The EMBO Journal vol.4 no.2 pp.297-307, 1985

An enzymatic assay reveals that proteins destined for the apicalor basolateral domains of an epithelial cell line share thesame late Golgi compartments

S.D.Fuller, R.Bravo and K.Simons

European Molecular Biology Laboratory, Postfach 10.2209,D-6900 Heidelberg, FRG

Communicated by K.Simons

The expression of viral envelope proteins on the plasma mem-brane domains of the epithelial cell line, NMDCK, is polar.Influenza virus infection of these cells leads to expression ofthe viral haemagglutinin and neuraminidase glycoproteins onthe apical domain of the plasma membrane while vesicularstomatitis virus (VSV) infection yields basolateral expressionof the sialic acid-bearing G protein. We have exploited theability of the influenza neuraminidase to desialate the G pro-tein of VSV to test for contact between these proteins duringtheir intracellular transport to separate plasma membranedomains. We were able to select for VSV-G protein expressionin doubly-infected cells because VSV protein production wasaccelerated in cells pre-infected with influenza virus. Duringdouble infection the envelope proteins of both viruses dis-played the same polar localization as during single infectionbut the VSG-G protein was undersialated due to the action ofthe influenza neuraminidase. Incubation of singly-infectedcells at 20°C blocked the transport of VSV-G protein to thecell surface and resulted in increased sialation of the proteinover that seen at 37°C. This suggests that G protein is held incontact with the sialyl transferase at this temperature. 20°Cincubations of doubly-infected cells also produced the under-sialated G protein characteristic of interaction with the neur-aminidase. We conclude that most of the newly synthesisedbasolaterally-directed G protein is in physical contact with themajority of the neuraminidase through the terminal steps ofGolgi processing.Key words: double infection/intracellular transport/MDCK/neuraminidase/polarity

IntroductionThe vectorial functions of a transporting epithelium are ac-

complished through the polarity of its plasma membranedomains. The development and maintenance of this polarityis an interesting phenomenon in its own right, as well as a

model for the sorting events which characterize the intra-cellular traffic of the animal cell. The Madin Darby caninekidney (MDCK) cell line provides a convenient model for thestudy of epithelial cell polarity because this line retains inculture many of the structural and functional properties of anin vivo epithelium (Misfeldt et al., 1976; Rindler et al., 1979;Simmons, 1981). Confluent monolayers of MDCK cells are

linked by circumferential tight junctions into an epithelium-like sheet. The tight junction defines the two plasma mem-

brane domains of the cell: the apical domain, correspondingto the luminal surface of the kidney tubule, and the baso-lateral domain corresponding to the serosal surface. Thepolarity of the epithelium is manifested in the composition ofthese plasma membrane domains; certain proteins, such as

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the aminopeptidase, being concentrated on the apical domainwhile others, such as the Na+K+ ATPase, are confined tothe basolateral domain (Louvard, 1980; Reggio et al., 1982;Richardson and Simmons, 1979).

Rodriguez-Boulan and Sabatini (1978) reported that thematuration of enveloped animal viruses is polar in MDCKcells. Influenza and parainfluenza viruses bud exclusivelyfrom the apical surface of the cells while formation of virionsof the rhabdovirus vesicular stomatitis virus (VSV) occursonly on the basolateral surface. Rodriguez-Boulan and Pen-dergast (1980) showed further that the polarity of virus matu-ration was preceded by a corresponding polarity of localiz-ation of viral envelope protein but a random cytoplasmicdistribution of the viral nucleocapsids. Roth et al. (1983) haveshown that the influenza haemagglutinin is apicallydistributed when expressed from cDNA in the absence ofother influenza proteins. Therefore the envelope protein itselfcontains the information necessary for its correct cellularlocalization. Together, these observations show that thephenomena of viral envelope protein localization and theresultant polarity of virus maturation are not peculiar to virusinfection but reflect the normal sorting mechanisms by whichan epithelial cell develops and maintains the polarity ofendogenous membrane proteins.

Plasma membrane proteins undergo several well-character-ized processing steps during their transport from their site ofsynthesis in the rough endoplasmic reticulum to the cell mem-brane. Intracellular transport of these proteins in a polarizedepithelial cell includes an extra level of complexity becauseapically-directed and basolaterally-directed proteins must besegregated and routed to the correct plasma membrane do-mains.Alonso and Compans (1981) reported that the Na+

ionophore, monensin, dramatically slowed the transport ofVSG-G protein, but left the transport of influenza virusglycoproteins to the cell surface unaffected. They interpretedthis as proving the existence of two distinct transportpathways in MDCK cells because the same concentration ofmonensin had equal inhibiting effects on the production ofVSV and influenza virus envelope proteins in the non-polarbaby hamster kidney (BHK) cell. This result would suggestdivergence of the pathways early during Golgi processingsince the inhibition of viral protein transport by monensin hasbeen shown in BHK cells to occur in the medial cistemae ofthe Golgi, prior to sialation (Quinn et al., 1983). The findingby Brown and Farquhar (1984) that the mannose-6-phosphate receptor involved in recognition and sorting oflysosomal enzymes is concentrated in the cis Golgi adds sup-port to the possibility that the recognition step in sorting ofglycoproteins may occur early in the Golgi processing.

Sabatini and co-workers (Sabatini et al., 1983; Rindler etal., 1982, 1984) used immunogold labelling of frozen thin sec-tions of MDCK cells infected with both VSV and influenzavirus to look for separation of the viral glycoproteins duringtheir transport to the cell surface. They observed the influen-

297

S.D.Fuller, R.Bravo and K.Simons

za virus haemagglutinin and the VSV-G protein occupying allthe cisternae of every Golgi stack in the cell, irrespective ofthe position of the stack within the cell. The distributions ofthe two glycoproteins were slightly different, the haemag-glutinin being restricted to the outer edges of the cisternaewhile the G protein was found in haemagglutinin-excludingaggregates throughout the cisternae (Sabatini et al., 1983).They concluded that G protein and haemagglutinin utilize thesame Golgi stack during their transport to the cell surface.They raised the possibility that the clustering of proteinsdestined for the same plasma membrane domain may reflecttheir physical segregation within a single cistema. Arguingagainst such a segregation was the observation of both G pro-tein and haemagglutinin in large intracellular vesicles betweenthe plasma membrane and the Golgi complex, althoughwhether these vesicles were of endocytotic or exocytotic originwas not determined.

