A. H. Maddy

a fibrous lamina are easily identifiable, they do not exhibit such a clear degree of organ-ization a9 that seen in the micrographs of Dwyer & Blobel (1976).

The chemical composition of the preparation (93-6% protein, 6% RNA, 0-4%phospholipid, DNA not present in detectable amounts - less than 1 %) differs some-what from that of Dwyer & Blobel, containing less DNA and rather more RNA.RNA accounted for 6% of purified pore-lamina (2% in the Dwyer & Blobel study)and this is despite extraction in conditions that completely remove DNA and theouter nuclear membrane with its associated ribosomes. There is some experimentalevidence to indicate that RNA is located in the pore complex (Mentre", 1969; Scheer,1972; Franke & Scheer, 1974; Agutter, Harris & Stevenson, 1977) but whether it isRNA or some other factor that contributes to the astonishing structural stability ofthe pore complex is unknown.

Polypeptide analysis

Comparison of the Coomassie brilliant blue and autoradiograph patterns of thepolypeptides from whole nuclei, of nuclear envelopes and the pore lamina fractionderived from labelled nuclei (Fig. 3) shows that the labelling of polypeptides is veryselective. In particular, major iodinated bands co-migrate with 2 of the major nuclearenvelope polypeptides (Ni and N2) but not with a third (N3). Both soluble andimmobilized lactoperoxidase were ineffective at labelling N3 in intact nuclei and thispolypeptide could only be labelled when nuclei were first broken open and the bulk ofthe nucleoplasm was removed. When this was done, N3 became highly labelled andthe overall pattern of labelling was greatly altered (Fig. 4).

For the following reasons it is concluded that the labelling pattern in Fig. 3 isspecific to the cytoplasmic surface of intact nuclei. (1) Insoluble lactoperoxidase isabsolutely impermeable. (2) Labelling was dependent upon the presence of lacto-peroxidase and of a peroxidase-generating system. (3) Lipid labelling was not detected,indicating the absence of diffusible I2. (4) Morphometric analysis of iodinated nucleishowed that approximately 88% of the surface of nuclei was covered by outer nuclearmembrane. (5) The pattern of labelling was highly selective and dependent uponnuclei being intact. When nuclei were broken open, further polypeptides were iodinatedand the overall iodination pattern was substantially altered. (6) All the polypeptideslabelled in the nucleus are retained in the envelope preparation - the 2 autoradiographsare virtually identical but the Coomassie blue patterns of the 2 preparations are quitedifferent. The pore lamina autoradiograph is very similar to the envelope. This isbecause the bulk of the protein of the envelope belongs to the lamina, the Triton-soluble fraction being quantitatively small.

Morphometric analysis has indicated that 88 % of the nuclear surface is coveredby the outer nuclear membrane (Table 2), and that 6% of the membrane profilesexposed to lactoperoxidase beads is unidentified single membranes: 97 % of radio-active counts were dependent on the presence of lactoperoxidase. Thus not less than80% (88 x 94 x 97%) of radioactive counts may with confidence be ascribed to thecytoplasmic surface of intact nuclei. It will be difficult to significantly improve upon

Polypeptides of rat liver nuclear envelope. I 263

N3 (58000)

Fig. 4. Fluorograph of a sodium dodecylsulphate electropherogram of crude nuclearenvelopes. The preparation was iodinated with immobilized lactoperoxidase afterthe nuclei had been swollen open at pH 85 and the bulk of the nucleoplasm removedby DNase digestion. Thus lactoperoxidase has access to the nucleoplasmic face of thenuclear envelope and polypeptide N3, previously inaccessible, becomes highlylabelled. The overall pattern of iodination is quite different from that achieved whenintact nuclei are iodinated (Fig. 3).

this figure since 2 major limiting factors, purity of the preparation and integrity ofthe outer nuclear membrane, are almost conflicting requirements.

The iodination patterns of isolated nuclei and their subfractions (Fig. 3) reveal theselectivity of the labelling method. The labelling patterns of nuclei and the purifiedpore-lamina fraction are, with the exception of 2 low-molecular-weight polypeptides,almost identical. The 2 low-molecular-weight components are coincident with

264 J. C. W. Richardson and A. H. Maddy

histones H2b and H4 and might represent iodination of chromatin in leaky nuclei.However, when chromatin is actually made accessible, an entirely different pattern oflabelling is seen where all the histones are labelled, although to varying extents (Fig. 4).The pore lamina material has been rigorously extracted in low and high salt solutionsand with detergent. Triton X-100 extraction, which removes the outer nuclearmembrane, removes only a minor portion of protein-bound label (~ 10%) althoughit removes more than 95 % of membrane phospholipid. Although precipitation ofouter nuclear membrane proteins onto the pore lamina during Triton extractionmight explain the low proportion of radioactive counts removed by this procedure,we think this improbable. The evidence that Triton effectively removes the outermembrane from the pore lamina is substantial (Aaronson & Blobel, 1974, 1975;Dwyer & Blobel, 1976; Kartenbeck et al. 1973; Tata, Hamilton & Cole, 1972). Itwould appear therefore that the labelling procedure places label predominantly inthe nuclear pore complex and to a rather lesser extent in the outer nuclear membrane.Such apparent selectivity may be explained by the fact that the pore complex sitswell proud of the outer nuclear membrane and its prominence may reduce the acces-sibility of membrane proteins to lactoperoxidase beads. Furthermore, Triton extrac-tion of highly purified nuclear envelopes (see subsequent paper) shows that verylittle protein may be extracted by Triton and that the bulk of nuclear envelopeprotein is associated with the pore complex and its lamina. Thus, not only does thepore complex present more protein to the nuclear surface than does the outer nuclearmembrane, but it also presents it in a more accessible manner.

