Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a

late stage of post-translational processing

JANIS K. BURKHARDT and YAIR ARGON*

Department of Microbiology' and Immunology, Duke University Medical Center, Box 3010, Durham, North Carolina 22710, USA

•Author for correspondence

Summary

The appearance of newly synthesized glycoprotein(G) of vesicular stomatitis virus at the surface ofinfected BHK cells is inhibited reversibly by treat-ment with carbonylcyanide m-chlorophenylhydra-zone (CCCP). Under the conditions used, CCCPtreatment depleted the cellular ATP levels by40-60%, consistent with inhibition of transport atenergy-requiring stages. The G protein that ac-cumulates in cells treated with CCCP is hetero-geneous. Most of it is larger than the newly syn-thesized G protein, is acylated with palmitic acid,and is resistant to endoglycosidase H (Endo H).Most of the arrested G protein is also sensitive todigestion with neuraminidase, indicating that it hasundergone at least partial sialylation. A minority ofG protein accumulates under these conditions in a

less-mature form, suggesting its inability to reachthe mid-Golgi compartment. The oligosaccharidesof this G protein are Endo-H-sensitive and seem tobe partly trimmed. Whereas sialylated G proteinwas arrested intracellularly, fucose-labelled G pro-tein was able to complete its transport to the cellsurface, indicating that a late CCCP-sensitive stepseparates sialylation from fucosylation. These post-translational modifications indicate that G proteincan be transported as far as the frans-Golgi in thepresence of CCCP and is not merely arrested in theendoplasmic reticulum.

Key words: intracellular transport, Golgi, CCCP, post-translational modifications, VSV, G protein.

Introduction

Two transitional stages are critical for efficient unidirec-tional protein transport: movement of proteins from theendoplasmic reticulum (ER) to the Golgi complex andmovement from the Golgi to the cell surface or othertarget membranes. The importance of these transitionalstages is demonstrated by several lines of evidence. First,sorting of proteins destined to reside in various cellularcompartments occurs during these stages (Farquhar,1985; Palade, 1975; Sabatini et al. 1982). Second, thesetransitional stages appear to be rate-limiting in thetransport of many proteins (Argon & Milstein, 1984;Melchers, 1971; Strous & Lodish, 1980). Membrane andsecreted proteins which travel at different rates within thesame cell often diverge at the ER-Golgi transition(Fitting & Kabat, 1982; Fries et al. 1984; Lodish et al.1983; Scheele & Tartakoff, 1985; Strous & Lodish,1980). Finally, perturbing the structure of transportedproteins often results in their failure to exit from the ERor the Golgi complex (Garoff, 1985; Guan & Rose,1984). These observations indicate that the traffic ofproteins into and out of the Golgi complex is tightlyregulated.

Journal of Cell Science 92, 633-642 (1989)Printed in Creat Britain © The Company of Biologists Limited 1989

While the transitional stages of protein transport havebeen of interest for some time, they remain ill-defined.This is partly because the vesicular compartments thatmediate these transitions are not structurally obvious. Asecond difficulty is that transported proteins reside in thetransitional compartments only briefly. If protein trans-port could be arrested at these stages, biochemicalanalysis of the arrested proteins could be used to assignenzymic activities to the transitional compartments.Moreover, localization of the arrested proteins could beused to identify the compartments by microscopy. Aninhibitor that acts during transport into or out of theGolgi complex could therefore be useful in examining thetransitional stages in much the same way that monensinhas helped to dissect transport through the Golgi stackitself (Griffiths et al. 1983; Quinn et al. 1983; Tartakoff& Vassalli, 1977).

The protonophore carbonylcyanide wj-chlorophenyl-hydrazone (CCCP) arrests protein transport at twostages. The first of these stages has been characterizedrelatively well and seems to be that of exit from the ER.Proteins arrested at this stage have undergone the ad-dition of N-linked oligosaccharides, initial trimming ofsugars (Tartakoff & Vassalli, 1979; Fries & Rothman,

633

1980; Godelaine et al. 1981; Argon & Milstein, 1984;Kabcenell & Atkinson, 1985), and oligomeric assembly(Argon & Milstein, 1984; Tartakoff & Vassalli, 1979).Their carbohydrates remain sensitive to endoglycosidaseH (Endo H), and are less completely processed thanthose arrested by monensin (Argon & Milstein, 1984),indicating that the first CCCP-sensitive site is proximal tothe medial Golgi. Immunofluorescence (Kaarianen et al.1980) and cell fractionation data (Godelaine et al. 1981;Geetha-Habib et al. 1985) indicate that the proteins arearrested in ER membranes.