Both of these approaches have advantages but suffer froma lack of sensitivity. They must be used at a point in viralinfection when large amounts of viral protein are being pro-duced and at which the cytopathic effects of viral infectionupon transcellular transport and on cell polarity must be care-fully considered (Lopez-Vancell et al., 1984). This is particu-larly true with MDCK cells grown on impermeable supportswhere the period during which viral envelope proteins ex-pression is polar is at most a few hours (Roth and Compans,1981; Rodriguez-Boulan, 1983).We have approached the question of the divergence of the

processing pathways for apically-directed and basolaterally-directed membrane proteins by exploiting the enzymaticactivity of the influenza virus neuraminidase. The ability ofthis glycosidase to remove sialic acid from the glycans of Gprotein provides a sensitive assay for contact between theseproteins during the late stages of Golgi processing and trans-port to the plasma membrane. This enzymatic approach iscomplementary to the immunocytochemical and virologicalapproaches. The sensitivity of the assay allows us to followsmall quantities of G proteins so that experiments can be per-formed at early times during infection when cytopathiceffects of virus infection should be minimal. The stage of thetransport pathway occupied by G protein can be determinedfrom pulse-chase experiments and the biochemical character-ization of modifications to the protein. Further, the assay issensitive to physical contact of the proteins rather than theirlocalization within the same morphological entity and hencegives a different view from the static one afforded by immu-nocytochemistry. The sensitivity of the approach is gained atthe expense of being able to quantitate only the fraction ofthe substrate, G protein, which sees the enzyme, neuraminid-ase. We cannot accurately define the fraction of the neuram-inidase responsible for this cleavage, but comparison ofresults at different temperatures shows that a large fraction ofthe neuraminidase is in contact with G. Our finding that mostof the newly synthesized G protein loses its sialic acid indoubly-infected cells, shows that it is in contact with someneuraminidase through the last characterized step of Golgiprocessing on its way to the basolateral membrane. Segre-gation of apical and basolaterally directed proteins is notcomplete at this stage in transport.

Results

Expression of viral proteins in MDCKOur studies of the interaction of VSV-G protein and the

298

influenza virus neuraminidase required only that the proteinswere synthesized in detectable quantities by the cells. Hencewe could work at much earlier than 5 h post-VSV superinfec-tion, the time needed to observe VSV production (Roth andCompans, 1981; Rindler et al., 1982). Further, an unexpectedfeature of the interaction of VSV and influenza virus inMDCK cells ensured that only the doubly-infected cells of themonolayer would be synthesizing significant amounts of Gprotein at early times after VSV superinfection.

Figure 1 displays a fluorograph of a SDS-urea polyacryl-amide gel of lysates of MDCK monolayers which were in-fected with either the avian influenza virus fowl plague virus(FPV) or with VSV for various times and then pulse-labelledwith [35S]methionine and prepared for electrophoresis. Theprogress of the infection is revealed by the appearance of viralproteins and the shut-off of host cell protein synthesis. Infec-tion with influenza virus alone produces an almost completeinhibition of cellular protein synthesis after 2 h. At that time,significant amounts of viral proteins can already be detected(Figure la). The progress of VSV infection is markedlyslower. Little viral protein synthesis is seen before 4 h post-infection and host cell protein synthesis is only marginallyaffected at this time. Double-infection experiments reveal avery different time course for VSV protein expression. Figurelb shows the relative levels of FPV-M protein and VSV-Mprotein synthesis in doubly-infected cells at 3 h post-VSVinfection. The M proteins were chosen for quantitationbecause they form sharp, well separated bands on 10% acryl-amide SDS-urea gels of doubly-infected cell lysates. The otherviral proteins show qualitatively similar behaviour. Twocomplementary effects are revealed. First, FPV protein syn-thesis is inhibited by VSV superinfection, unless the FPVinfection has been allowed to proceed for at least 2 h. Second,VSV protein synthesis reaches significant levels earlier than insingly-infected cells when the VSV superinfection follows atleast 3 h of FPV infection. This 3-h time point corresponds tothe first appearance of FPV haemagglutinin on the cell sur-face as revealed by immunofluorescence (data not shown).

This acceleration of VSV protein synthesis allowed us toselect for VSV-G protein expression in the doubly-infectedcells of the monolayer by working prior to the start of signifi-cant G protein synthesis in singly-infected cells. All subse-quent double infection experiments in this paper were per-formed by infecting with 10 p.f.u./cell of FPV at 37°C for4 h and superinfecting with 10 p.f.u./cell of VSV for 2 h at31°C (van Meer and Simons, 1982) before beginning pulse-chase experiments. Almost all G protein synthesis must occurin doubly-infected cells under these conditions, since onlytrace levels of G protein are detected before 4 h of single VSVinfection. For comparison, the levels of M protein expressionunder these standard double infection conditions are shownat the right side of Figure lb.The expression of viral envelope proteins was polar under

these double infection conditions. Figure 2 displays the resultsof indirect immunofluorescent labelling with anti-VSV-Gglycoprotein antibody. No G protein was detected on theapical surface (Figure 2a). Fluorescence corresponding to Gprotein was observed only after opening the Ca2+ -sensitivejunction by EGTA treatment to allow access to thebasolateral surface of the cell (Figure 2b). The polarity of theinfluenza virus proteins was examined using an affinitytechnique (Burke et al., 1982) to resolve a polyclonal antibodywhich reacted with all the FPV proteins into fractionsdirected against individual viral proteins (Figure 3). The