From the iodination pattern (Fig. 3), bands Ni (70000 mol. wt) and N2 (67000mol. wt) of the major triplet may be identified as being externally disposed proteins ofthe cytoplasmic surface of the nuclear pore complex. As such, these are the firstpolypeptides in the mammalian cell nuclear pore complex as distinct from pore-lamina, to be identified. Pore-complex polypeptides may also be identified at 200000,160000, 118000, 97000, 88000, 51000, 47000, 38000, 36000, and 33000 mol. wt.The specific activity of these latter polypeptides is greater than for Ni or N2 whichsuggests that these are more highly exposed than Ni and N2. Significantly, N3, oneof the major polypeptides of the pore-lamina fraction remains unlabelled. It couldperhaps be a pore-lamina polypeptide which, buried deep within the pore complexremains inaccessible to the lactoperoxidase bead labelling system. This is unlikelyto be the case however for, although N3 is labelled neither by free nor by immobilizedlactoperoxidase if nuclei are intact, it is heavily labelled when lactoperoxidase beadshave access to the nucleoplasmic surface of the envelope after breakage of the nucleiand removal of the bulk of the nucleoplasm (Fig. 4). Since laminal material is the maincomponent of this surface, and because the great size of the lactoperoxidase beadswould preclude their gaining access to the interior of the pore complex via thenucleoplasmic side, it is likely that N3 is a polypeptide of the fibrous lamina. It isnoteworthy in this respect that this component is clearly enriched in pellets of fibrillarmaterial detached from nuclear membranes by homogenization and centrifugation(Krohne et al. 19786).

The maturing amphibian oocyte contains an unusually high number of pore

Polypeptides of rat liver nuclear envelope. I 265

complexes in very close packing. Thus nuclear envelope fractions, extracted withTriton X-iooo and high salt provide for a remarkable enrichment in nuclear porecomplex material (Krohne et al. 19786). Such material is greatly enriched in a poly-peptide recognizable as N2 and in a polypeptide at 150000 mol. wt. Nr is apparentlyabsent from such fractions and may be specific to preparations made from liver(Krohne et al. 19786). Counterparts to the high mol. wt (150000) component detectedby the latter authors exist in the iodination pattern of nuclei labelled with lacto-peroxidase beads (Fig. 3), and identified as pore complex components, at 160000and 200000 mol. wt. We believe therefore that our data are closely compatible andcomplementary to that of Krohne et al. (19786).

Recently, a number of workers (Gerace, Blum & Blobel, 1978; Ely, D'Arcy &Jost, 1978; Krohne et al. 1978a) have eluted Ni, N2 and N3 of rat liver pore-laminafrom SDS polyacrylamide gels and raised antibodies to these polypeptides. Usingimmunofluorescence localization, they found that antibody to Ni, N2 and N3 boundexclusively to the nuclear periphery. Indirect immunoperoxidase staining showed thatantibodies to Ni, N2 and N3 bound only the fibrous lamina and not to the porecomplex (Gerace et al. 1978). From this it was concluded that these polypeptides arenot present, or concentrated, in the pore complex in an immunologically reactiveform; and it was suggested that Ni, N2 and N3 are the major structural componentsof the fibrous lamina.

The absence of Ni and N2 from the pore complex is contrary to our findings, and,with regard to N2, the report of Krohne et al. (19786) as well. The binding of antibodyto a site might indicate the presence of its hapten, but failure to bind does not neces-sarily exclude its presence. As Gerace et al. point out their antisera were raised toSDS-denatured polypeptides so that the antibodies may be directed towards deter-minants buried in the proteins and not exposed at the pore complex surface, and all 3antibodies cross-reacted.

The lactoperoxidase labelling studies have indicated that Ni and N2, both majorcomponents of the nuclear envelope, are located in the nuclear pore complex (althoughnot necessarily exclusively so) along with at least 10 other more minor, though moreexposed polypeptides. It seems improbable, in view of the regular architecture ofthe pore complex and the high proportion of polypeptides Ni and N2 in the nuclearenvelope, that these are other than skeletal components, whose gross and dynamicorganization is dependent on other, quantitatively minor, envelope components. TheCoomassie brilliant blue pattern of the pore-lamina fraction reveals approximately90 bands to the naked eye (rather more than can be seen in Fig. 3) so that there is noshortage of polypeptides whose function might be to organize and control the activityof the pore complex. The radioactive peptides assigned in this paper to the pore-lamina fraction of the envelope are absent from the Triton-soluble fraction. TheTriton-soluble polypeptides are described together with a consideration of theirrelationship with polypeptides of the endoplasmic reticulum in a later publication.

18 CEL43

266 J. C. W. Richardson and A. H. Maddy

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(Received 31 August 1979)

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