Other data suggest that CCCP blocks transport at asecond transitional stage, between the trans-Go\gi andthe plasma membrane (Argon & Milstein, 1984; Argon etal. 1989). Immunoglobulin (Ig) arrested at this late stagebears galactose (Argon & Milstein, 1984), demonstratingthat it has reached the trans-Go\gi (Roth & Berger,1982). During Ig transport both the early and late CCCP-sensitive stages are present. The existence of two CCCP-sensitive transport stages complicates the precise defi-nition of the affected compartments, particularly the laterone.

We have attempted to dissociate the two CCCP-sensitive compartments using the transport of the glyco-protein (G) of vesicular stomatitis virus (VSV). Gprotein is inserted co-translationally into the rough ER,where it receives two Asn-linked oligosaccharides (Roth-man & Lodish, 1977). Shortly after synthesis, palmiticacid is added to a cysteine residue in the cytoplasmicdomain of G (Schmidt & Schlesinger, 1979, 1980; Roseet al. 1984). The protein then passes to the medial Golgi,where both oligosaccharides are converted to the complextype (Tabas et al. 1978). Finally, G protein reaches thecell surface, where it mediates assembly and subsequentbudding of virions (Knipe et al. 1977a). In Chinesehamster ovary cells, the transit time of G protein to thesurface is about 50min (Knipe et al. 19776).

In this study, we show that the transport of G proteinis blocked by CCCP at a very late stage. In CCCP-treatedcells, G protein acquires most of its normal post-translational modifications, but the expression of G onthe surface is inhibited. Since the enzymes responsiblefor some of these modifications reside in the trans-Golgi,this suggests that G protein is arrested by CCCP at theGolgi complex—plasma membrane transition. In theaccompanying paper (Burkhardt et al. 1989), we showthat G protein indeed accumulates in the Golgi region inthe presence of CCCP.

Materials and methods

CellsBHK-21 cells were obtained from the American Tissue CultureCollection (Rockville, MD). They were maintained in Dulbec-co's modified Eagle's medium (DMEM) supplemented withglutamine, penicillin, streptomycin (all from Grand IslandBiological Co., Grand Island, NY), and 5% foetal calf serum(FCS; KC Biologicals, Lenexa, KS).

Virus infectionsBHK-21 cells (106) were seeded in 60 mm Petri plates and

allowed to grow overnight. Subconfluent cultures were washedtwice with Earle's balanced salts solution (BSS, GIBCO) andcovered with DMEM/0-5 % FCS. VSV, (Indiana serotype, agift from Dr J. Keene, Duke Medical Center) was added to givea multiplicity of infection of 10. Following adsorption of thevirus for 30min at 37°C, the inoculum was replaced withcomplete medium and the cells incubated for 3-5-4h at 37CC.

Drug treatmentsCCCP, monensin and tunicamycin were prepared and used asdescribed by Argon & Milstein (1984).

ATP measurementsBHK-21 cells (2x 106 to 5 X 106) were incubated in the presenceof various concentrations of CCCP for 60min, or in 20/iM-CCCP for various times. The cells were then washed quickly inice-cold PBS (phosphate-buffered saline)/BSA (bovine serumalbumin) and pelleted in a microfuge (Eppendorf). The cellswere extracted with perchloric acid according to Lowry &Passonneau (1972), neutralized with potassium carbonate andthe lysates were stored at —70°C until use. Portions of thelysates were analysed by HPLC on a Whatman strong anionexchange resin. The column was run with an isocratic gradientof 60% of 750mM-(NH4)H2PO4, pH3-8, 40% of 30mM-(NH4)H2PO4, pH2-8, at a flow rate of 2 ml min"1. Nucleotideswere identified and quantified by calibration with knownstandards. The absolute ATP pools varied from experiment toexperiment (« = 3), but the extent of depletion was consistentwithin each experiment. Duplicate samples did not vary bymore than 10%.

Metabolic labellingBefore labelling with methionine, the cells were washed withBSS and starved for 20 min in DMEM lacking methionine andcontaining 3 % dialysed FCS. [35S]methionine (>600Cimmol"1, Amersham International, Arlington Heights, IL) wasadded to a final concentration of 150jiCiml~'. The duration ofradioactive labelling was 3-5 min. Chase medium (completeDMEM supplemented with 2mM-methionine and 5% undia-lysed FCS) was added, and the incubations continued asdescribed in the figure legends. Palmitic acid labelling wasperformed on cells washed twice with BSS and incubated for30min in serum-free DMEM. [3H]palmitate (SOCimmor1,Amersham), from a stock solution of \QjiC\jA~x in 80%ethanol, was added to 80^Ciml~ , and the incubation con-tinued for 45 min.