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Fig. 1. Time course of infection. (a) Fluorogram of a 10% acrylamideurea-SDS gel of lysates of singly-infected MDCK monolayers at 1, 2 and4 h post-infection. The bands corresponding to the FPV haemagglutinin(HA) and the NP and M proteins as well as the VSV-L, G, N, NS and Mproteins are marked. Note that FPV infection progresses much morerapidly than VSV infection. (b) Levels of VSV and FPV-M proteinsynthesis under single and double infection conditions. Monolayers wereinfected with VSV (V) or FPV (F) at 37°C (unless noted) for the timeshown, pulsed with [35S]methionine for 10 min, chased for 40 min, lysedand analyzed by electrophoresis on a 10% acrylamide urea-SDS gel. Thebands corresponding to M proteins are well separated by this system andtheir integrated intensities are displayed in arbitrary units. The increase inVSV-M protein synthesis with increased time of FPV pre-infection revealsthe acceleration of VSV protein expression in FPV-infected cells.

results of immunofluorescence with the anti-neuraminidasefraction (Figure 2c) and with an anti-haemagglutinin an-tibody (Figure 2d) were identical and the same as thatreported for the total influenza envelope proteins in MDCKcells (Rodriguez-Boulan and Pendergast, 1980). These im-munofluorescence results also show that each virus was ex-

Fig. 2. Surface expression of viral proteins. Immunofluorescencemicrographs of VSV and FPV envelope proteins during double infection.Confluent MDCK cells were doubly-infected with VSV and FPV fixed andprocessed for immunofluoresnce 2 h after VSV infection. The basolateralexpression of G protein is shown by comparison of (a) which shows thefluorescence resulting from application of anti-G antibody to the apicalsurface of the intact monolayer, with (b) which shows fluorescence fromcells labelled with anti-G after opening of the tight junctions of themonolayer by EGTA treatment. The Nomarski image of the field shown in(a) is displayed in (a-N) below. The apical nature of the influenza envelopeprotein expression is shown by staining with anti-neuraniinidase (c) or anti-haemagglutinin (d). Bar indicates 10 ym.

pressed in at least 95%o of the population under these condi-tions and confirm that co-infected cells synthesized the bulkof G protein as argued above.The FPV neuraminidase cleaves sialic acidfrom the VSV-Gprotein in vitroThe specificity of the influenza neuraminidase and the glyco-sylation of G protein are complementary. The neuraminidasecleaves N-acetylneuraminal (2-3) and (2-6) ,B-D-galactosyllinkages generating N-acetylneuraminic acid and free ter-minal galactose from a wide range of protein substrates(Bucher and Palese, 1975; Meindl et al., 1974; Sutajitt andWinzler, 1971; Rafelson et al., 1966). The structures of thecarbohydrate of both VSV-Indiana (Etchison et al., 1977)and VSV-New Jersey (Reading et al., 1978) have been deter-mined. The mature G protein carries two asparagine-linkedcomplex tri-antennary carbohydrates which terminate in:

NA (a2-3) Gal (13 1-4) Glc NAc.These 2-3 linkages should serve as substrates for the viralneuraminidase unless steric factors hinder access to them.We tested for the activity of FPV neuraminidase on VSV-G

protein by mixing [35S]methionine-labelled VSV with un-

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Fig. 3. Resolving an anti-FPV antibody. 5 mg of FPV were run on a12 cm wide 6 M urea, SDS, 1001 acrylamide gel and transferred tonitrocellulose as described in Materials and methods. Strips were cut afterPonceau S staining (a) and used to absorb antibodies against theneuraminidase and the two haemagglutinin subunits from an anti-FPVserum. The antibodies were eluted from the strips and used to stain totalviral protein transferred to another filter. The staining of theneuraminidase component for 40 itg, 4 itg and 0.4 yg of FPV is shownin (b). The staining of 40 yg of FPV with the HAI (c), HA2 (d) andabsorbed serum (e) which retains anti-M protein activity.

labelled FPV in the presence of solubilizing levels of non-

ionic detergent. The increased mobility ofG protein on a one-

dimensional SDS-urea gel after incubation with the influenzaviral proteins is consistent with the loss of sialic acid (Figure4a). This shift is small relative to that seen for VSV derivedfrom other cells (Knipe et al., 1977) but it can be readilyvisualized when two-dimensional electrophoresis is employedto show the change in the charge ofG protein. The spots cor-

responding to G protein (Figure 4b) shift towards the negativepole after incubation with FPV proteins (Figure 4c) indicatinga loss of negative charge consistent with the removal of sialicacid. The charge heterogeneity of G protein is also decreasedshowing that incomplete sialation and not other modifi-cations, such as sulfation, are the primary source of thecharge heterogeneity of the untreated virus (Hsu and Kings-bury, 1982; Pinter and Compans, 1975). Incubation ofMDCK-derived VSV with the soluble neuraminidase fromClostridium perfringens (Figure 4d) results in a further loss ofnegative charge showing that a fraction of the sialic acid isinaccessible to digestion by the FPV neuraminidase. The con-figuration of charged species shown in Figure 4c correspondsto limit digestion with the FPV neuraminidase since increasingthe temperature or length of the digestion did not result infurther cleavage (data not shown). Finally, the generation offree terminal galactose residues by the action of the FPVproteins was assayed by affinity chromatography on the lec-tin, Ricinus communis agglutinin. Baenziger and Fiete (1979)showed that peptides carrying sialic acid in a 2-6 linkage or

with a single terminal galactose bound to this lectin tightly(Ka >2 x 106/M) while peptides bearing galactose shieldedby a 2- 3 linkage with sialic acid or bearing only N-acetyl glu-cosamine showed no detectable binding. Only 2- 407o of

300

Fig. 4. Neuraminidase action on G protein. (a) Fluorograph of 12.501oacrylamide urea-SDS gel of 35S-labelled VS virions produced in MDCKcells. The isolated virus was incubated for 12 h at 37°C in 1Plo (w/v)Triton X-100. 0.1 M sodium acetate pH 5.0, 1 mM CaCl2 in the presence(+) or absence (-) of 140 itg/ml of unlabelled isolated FP virions.(b,c,d) Fluorographs of two-dimensional gels of isolated VS virions after48 h incubation at 4°C in I%o (w/v) Triton X-1 14. 0.1 M sodium acetatepH 5.0, 1 mM CaC12 alone (b), with 140 Agg/ml of FP virions (c), or with5 U/ml Clostridium perfringens neuraminidase (d). The spotscorresponding to the different charged forms of G protein are labelled GI(most positive species) to G7 (most negative species) by reference to Nprotein and the interspot spacing.