Immunoprecipitations and gel electrophoresisExcept where indicated, the following techniques were done asdescribed (Argon & Milstein, 1984): lysis of cells, immuno-precipitation, digestions with endoglycosidase H (Miles Scien-tific, Naperville, IL) and with Vibrio cholerae neuraminidase(CalBiochem, La Jolla, CA), SDS-polyacrylamide gel electro-phoresis (SDS-PAGE) and fluorography. G protein was preci-pitated quantitatively with a mouse IgG2a monoclonal antibody(MAb) H14D5 (Lefrancois & Lyles, 1982). This MAb, whichrecognizes all biosynthetic intermediates of G protein, was agenerous gift from Dr Doug Lyles (Bowman Gray School ofMedicine, Winston-Salem, NC). Gels were 24cm long, andcontained a linear 9% to 15% acrylamide gradient. Molecularweights were calculated by plotting relative mobilities of thevarious G protein intermediates against known markers. Mol-ecular weight values are averages of five separate gels. Auto-radiograms were quantified by scanning with a Zeineh soft laserdensitometer (Biomed Industries, Fullerton, CA). Several

634 J. K. Burkhardt and Y. Argon

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Fig. 1. Representative experiments showing the effects ofCCCP on ATP levels and protein synthesis. Each pointrepresents an average of duplicate samples. Duplicates didnot vary by more than 10%. Control ATP levels ranged from12 to 30nmol/l06 cells in three separate experiments.A. Relative levels of ATP following a 1-h incubation with theindicated concentrations of CCCP. B. Time course of ATPdepletion using 20/iM-CCCP. C. Relative levels of proteinsynthesis in the presence of the indicated concentrations ofCCCP. Cells were pretreated for 20 min prior to a 1-hlabelling in the continued presence of the drug. Proteinsynthesis was measured by the incorporation ofr/5S]methionine into TCA-precipitable material. D. Theeffect of pretreatment with CCCP on protein synthesis. Cellswere pretreated with 20/iM-CCCP for the indicated times,and labelled for 15 min with [35S]methionine in the continuedpresence of the drug.

exposures were used to ensure that measurements were withinthe linear range.

Flow cytometryMAb H14DS was coupled to dichlorotriazinylamino fluorescein(DTAF; Sigma, St Louis, MO) to give a fluorescein: proteinratio of 3-5 (Goding, 1976). Culture dishes were incubated with0 6 m g m l - 1 proteinase K (Boehringer Mannheim, Indianapo-lis, IN) for 15 min at 37°C, a treatment that both detaches thecells and removes most of the surface G protein. A controlsample, permitting measurement of surface G protein levelsprior to protease treatment, was acquired by harvesting cellsdetached due to the cytopathic effects of VSV infection.Samples in suspension were treated with inhibitors as describedin Fig. 1. Re-attachment of cells was prevented by periodicrocking in bacteriological grade dishes. Following treatment,cells were washed twice with phosphate-buffered saline contain-ing 5% horse serum (PBS/HS), and incubated withDTAF-H14D5 at 15/ igmr1 for l h on ice. Cells were thenwashed twice with PBS/HS, once with PBS, and fixed in 2%paraformaldehyde/PBS overnight at 4°C. Cell-associated fluor-escence was measured using an Ortho 50H flow cytometer gatedto analyse 104 single cells as described (Sturtevant & Balber,1986).

Results

CCCP depletes cellular ATP and reduces proteinsynthesisPrevious studies by us and others have shown that CCCPaffects not only protein transport, but other metabolicprocesses as well (Heytler, 1963; Poole & Okhuma, 1981;McKinnon et al. 1988; Argon et al. 1989). We thereforedetermined the magnitude of these effects in BHK cells.Cells were treated for 1 h with the concentrations ofCCCP indicated in Fig. 1A, or with 20 /ZM-CCCP for thetimes indicated in Fig. IB. At the end of each treatment,ATP concentrations were determined. As shown inFig. 1A, lOjiM-CCCP is sufficient to deplete cellularATP to 68 % of control levels. Over the time course of theexperiments, this dose did not affect cell viability.Although IOOJIM-CCCP lowered ATP levels slightlyfurther, this dose was toxic to cells after several hours.The depletion of ATP is a rapid process (Fig. IB), withmaximal depletion evident already by 5 min in thepresence of the drug.

We next examined the effects of CCCP on proteinsynthesis. Cells were pretreated with varying concen-trations of CCCP for 20 min, then metabolically labelledfor 1 h with [35S]methionine in the continued presence ofthe drug. The incorporation of [35S]methionine intotrichloroacetic acid (TCA)-precipitable material was de-termined for each sample. At 20 /XM, CCCP inhibitedprotein synthesis by 40-50%, with higher doses havinglittle further effect (Fig. 1C). Under the same con-ditions, 100 ng ml"1 cycloheximide inhibited protein syn-thesis by 93% (data not shown). Protein synthesis isinhibited at the earliest measurable times after CCCPaddition, and no further inhibition occurs over a timecourse sufficient for our experiments (Fig. ID). Theconcentration dependence of the inhibition of proteinsynthesis resembles the concentration dependence of thedepletion of ATP. In addition, both effects of CCCPoccur rapidly. In the light of these experiments, we chose20fiM-CCCP as the dose that optimally depletes ATP andreduces protein synthesis without compromising cellviability.