VSV-G protein [isolated from the other viral proteins byTriton X-1 14 (TX1 14) partitioning; Bordier, 1981, and Figure7a] bound reversibly to R. communis agglutinin coupled toSepharose 4B before incubation with FPV proteins, while>95'70 of G protein bound to the column after incubationwith influenza proteins. Free terminal galactose is clearly un-covered by this incubation. Etchison et al. (1974) found that,B-galactosidase treatment of G removed few galactose resi-dues unless it was preceded by neuraminidase treatment, sug-

gesting that few free terminal galactoses exist in the matureviral protein. Our observation that the charge heterogeneityin the mature virus (Figure 4b) is accompanied by smallamounts of binding to the lectin is consistent with this in sug-

gesting that sialation of galactose is almost complete and thatthe charge heterogeneity reflects variability at the level ofgalactose addition.G protein produced in double infections is less sialated thanthat produced in single infectionsThe sialation ofG protein produced during FPV/VSV doubleinfection was compared with that produced after 5 h of single

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Fig. 5. Pulse-chase of singly- and doubly-infected MDCK cells at 37°C. Cells were infected with VSV after 4 h of FPV infection as described in Materials

and methods and pulsed with [35S]methionine for 10 min after 2 h of VSV infection, or singly-infected for 5 h and then chased for either 10 or 40 min at

37°C before lysis and processing for electrophoresis. (a) 12.50%o acrylamide urea-SDS gel of VSV (V), FPV (F) or doubly-infected (V/F) cells. (b-d) Two-

dimensional gels of VSV-infected (V-b,c) or doubly-infected (V/F-d,e) cells after 10 min (b,d) or 40 min (c,e) of chase. The shift in mobility of G

protein which corresponds to sialic acid addition is seen in the VSV-infected samples but not in the doubly-infected samples.

infection with VSV using the assays described in the last sec-

tion. Figure 5a shows an SDS-urea gel of the cell lysates ofVSV-infected and of FPV/VSV-co-infected cells which havebeen pulsed with [35S]methionine for 10 min and chased forvarious times at 37°C. The VSV-G protein in singly-infectedcells gradually shifts to a slightly higher apparent mol. wt.over 40 min of chase at 37°C, by which time it begins toappear at the surface. Knipe et al. (1977) first described thisshift as the maturing of an unsialated GI form of the proteinto a sialated G2 form. This GI to G2 maturation of theproteins is not seen in the proteins of FPV/VSV doubly-infected cells. The same result is obtained whether cells were

lysed by 207o SDS at 95°C, by TX1 14 in the presence of theneuraminidase inhibitors EDTA and 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, or with the urea-NP40-ampholine mixdescribed by O'Farrell (1975). Each of these treatments com-

pletely inhibited FPV neuraminidase activity in pilot experi-ments so that action of the neuraminidase on G protein aftercell lysis can be excluded. Two-dimensional gels (Figure 5b - e)show this phenomenon more dramatically as a shift from a

few species of similar charge (10 min chase, Figure 5b) to an

array of more heterogeneous and negatively charged species(40 min chase, Figure 5c) in the single infection samples. Thedoubly-infected samples display neither this increase in nega-tive charge nor the charge heterogeneity of VSV-G protein.The 40 min chased G from doubly-infected cells (Figure 5e)has a pattern similar to the limit-digested G protein of Figure

4c. Three percent of the G protein produced in singly-infectedcells and 3907o of the protein produced in doubly-infected cellsbound reversibly to R. communis agglutinin-Sepharose show-ing that the loss of sialation has been accompanied by expos-

ure of free terminal galactose residues. The influenza glyco-proteins make no contribution to this binding since they are

removed by the TXl 14 partitioning used to isolate G protein(see Figure 7a). Experiments in which the chase was perform-ed at 31 C also show a decreased sialation of G duringVSV/FPV double infection relative to G produced in singleinfection at 31 'C. However, the cleavage is not as extensiveas at 37°C. Spots G5 and G6 retain some intensity even whenchase is prolonged to 60 min (data not shown). Hence G pro-tein is not completely desialated at 31 'C although it has ap-peared at the surface.

Cleavage ofsialic acidfrom VSV-G protein occurs within thecellIncubations at reduced temperatures slow the transport of in-fluenza haemagglutinin to the plasma membrane of MDCKcells (Matlin and Simons, 1983; Rodriguez-Boulan et al.,1984). The protein appeared to be blocked at some post-endoplasmic reticulum stage because it had acquired endo-glycosidase H (Endo H) resistance. These observations sug-gest that reduced temperature could provide a useful way oflocalizing the interaction between the FPV neuraminidaseand VSV-G protein, which causes the loss of sialation de-

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Fig. 6. [1251]protein A binding assay for surface G protein expression. (a,b) Confluent MDCK monolayers were infected with FPV for 4 h and theninfected with VSV for various times at 31°C and 20°C before fixation and processing for protein A binding assay as described in Materials and methods.The binding is expressed as a percentage of the total applied protein A counts and is linear with bound anti-G protein at the concentrations used. Abackground (70%o) corresponding to bound input VS virions has been subtracted from all values. The bars show the standard error of the mean. (a) Surfaceexpression at 31°C. The amount of G protein on the apical and basolateral surfaces was evaluated by comparing binding to the apical surface in intact cells(0) with that to the apical plus basolateral surface in EGTA-treated cells (0). Note that >90% of the G protein is basolateral at 2.5 h post-infection. (b)Surface expression of G protein at 20°C. The total amount of surface G protein is shown by the [1251] protein A binding assay of EGTA-treated cells. After1.5 h of VSV superinfection at 31°C, a set of samples was incubated at 31°C (v) while a parallel set was shifted to 20°C for 2 h and then shifted to 37°C(0). Note that G protein is held internally at 20°C but is brought to the surface within 30 min after shifting to 37°C. (c,d) Immunofluorescence of Gprotein at 20°C. MDCK monolayers were infected as for (b) and fixed after 2 h incubation at 20°C and processed for immunofluorescence afterpermeabilization with 0.20%o Triton X-l00 as described in Materials and methods to show internal G protein (c) or after opening of tight junctions withEGTA to show total surface G protein (d). The Nomarski image (c-N) of the field in (c) is shown. Bar represents 10 ltm.

scribed above. Therefore, we used reduced temperature incombination with pulse-chase experiments to block VSV-Gprotein transport to the cell surface and localize this inter-action. Figure 6 shows the effect of reduced temperature(20°C) on the transport of G protein to the cell surface.

Figure 6a also shows the results of indirect radiometric assay

of the amount of G protein on either the apical surface or theapical plus basolateral surface after the opening of junctionswith EGTA. The curves clearly show that VSV-G protein sur-

face expression was polar (i.e., basolateral). Since the

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Fig. 7. Pulse-chase of singly- and doubly-infected cells at 20°C. Confluent monolayers were infected and pulsed as described for Figure 5 and then chasedat 20°C for 45 min or 120 min before lysis and processing for electrophoresis. (a) 12.5% acrylamide urea-SDS gel of VSV (V), FPV (F) or doubly-infected(V/F) samples. The Triton X-1 14 extractable material from the doubly-infected sample chased for 120 min is shown at the right. This is the material whichwas used for the affinity chromatography shown in Figure 9. (b) Two-dimensional gel electrophoresis of VSV-infected (b,c) or doubly-infected (d,e) samplesafter 45 min (b,d) or 120 min (c,e) chase at 20°C. The shift of G protein towards the anode seen in VSV-infected cells at 20°C is greater than that seen at37°C and both are greater than that seen in doubly-infected samples.

[1251I]protein A binding assay, as well as the immuno-fluorescence, detect the total amount of surface G protein, webegan our reduced temperature incubations prior to theemergence of G at the plasma membrane to see the effect ofthe incubation in the absence of a surface G proteinbackground. This made the absolute values seen in the [1251]protein A assay relatively low in comparison with the 31°Cresults. Figure 6b shows that the appearance of G protein wasblocked when the infected cells were held at 20°C. G proteinwhich had been held within the cell by 20°C incubation couldthen be brought rapidly to the cell surface by returning thecells to 370C. The amount of protein brought to the surfacewithin 15 min of returning the cells to 37°C corresponds toroughly half of that synthesised between 1.5 and 2.5 h at31 °C (Figure 6a). A similar amount is brought to the surfacein the presence of cycloheximide (data not shown). Immuno-fluorescence (Figure 6c, focused at the level of the nucleus)shows that the G protein, which had accumulated within thecell at 20°C, appeared to occupy vesicles in the Golgi regionof the cell, and that no fluorescence corresponding to plasmamembrane G protein is seen when the surface is stained (Fig-ure 6d, focused at the base of the cell).The ability to use reduced temperature to hold the G pro-

tein within the cell enabled us to localize the contact betweenG protein and the neuraminidase more precisely. Pulse-chaseexperiments with VSV-infected cells held at 20°C during the

chase showed that the GI - G2 shift and the increase in nega-tive charge (Figure 7) which indicate G protein sialation,occurred at 20°C. Comparison of the two-dimensional gels ofFigure 7 with those of the singly-infected cells in Figure 5reveals that 20°C incubations yield a greater degree of Gprotein sialation than is seen at 37°C. Quantitation of theintensity distribution of the spots corresponding to the differ-ent sialated species of G protein (Figure 8) reveals the increasein sialation between the 45 min chase sample and the 120 minchase sample, a consequence of the protein remaining internalduring the 20°C incubation. The effect of double infection onthe sialation of G protein is more striking in comparison. Theprolonged 20°C incubations which result in increasingmobility changes in the singly-infected case result in nomobility shift in the doubly-infected case. Quantitation of thechange in the relative intensities (Figure 8) shows that > 9807oof the highly sialated species (spots 5, 6 and 7) have beencleaved during the 20°C incubation, hence this fraction of theG protein must have been in contact with the neuraminidase.Only 207o of the G protein produced in single infections at20°C bound reversibly to the R. communis agglutinin-Sepha-rose column while 57%0 of G produced in double infectionsbound reversibly to the lectin (Figure 9). Loss of sialic acid isaccompanied by exposure of galactose under these conditionsas it was at 37°C.

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Fig. 8. Quantitation of the relative number of 5S counts associated witheach species of G protein for the samples shown in Figure 7b - e was

determined as described in Materials and methods. The relative amounts ofradioactivity in each species is shown for VSV-infected cells which havebeen chased at 20°C for 45 min (V 45) and 120 min (V 120) and fordoubly-infected cells chased at 20°C for 45 min (F/V 45) and 120 min(F/V 120). The increase in G protein sialation during prolonged incubationat 20°C is seen by comparing V 45 with V 120. The complete loss of thehighly sialated species is seen by comparing the doubly-infected (FV)samples to the singly-infected (V) samples at either time point. Abackground equal to that of an equal area of adjacent unexposed film hasbeen subtracted and the total counts for each sample has been normalizedto I100b.

DiscussionThe central result of this study is the demonstration that an

apically directed protein, influenza virus neuraminidase, anda basolateral directed one, VSV-G protein, are in physicalcontact during their passage through the Golgi complex ofdoubly-infected MDCK cells. The cleavage of the VSV-Gprotein indicates its contact with the influenza virusneuraminidase.The enzymatic nature of the assay gives us great sensitivity.