CCCP blocks appearance of G protein at the cell sutfaceTo determine whether CCCP arrests the intracellulartransport of G protein, we asked if the surface expressionof G protein is inhibited by treatment with CCCP. Fourhours after infection with VSV, BHK cells were treatedwith proteinase K to remove the existing surface Gprotein. After several washes, the cells were divided intothree portions. One was labelled immediately withDTAF-anti-G, to determine the level of surface Gprotein that remained after the protease treatment. Thetwo other portions were incubated for 90 min in thepresence or absence of CCCP, to allow G protein torepopulate the cell surface. At the end of this incubation,the cells were labelled with DTAF-anti-G and analysedby flow cytometry. Uninfected cells served as negativecontrols, while infected cells that were removed from theculture dish without protease treatment served as positivecontrols. Digestion with proteinase K decreased the

Late-Golgi inhibition of G-protein processing 635

Fig. 2. The effect of CCCP on the regeneration of surface Gprotein expression, after protease treatment. VSV-infectedBHK cells were labelled with DTAF-H14 (anti-G) and thelevels of surface G protein were quantified by flow cytometry,all as described in Materials and methods. A. Cells thatdetached in the course of viral infection, harvested withoutprotease treatment; B, cells following digestion of surface Gprotein with proteinase K (0-6mg ml"1 for 15 min at 37°C);C, cells digested with protease and allowed to recover for90min; D, cells digested with protease and allowed to recoverfor 90 min in the presence of 20/iM-CCCP; E, uninfectedBHK cells. Mean, the mean fluorescence channel for eachdistribution.

mean surface fluorescence to 21 % of control levels(Fig. 2A and B). After chasing for 90min in mediumalone, the mean surface fluorescence recovered to 81 % ofcontrol levels (Fig. 2C). When chased in medium con-taining CCCP, however, surface fluorescence did notrecover significantly, reaching only 32% of control(Fig. 2D). Because CCCP inhibits protein synthesis aswell as transport, parallel samples were proteolized andallowed to recover in the presence of cycloheximide.Recovery of surface G protein in cycloheximide-treatedcells was almost twice that in cells treated with CCCP(either alone or in combination with cycloheximide, datanot shown). Therefore, the failure of G protein torepopulate the cell surface in the presence of CCCPcannot be explained by the partial inhibition of proteinsynthesis. Our data thus show that treatment with CCCPinhibits the transport of the viral glycoprotein to the cellsurface.

The presence of G protein at the plasma membrane ofinfected cells is required for the assembly and budding ofVSV virions (Knipe et al. 1977a). When sufficient Gprotein accumulates at the plasma membrane, it nu-cleates the assembly of the other viral proteins (Knipe etal. 1977'a), all of which are synthesized on free ribosomes(Morrison & Lodish, 1975). We reasoned that if CCCPinhibits the arrival of G protein at the cell surface, itshould as a consequence inhibit the release of virionsfrom infected cells. To test this, infected BHK cells werepulsed for 3 min with [35S]methionine and chased in thepresence or absence of CCCP. Culture supernatants werecollected after one hour of chase and again after 4h.Virions were isolated by centrifugation and analysed bySDS-gel electrophoresis. The results are shown in Fig. 3and quantified in Table 1. CCCP treatment reduces thetotal amount of labelled viral protein released during thechase (Table 1). In particular, labelled G protein isdepleted more than the other viral proteins (comparelanes 1 and 2 of Fig. 3). Because G protein is required forbudding, the virions that are released from the cellsshould contain unlabelled G protein assembled withlabelled L, N, NS and M proteins. Indeed, when the gelshown in Fig. 3 was analysed by Coomassie Blue stain-ing, rather than autoradiography, this unlabelled Gprotein was detected (data not shown). Virions collectedfrom the supernatants after the second chase period(60-240 min, lane 4) reveal the longer-term effects ofCCCP treatment on virus production. In virions released

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636 jf. K. Burkhardt and Y. Argon

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Fig. 3. SDS-PAGE analysis of labelled virions produced byCCCP-treated cells. Infected BHK cells were pulsed for3 min with [35S]methionine and chased in the presence (lanes2 and 4) or absence (lanes 1 and 3) of CCCP. Supernatantswere collected at 60 min (lanes 1 and 2) and 4h (lanes 3 and4) of chase, and centrifuged at 50 000£ for 1 h to collectvirions. In order to reverse the effects of the drug, cellstreated as in lane 2 were washed and chased again withoutCCCP for 2-5 h. Virions collected during this second chaseperiod are shown in lane 5. In order to resolve better the Nand NS proteins, samples were separated using a gelcontaining 0 2 7 % bisacrylamide rather than the usual 0 '8%.

from CCCP-treated cells, the radioactivity in each viralprotein is decreased by 80% (Fig. 3, lanes 3 and 4, andTable 1). We interpret this result to indicate that surfaceG protein has become limiting during this interval(Knipe et al. 1977a), thus inhibiting budding of labelledvirions.