Cleavage of a single sialic acid from G protein by the neuram-inidase produced a charge shift which was easily detectable bytwo-dimensional gel electrophoresis and exposed terminalgalactose residues which could be detected by binding to a

ricin column. This sensitivity allowed us to work at very earlytimes of infection when cytopathic effects of infection were

minimal. This is critical when studying a phenomenon such as

cell polarity which is destroyed during the later stages of viralinfection. The information provided by these experiments atearly times of infection is, therefore, complementary to thatderived by immunocytochemical approaches which are oflower sensitivity and hence must be applied to the later stagesof infection.The general approach of using the virally encoded, mem-

brane neuraminidase to act upon other viral proteins should

304

o

a)

0U

C-E

0

0

Fraction number

Fig. 9. Ricin-Sepharose chromatography of G protein from doubly- andsingly-infected cells. Cells were infected with VSV for 5 h or with VSV for2 h at 31°C after 4 h of FPV infection, pulsed with [35S]methionine for10 min at 37°C and chased at 20°C for 2 h. The pulse-chased cells werelysed with Triton X-1 14 containing lysis buffer and the G protein isolatedby two cycles of phase separation as described in Materials and methods.The isolated G protein was applied to a ricin-Sepharose column in 0.5%Triton X-100 in PBS lacking Ca2+ and Mg2+, washed with five columnvolumes and the reversibly bound G protein eluted with 0.1 M galactose in0.5% Triton X-100 in PBS lacking Ca2+ and Mg2+. The amounts areexpressed as a fraction of the total counts which either did not bind to thecolumn or which were eluted with galactose.

also find application to other systems in which a sensitiveassay for contact between membrane proteins or membrane-bound compartments is desired. The ability to introduce theneuraminidase into one cell population by viral infection andthe high sensitivity afforded by the enzymatic nature of theinteraction should provide a basis for in vitro assays of intra-cellular transport. This approach would complement the oneintroduced by Rothman and co-workers (Rothman et al.,1984) which is dependent on mutant cell lines for assayingtransport between compartments.

Co-expression of G protein and neuraminidase results in areduction of the sialic acid content of G protein as assayed byelectrophoresis and the accessibility of the normally penulti-mate galactose residues for binding to R. communis lectin.These effects are completely consistent with the activity of theFPV neuraminidase on the VSV-G protein demonstrated invitro and therefore with contact between these two proteins.Any alternative explanation of these results must postulate thatthe addition of sialic acid to G protein simply does not occurin doubly-infected cells. This could result from competitionfor sialyl transferase between VSV-G protein and other pro-teins or the saturation of the capacity of the enzyme by thehigh level of G protein synthesis during double infection.Three considerations argue against this possibility. First, ourexperiments were performed at very early times in VSV infec-tion. Virus harvested from MDCK cells after long times ofsingle VSV infection is properly sialated (Figure 4), althoughit was produced while the level of G protein synthesis wasgreater than the total glycoprotein synthesis in the doubly-infected cell under our conditions (Figure lb). Second,holding VSV-G protein in contact with a sialating compart-ment at 20°C greatly increases the sialation of the proteinduring single infection, but causes no increase in the sialationof G protein during double infection. Third, comparison ofthe two-dimensional gel results of Figure 4c and d with those

ApicaDly and basolaterafly directed proteins share Golgi compartments

of Figures 5 and 7 show that the G protein retains the samesialation as the FPV neuraminidase-digested G protein andmore than that digested with C. perfringens neuraminidase.This shows that some sialation has occurred in the doubly-infected cells.The results from our 20°C experiments define the site of

interaction between neuraminidase and G protein. The trans-port of the haemagglutinin of the influenza virus N to the cellsurface is slowed at 20°C and the glycoprotein acquires EndoH resistance at this reduced temperature (Matlin and Simons,1983). We have extended these observations to another viralglycoprotein, the G protein of VSV, showing that it too isblocked inside the cell for at least 2 h by 20°C. We have fur-ther characterized this compartment by showing that anenvelope protein which is blocked in its transport to the cellsurface remains in contact with sialyl transferase at 20°C.VSV-G protein acquires its terminal sialation and is indeedover-sialated, relative to the protein produced at 37°C, whenthis 20°C incubation is prolonged. Together with the obser-vation that G protein, although heterogeneously sialated,contains few terminal galactose residues when produced ateither 37°C or 20°C, this argues that galactosylation mustalso continue in the compartment in which protein is blockedat 20°C. The presence of these two activities define this 20°Ccompartment more precisely. Roth and Berger (1982) havelocalized the galactosyl transferase to the trans cisternae ofthe Golgi by immunoelectron microscopy. Attempts tofractionate the Golgi (Bretz et al., 1980) showed that galac-tosyl and sialyl transferase co-localize during fractionation.This co-localization was also seen by Goldberg and Kornfeld(1983) and by Dunphy and Rothman (1983) in which fivecompartments of the Golgi, each with characteristic enzymeactivities, were resolved. We suggest that at 20°C the G pro-tein is blocked in a compartment which is in equilibrium withthe trans cisterna which contains the galactosyl and sialyltransferases. The lack of sialic acid on G protein which hasbeen blocked at 20°C in this compartment during double in-fection shows that G and neuraminidase are in contact withthe most distal identified Golgi compartment.The compartments referred to in this discussion are kin-

etically defined. A compartment in this sense need not referto a single morphological unit but could encompass a varietyof elements which are in equilibrium during the time courseof the observations (Jacquez, 1972). For this reason thedirectedness of transport through the Golgi is important.Return of protein from the sialating compartment to, forexample, the mannosidase-containing compartment wouldallow interaction of neuraminidase and G protein eventhough they are separated in later Golgi compartments. Thework on Semliki forest virus-infected and monensin-treatedcells in which ricin binding components were seen to be chas-ed through the trans cisternae of the Golgi in the presence ofcycloheximide argues against this possibility (Quinn et al.,1983; Griffiths et al., 1983a, 1983b) by showing that proteinwhich has been galactosylated does not later return to the cisside of the Golgi. Goldberg and Kornfeld (1983) also showedthat movement of proteins through the Golgi is directional byassaying the carbohydrates on proteins in the same Golgifractionation which separated the glycosylation enzymes.Pulse-chases of glycosylation intermediates showed that theintermediates were found only in the same and later fractionsas the enzyme responsible for their formation. Galactosyltransferase product was not found, for example, in the samefractions as GlcNAc transferase. Movement through the