We used the virus-budding assay to test whether the

Table 1. Densitometric analysis of the effects of CCCPon production of [35SJmethionine-labelled virions

Protein

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inhibition of surface G protein expression is reversible.Lane 5 of Fig. 3 shows the protein composition of virionscollected from cells that were first chased for 60 min inthe presence of CCCP, then washed and chased foradditional 2-5 h in the absence of the drug. Budding oflabelled virions resumes, and these virions contain thelabelled G proteins that had accumulated during thechase in CCCP. T h e radioactivity recovered in each viralprotein is increased to a similar extent (Table 1). Thus ,G protein that was previously arrested with CCCP isavailable for virus budding after the protonophore isremoved.

These two experiments demonstrate that CCCP in-hibits the arrival of newly synthesized G protein at thecell surface, and that this inhibition is reversible. Inaddition, these experiments show that CCCP does notaffect virus budding per se, because other labelled viralproteins can assemble and bud with pre-existing, un-labelled G protein.

CCCP arrests G protein transport at a late stage ofprocessing

Having determined that CCCP inhibits the arrival of Gprotein at the cell surface, we asked at what stage intransport it is arrested. One test was to determine thestate of oligosaccharide processing undergone by CCCP-arrested G protein. Infected B H K cells were pulse-labelled for 3 min with [35S]methionine and either lysedimmediately or chased for 90 min in the presence ofCCCP, monensin or control medium. G protein wasisolated from cell lysates, and analysed by S D S - P A G Ebefore (Fig. 4, lanes 1-4) and after (lanes 5-8) digestionwith Endo H. Newly synthesized G protein isolated

1 8

Fig. 4. Comparison of CCCP-arrested G protein with otherintracellular forms. Infected cells were pulse-labelled for3 min with [3sS]methionine. They were lysed immediately(lanes 1 and 5) or chased for 90 min in the presence of CCCP(lanes 2 and 6), in the presence of monensin (lanes 3 and 7),or without drugs (lanes 4 and 8). G protein wasimmunoprecipitated from each sample, and half of eacheluate was digested with Endo H (lanes 5-8). Only thatportion of the gel around the G protein is shown.

Late-Golgi inhibition of G-protein processing 637

immediately after the pulse has the mobility of anapproximately 64-5xlO3Afr protein (lane 1). Note thatthere is also a minor species in lane 1 that comigrates withunglycosylated G and disappears during the subsequentchase. This species may be the secreted form of G(Garreis-Wabnitz & Kruppa, 1984). G protein chased inthe presence of CCCP (lane 2) is larger than the newlysynthesized G. It is similar in size to the mature form(67-5xlO3Mr, lane 4), but often migrates as a broaderband. In contrast, the G protein isolated from cellschased with monensin (lane 3) is slightly smaller than thenewly synthesized G (about 64xlO3Mr). When newlysynthesized G and monensin-arrested G are digestedwith Endo H (lanes 5 and 7 respectively), they arereduced to the size of unglycosylated G (60-5X 10 Mt).This confirms that both of these species bear high-mannose oligosaccharides. In contrast, if G protein ischased without drugs, it becomes fully resistant to EndoH digestion (lane 8), indicating conversion of its oligosac-charides to the complex type. Digestion of CCCP-arrested G with Endo H reveals a mixed pattern ofsensitivity (lane 6). Approximately 25 % of the label is ina fully resistant form, 25 % in a fully sensitive form, andabout 50% in an intermediate form. This intermediatespecies probably bears one complex and one high-mannose oligosaccharide.

This analysis demonstrates that the CCCP-arrested Gprotein is heterogeneous. Most of the arrested G has beentransported at least as far as the mid-Golgi, where EndoH resistance is conferred, and it is more mature thanmonensin-arrested G. A minor portion bears less-matureoligosaccharides, indicating that there may be a secondCCCP-sensitive step at a distinct, pre-Golgi site (seebelow).