Golgi appears unidirectional in the sense that the products oflate compartments do not enter earlier ones. A similar con-clusion has been reached in recent work by Rothman et al.(1984).Our demonstration of physical contact between the neu-

raminidase and G protein must be viewed in terms of thiskinetic defintion of a compartment. The basolaterallydirected G protein of VSV and the influenza neuraminidasedo not traverse separate routes during their transit throughthe Golgi because they are in contact with the last biochemi-cally characterized compartments of that organelle. Ourobservations show that the separate clustering of apically andbasolaterally directed proteins in Golgi cisternae observed byRindler et al. (1982, 1984), does not represent segregation of asignificant fraction of these molecules since > 987o of G pro-tein contacts the influenza neuraminidase after sialation. Wecannot make the corresponding statement about the segre-gation of the neuraminidase. Calculation of the amount ofneuraminidase in contact with G protein requires an accuratedetermination of the activity of the enzyme in situ. This, inturn, depends on unknown parameters such as the pH, di-valent ion concentration and the concentration of competingsubstrates and hence cannot be evaluated. A fraction of theneuraminidase might be sufficient to produce the cleavage ofG protein which we observe. This is a significant consider-ation in light of reports that a fraction of some predomi-nantly apical proteins can be found on the basolateral surfaceof MDCK (Fuller et al., 1984; Balcarova-Stander et al.,1984). An important question is whether the observed cleav-age of G protein could result from a misdirected componentof the neuraminidase, the small fraction of the enzyme whichemerges basolaterally. This component might remain with theG protein during the late stages of Golgi processing eventhough the apically directed neuraminidase has been segre-gated from G. Our 31°C results allow us to rule out thispossibility. The fact that the influenza virus infectionprecedes the VSV infection means that the ratio of themisdirected component of neuraminidase to G on thebasolateral surface will be higher than it is within the cell. Theactivity of the basolateral neuraminidase on basolateral Gprotein serves as an upper limit for the activity of themisdirected component of the intracellular neuraminidase.Pulse-chase experiments at 37°C produce limit digested Gprotein so that the activity of the surface neuraminidase onsurface G cannot be evaluated. During chases at 31°C,however, the intracellular desialation of G protein is not com-plete. If the basolateral component of the neuraminidase hadan activity comparable with that of the intracellularneuraminidase responsible for the cleavage, a further loss ofsialic acid should occur after the appearance of G at the sur-face. Further cleavage of G protein was not observed afterchase times of up to 60 min. Hence, the cleavage we observeis accomplished by an apically directed component of theneuraminidase which is later segregated from the basolateral-ly directed G. The segregation of newly synthesized mem-brane proteins which is responsible for the establishment ofepithelial cell polarity occurs after passage through the Golgicomplex.

Materials and methodsCells and virus infectionThe cloned line of MDCK described by Louvard (1980) was used for all exper-iments. Cells were grown and passaged as described by Matlin et al. (1981).Infections were performed with cells which were seeded at a density of105

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cells per 25 mm diameter plastic dish (Flacon Plastics, Oxnard, CA) andgrown for 60 h in growth medium: Eagle's minimum essential medium withEarle's salts (Earle's MEM) supplemented with 10 mM Hepes pH 7.3, 5%7o(v/v) foetal calf serum (Gibco Europe, UK), penicillin (110 U/ml), strepto-mycin (100 Ag/ml) and Fungizone (0.025 Ag/ml). Infections were performedby rinsing the MDCK monolayers with infection medium: Earle's MEM sup-plemented with 0.2%7o (w/v) bovine serum albumin, 10 mM Hepes pH 7.4,penicillin (110 U/ml) and streptomycin (100 Ag/ml) and applying 10 p.f.u./cell of virus to a volume of 200 Al in infection medium. After absorption ofvirus to the cells at 37°C for 1 h, the monolayers were rinsed with growthmedium and held at 37°C or 31IC in growth medium to allow the infection toproceed. The standard double infection was performed by infecting mono-layers with 10 p.f.u./cell of FPV, absorbing for 1 h at 37°C and transferringthe monolayers to growth medium at 37°C for a further 3 h, rinsing with in-fection medium, absorbing with 10 p.f.u./cell of VSV at 31°C for 1 h, andtransferring to growth medium for 1 h at 31°C before use in pulse-chase ex-periments. Pilot experiments showed that incubating the cells at 31°C (asdescribed by van Meer and Simons, 1982) and in the presence of serumsignificantly mitigated the cytopathic effects of VSV infection.

Isolates of fowl plague virus (A/FPV/Rostock, Havl NI) were obtainedfrom H.-D.Klenk, Justus Liebig University, Giessen, FRG, plaque purifiedon MDCK, grown in eggs as described by Matlin and Simons (1983) and titredon MDCK as described by Matlin et al. (1981). VSV was grown in BHK-21cells and titred on MDCK as described by Matlin et al. (1982).[35S]Methionine-labelled virus was obtained as described by Matlin et al.(1981, 1982).Pulse-chase experimentsMonolayers were rinsed twice in warm Earle's MEM lacking methionine con-taining 10 mM Hepes pH 7.3 and then pulse-labelled at 37°C in a 5%o CO2 at-mosphere with 0.5 ml of 100 itCi/mil of [35S]methionine (Amersham,>800 Ci/mmol) in methionine-free Earle's MEM for 10 min. The mono-layers were then chased either with 2 ml of warm chase medium (growthrMedium containing 150 mg/l of unlabelled methionine) at 37°C in a 5% CO2incubator or with Earle's MEM lacking bicarbonate and supplemented with10 mM MOPS, 2 mM NaH2PO4, 10 mM TES, 15 mM Hepes adjusted to pH7.4 (Eagle, 1971) as well as 5%o (v/v) foetal calf serum at 20°C. Chase washalted by transfer to an ice bath and rinsing the individual plates with ice-coldDulbecco's PBS lacking Ca2+ and Mg2+. Monolayers were lysed on ice with0.5 ml per dish of 2070 (w/v) TXI 14 in 50 mM Tris-HCl pH 7.4 containing1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, 1 mMiodoacetamide (IAA) and 10 mM 2,3-dehydro-2-deoxy-N-acetyl-neur-aminic acid (DDNA) (Boehringer-Mannheim) an inhibitor of the viralneuraminidase (Meindl et al., 1974). In some experiments, parallel sampleswere lysed with the urea-NP40-ampholine buffer described by O'Farrell(1975) and the degree of sialation of G protein compared with that of sampleslysed with TXl 14 mixture by two-dimensional electrophoresis. No differencesin sialation were observed between parallel samples lysed by these twomethods indicating that the viral neuraminidase is inactive after lysis. Sampleswere prepared for one-dimensional gel electrophoresis directly from theTX114 lysis mixture or the urea-NP40-ampholine-mixture by adding 20%o(w/v) SDS to a concentration of 407, heating at 95°C for 3 min, making thesolution 10 mM in dithiothreitol, 0.0207o bromophenol blue, 907o sucrose andheating again for 3 min at 95°C. Samples were cooled to room temperature,IAA added to 30 mM and incubated at 37°C for 15 min. One-dimensionalelectrophoresis and fluorography were performed as described by Fuller et al.(1981) except that a 12.5%o acrylamide gel containing 8 M urea was used toobtain the optimal visualization of the mobility shift due to sialation ofG pro-tein as well as the resolution of the other viral proteins from each other.