Other investigators have found that CCCP arrests Gprotein in an Endo-H-sensitive form (Fries & Rothman,1980; Kabcenell & Aktinson, 1985). One possible expla-nation for the discrepancy with our results is that underthe conditions we use, the early CCCP block is incom-plete. However, increasing the concentration of CCCP to120 fiM did not increase the proportion of G proteinarrested in an Endo-H-sensitive form (data not shown).Therefore, the dose used in our studies (20 (JM) issufficient. To ensure that the pulse-labelled G proteindoes not escape the effect of CCCP before the drug hastime to block transport, we pretreated cells for 1 h withCCCP before labelling with [35S]methionine. This pre-treatment did not alter the size of the arrested G protein,nor its heterogeneity with respect to Endo H sensitivity(Fig. 5, lanes 2 and 8). We could also show that theheterogeneity of sugar processing is not affected byongoing protein synthesis. Pulse-labelled G protein waschased in the presence of cycloheximide, CCCP, or acombination of the two. Like G protein that was chasedwithout drug (lanes 5 and 11), G protein chased withcycloheximide became fully resistant to Endo H digestion(lanes 6 and 12). However, G protein chased in thepresence of both cycloheximide and CCCP (lanes 4 and10) was identical to that chased with CCCP alone (lanes 3and 9), with most of it becoming resistant to Endo H. Infurther attempts to arrest all of G at an Endo-H-sensitive

1 2 3 4 5 6 7 8 9 10 11 12

Fig. 5. The forms of G protein synthesized in the presenceof CCCP and cycloheximide. Cells were pulse-labelled for5min with [35S]methionine and chased for 90min, asdescribed below. CCCP was always used at 20 jifA andcycloheximide at 100 ng ml" . Lanes 1-6 showimmunoprecipitated G without Endo H digestion. Lanes7-12 show the equivalent samples after digestion with EndoH. Lane 1 and 7, pulse only. Lanes 2 and 8, pretreatmentwith CCCP for 1 h and chase in the continued presence ofCCCP. Lanes 3 and 9, pulse label without pretreatment, andchase with CCCP. Lanes 4 and 10, chase with both CCCPand cycloheximide. Lanes 5 and 11, chase without drugs.Lanes 6 and 12, chase with cycloheximide only.

stage, we varied the temperature of the pulse-chase(30°C versus 37°C), and also used higher doses of CCCPin conjunction with low-glucose medium. Even underthese conditions the arrested G protein was hetero-geneous, with only a minority in an Endo-H-sensitiveform. We conclude that if CCCP does inhibit an early,pre-Golgi transport step in this system, the inhibition isvery leaky, allowing most of the molecules to proceed tothe late Golgi arrest site.

Since the above results indicate that some of G mightbe arrested in an early transport compartment, we askedif both the Endo-H-resistant and the Endo-H-sensitivepopulations of G undergo an early post-translationalmodification, acylation with palmitic acid. This modifi-cation normally occurs soon after G protein is syn-thesized and before it reaches the medial Golgi (Schmidt& Schlesinger, 1980). When infected BHK cells werelabelled with [3H]palmitate, the incorporation of labelinto G protein in the presence of CCCP was 80 % of thatobserved in untreated cells (3000ctsmin~ 106 cells ver-sus 3700ctsmin~ 106cells, respectively). The decreasein the incorporation of palmitate can be accounted for byinhibition of protein synthesis, because similar incorpor-ations are obtained in cells treated with CCCP and withcycloheximide. The 3H-labelled G protein fromuntreated cells is fully resistant to Endo H digestion(Fig. 6, lanes 1 and 2). In contrast, the acylated Gprotein from CCCP-treated cells exhibits a pattern ofpartial resistance, similar to the pattern seen with[35S]methionine labelling (compare Figs 6 and 4). Thus,treatment with CCCP does not inhibit the acylation of Gprotein, but does inhibit the transport of acylated ma-terial. We can also conclude that if there is an earlyCCCP-sensitive stage, it must occur after palmitateaddition.

We next wished to characterize the late stage of CCCParrest with respect to the terminal glycosylation of Gprotein. The two species of arrested G that exhibit partialor complete resistance to Endo H can be shown to bearsialic acid by neuraminidase digestion (Fig. 7). Like thefully mature form of G (lanes 5 and 6), the Mr of thelargest CCCP-arrested species (lanes 3 and 4) decreases

638 J. K. Burkhardt and Y. Argon

CCCP: -EndoH: -

Fig. 6. Acylation of G protein in the presence of CCCP.Infected cells, starved in serum-free medium, were pretreatedfor lOmin with 20/iM-CCCP or mock treated. [3H]palmitatewas then added, and cells were labelled for additional 90min(±CCCP). Intracellular G protein was isolated and a portiondigested with Endo H, as indicated. An overexposedautoradiogram is shown to reveal the minor species.

1

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Fig. 7. Sialylation of CCCP-arrested G protein. Infectedcells were pulse-labelled as described for Fig. 2, and lysedimmediately (lanes 1 and 2) or chased in the presence (lanes3 and 4) or in the absence (lanes 5 and 6) of CCCP. Afterimmunoprecipitation of G protein, all samples were digestedwith Endo H, and those shown in lanes 2, 4 and 6 weresubsequently digested with 5-10munits of neuraminidase for2hat37°C.

by about 1-5X103 after neuraminidase digestion. Themiddle band also shifts after digestion with neuramini-dase, but by only about 0-5X 103. This is consistent withthe sialylation of only one of the two N-linked glycans.Finally, the smallest form of CCCP-arrested G proteinresembles the newly synthesized form (lanes 1 and 2) inthat it is unaffected by neuraminidase. These data showthat in the presence of CCCP most of G reaches thetrans-Golgi, where sialic acid is added (Roth et al. 1985).

Fucose is reportedly added to N-linked oligosacchar-ides very late in transport, after the addition of sialic acid(Melchers, 1971). To ask whether fucosylated G proteinis arrested by CCCP or released into virions, we labelledcells for 60min with [3H]fucose in the presence or

absence of CCCP. Virions were then collected from themedium and G protein was isolated from cell lysates. Asshown in Table 2, 95 % of the fucosylated G protein fromCCCP-treated cells was found in virions, compared with69 % from control cells. This indicates that the export offucosylated G protein is not arrested by CCCP. It shouldbe noted that the incorporation of [3H] fucose in CCCP-treated cells was much lower than that in control cells.This decrease is not due solely to the inhibition of proteinsynthesis by CCCP, since greater incorporation of fucoseis observed in cycloheximide-treated cells than in CCCP-treated cells (data not shown). We interpret these resultsto show that CCCP arrests transport just proximal to thefucosylation site. Thus, the pool of G protein availablefor fucosylation is depleted, while G protein in or distal tothe fucosylation site is released from the cell.

Together, these results indicate that in the presence ofCCCP, the majority of G protein reaches a distaltransport compartment where terminal glycosylationtake9 place. Glycosylation is incomplete, however, asfucose is not added and some molecules bear one or twohigh-mannose oligosaccharides.

Discussion

In this study, we show that the protonophore CCCPinhibits the transport of the G protein of VSV at a verylate stage in its post-translational processing.

In the presence of CCCP, G protein does not reach thecell surface. Analysis of virions produced by CCCP-treated cells indicates that G, the only membrane proteinof VSV, is the only protein whose incorporation intovirions is directly affected by the drug. Since virions arereleased for at least one hour in the presence of CCCP,the drug does not inhibit virus budding per se. Theappearance of G protein at the cell surface, and sub-sequent budding of virions, resume if CCCP is removedfrom the culture medium, showing that the effects ofCCCP are reversible. The inhibition of G protein exportis consistent with studies showing CCCP-induced inhi-bition of surface expression or secretion of other proteins(Tartakoff & Vassalli, 1979; Kaarianen et al. 1980; Fries& Rothman, 1980; Godelaine et al. 1981; Argon &Milstein, 1984; Kabcenell & Atkinson, 1985).

The majority of G protein molecules that accumulatein the presence of CCCP is at least partially resistant todigestion by Endo H. From the standpoint of proteintransport, it is significant that even one glycan on theCCCP-arrested G protein is processed to the complexform. This indicates that these molecules, like those withtwo complex glycans, have been transported at least as far

Table 2. Secretion of [3Hjfucose-labelled G protein in the presence of CCCP

Treatment*Cell lysate

(ctsmin~')tSupernatant(ctsmin"1)

Total(ctsmin" )

Secreted

NoneCCCP

2084 ±174199 ±20

4737 ± 4703820 ± 469

68214019

69-495-1

• 107 infected cells were starved lOmin for glucose and labelled for 60min with [3H]fucose, in the presence or absence of 20/iM-CCCP.I Mean and S.D. of three experiments.

Late-Golgi inhibition of G-protein processing 639

as the mid-Golgi. Both of the mature species of G proteinare sensitive to neuraminidase digestion, indicating thattheir complex oligosaccharides have undergone sialyla-tion. This shows that the majority of CCCP-arrested Gprotein has reached the trans-Go\g\, where sialyltransfer-ase resides (Roth et al. 1985).

The presence of a species bearing only one Endo-H-resistant glycan cannot be explained by simple arrest of Gprotein transport, since both carbohydrates carried by asingle G protein molecule should be available to the sameprocessing enzymes. It is likely that incomplete sugaraddition results from another effect of CCCP, perhapsdepletion of the nucleotide sugar substrates for terminalglycosylation. It is less likely that CCCP directly inhibitsan enzyme required for terminal glycosylation, because itdoes not inhibit galactosyltransferase activity in vitw (D.Wiest, unpublished). In any case, it is clear from theirpartial terminal glycosylation that these molecules havereached a distal transport compartment.