Quantitation of the relative levels of VSV-M and FPV-M protein expressedfrom one-dimensional gels was performed as described by Fuller et al. (1981)using a Quick Scan R&D scanner (Helena Laboratories) with the tungstenlamp and a 5 mm slit width. A 100/o acrylamide urea-SDS gel was usedbecause it gave good separation of the M proteins. The 12.5% acrylamideurea-SDS gel displayed in Figures 5 and 7 show the G mobility shift well butdid not clearly resolve the M proteins. Two-dimensional gel electrophoresiswas performed by the method of O'Farrell (1975) as modified by Bravo et al.(1984).

Quantitation of the intensities of spots on autoradiographs of two-dimen-sional gels was performed by eluting the silver from the spots and measuringits absorbance. The array of spots as well as an area of background fromeither side was excised from the autoradiograph and divided into pieces con-taining single spots. Each piece was weighed, and then shaken with 0.5 ml of0.22,m filtered 1 M sodium hydroxide for 8 h to elute the silver (Suissa,1983). The eluted silver was quantitated by reading the absorbance at 690 nmin a spectrophotometer. Since the area covered by each spot was different, thebackground absorbance was normalized by the area of the film containingeach spot before subtraction from the spot absorbance. The background cor-

rected absorbances were normalized so that their sum equalled 100%7o beforeplotting to allow easier comparison.Triton X-114 purification ofG proteinPulse-chased MDCK monolayers were extracted with 2.0070 (w/v) TX1 14(Bordier, 1981) in 50 mM Tris-HCl pH 7.4 containing 1 ItM PMSF, 5 mMEDTA, I mM IAA and 10 mM DDNA. The lysate was centrifuged for15 min at 10 000 g at 4°C, the supernatant removed and warmed to 37°C for5 min. The cloudy solution was centrifuged for 5 min at 1000 g at 37°C, thetop phase removed and the Triton pellet dissolved in Ca2+-, Mg2+-free Dul-becco's PBS on ice. The 37°C-centrifugation-4°C cycle was repeated twiceand the pellet prepared for R. communis agglutinin Sepharosechromatography or one-dimensional gel electrophoresis. Material to be usedfor two-dimensional gel electrophoresis was subjected to only one cycle ofphase separation, washed with water, lyophilized and urea-NP40-ampholinebuffer aded. This allowed visualization of the VSV-N protein which aidedalignment.Lectin affinity chromatographyG protein was isolated from the other viral proteins in pulse-chase samples byphase separation with TX1 14 (Bordier, 1981) and diluted to a TXl 14 concen-tration of 0.17I with I%o Triton X-100 in Dulbecco's PBS lacking Ca2+ andMg2+. This solution was applied to a 5 x 0.5 cm column of R. communis ag-glutinin (Boehringer-Mannheim) coupled to CNBr Sepharose 4B (PharmaciaFine Chemicals) equilibrated with Ca2+-, Mg2+-free Dulbecco's PBS contain-ing 0.10o Triton X-100 and 0.04%7o sodium azide. The column was washedwith 10 ml of this buffer and eluted with the same buffer containing 0.1 MD( + ) galactose. 500 Id fractions were collected and counted after TCA precipi-tation on Whatman 3MM discs as described by Mans and Novelli (1961) toremove unincorporated [35S]methionine.[1251]Protein A binding assayQuantitation of the amount of surface VSV-G protein was performed asdescribed by Pesonen and Simons (1983) except that incubations of mono-layers with 5 mM EGTA in Ca2+-, Mg2+-free PBS to open tight junctionsand expose the whole cell surface were performed at 20°C for 8 min for theexperiments involving reduced temperature. Background values were deter-mined by using monolayers which had been incubated with VSV under iden-tical conditions but in the presence of 10 ltg/ml cycloheximide to stop viralprotein synthesis.Immunofluorescence and antibodiesThe anti-neuraminidase antibody was prepared from a rabbit antiserumdirected against all the proteins of the fowl plague virion by affinity purifi-cation as described by Burke et al. (1982). The rabbit anti-VSV-G protein andthe rabbit anti-FPV-haemagglutinin were as described in Fuller et al. (1984).Immunofluorescence was performed as described in Louvard (1980).

AcknowledgementsThe authors wish to express their thanks to Hilkka Virta for expert technicalassistance in the provision of virus stocks and antibodies and to HeatherMacDonald-Bravo and Pat Blundell for assistance in the running of two-dimensional gels. The help of Dr Marja Pesonen (Recombinant DNA Lab-oratory, Helsinki, Finland) with the lectin affinity chromatography and of DrBrian Burke (EMBL) is gratefully acknowledged. We also wish to thank DrPaul Quinn (EMBL), Dr Steven Pfeiffer (University of Connecticut, MedicalSchool, Farmington) and Dr Graham Warren (EMBL) for very valuablecriticism of the manuscript and Mrs Annie Steiner for typing the many draftswhich evolved as a result of their comments. SDF is a Helen Hay WhitneyFoundation fellow.

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Received on 2 November 1984

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