There is evidence that the last sugar to be added tocomplex oligosaccharides is fucose (Melchers, 1971), butthe site of fucosylation has not been determined. Meta-bolic labelling with [3H]fucose shows that fucosylated Gprotein is incorporated efficiently into virions in thepresence of CCCP. This suggests that the site of fucosyl-ation is distal to the site where G protein is arrested. Thissituation is similar to that observed with immunoglobulin(Argon et al. 1989). Because glycosylation is thought tobe completed in the Golgi complex, it is usually assumedthat the fucosyltransferase is present along with sialyl-transferase in the most distal Golgi cisternae. Our resultsfrom two independent systems show that these twoactivities can be separated, and raise the interestingpossibility that fucosylation occurs at a site distal to thetrans-most Golgi cisterna.

Our analysis of the major portion of CCCP-arrested Gprotein indicates that it is arrested in a distal transportcompartment. The oligosaccharides have been sialylatedbut not fucosylated, and the G protein does not reach thecell surface. These data are consistent with arrest oftransport through trans-Go\gi subcompartments, or withinhibition of subsequent transport to the cell surface.The pattern of glycosylation of G protein arrested byCCCP (including the presence of a minority of moleculesbearing immature oligosaccharides) bears a striking re-semblance to the phenotype of G protein arrested in thetrans-Go\gi reticulum at 20°C (Griffiths et al. 1985).

A minor portion of the CCCP-arrested G protein isfully sensitive to Endo H, indicating that both oligosac-charides are in the high-mannose form. CCCP-inducedarrest in an Endo-H-sensitive form has been described fora number of proteins (Godelaine et al. 1981; Kaarianen etal. 1980; Tartakoff & Vassalli, 1979), including G (Fries& Rothman, 1980; Kabcenell & Atkinson, 1985). Thearrested proteins bear oligosaccharides trimmed to theMan9GlcNAc-Man7GlcNAc intermediates (Godelaineet al. 1981; Kabcenell & Atkinson, 1985). In agreementwith these examples, we have described Ig arrested inCCCP-treated myeloma cells in an analogous stage(Argon & Milstein, 1984), under the same conditions weused here to examine G protein transport. We therefore

attempted to demonstrate a distinct early arrest site for Gprotein. To do so, we varied the time of CCCP addition,the concentration of CCCP, the temperature of thepulse-chase and the level of glucose. In particular, wetested the conditions used by Kabcenell & Atkinson(1985), i.e. 100J IM-CCCP with low-glucose medium, inour system. None of these conditions significantlyincreased the proportion of the high-mannose form of G.Thus, we are unable to find conditions where all the Gprotein is blocked before the Golgi complex, as describedby others. Because of this, the existence of a distinctCCCP-sensitive block to G transport between the ER andGolgi cannot be ascertained. If such a distinct blockexists, it must be very leaky. Alternatively, the accumu-lation of a high-mannose form of G may be a consequenceof the more distal Golgi block, which causes an accumu-lation of G protein along the secretory pathway. If this isthe case, CCCP affects G protein transport in a similarway to the mutation del-1554 (Gabel & Bergmann, 1985).

The failure to arrest G in an Endo-H-sensitive form isnot a function of the host cell. We find that the majority ofG protein is arrested in a mature form not only in BHKcells, but also in VSV-infected hybridoma cells. Arrest atthe late stage is also observed in Cos-7 cells transfectedwith G protein cDNA (J. Dul, unpublished). In anattempt to reproduce the results of Balch et al. (1986),who found that G protein is arrested in the ER of Chinesehamster ovary cells depleted of oxygen, we examined thetransport of G protein in CCCP-treated CHO cells.Again, arrest was primarily at the late transport stage.Thus, the insensitivity to CCCP at the ER-to-Golgitransition appears to be protein-specific rather than cell-specific. Indeed, our preliminary data show that Ig and Gprotein are differentially sensitive to CCCP at thistransition, even within the same cell. Perhaps this reflectsfundamental differences between proteins in their meta-bolic requirements for efficient transport. Such require-ments could determine the differences observed in thetransport kinetics of individual proteins within the samecell (Strous & Lodish, 1980).

Because CCCP uncouples oxidative phosphorylation(Heytler, 1963), it is commonly assumed that it inhibitstransport by depleting ATP. Both the transition from theER to the Golgi (Balch et al. 1986; Jamieson & Palade,1968; Tartakoff, 1986) and the transition from the Golgito secretory granules and to the cell surface (Balch &Keller, 1986; Jamieson & Palade, 1968; Tartakoff, 1986)have been shown to be particularly sensitive to ATPdepletion. Balch et al. (1986) found that the transfer of Gprotein to the m-Golgi in CHO cells, as assayed bymannosidase I activity, requires at least 80 % of normalATP levels. Kabcenell & Atkinson (1985) have shownthat G protein is arrested in the ER of MalO4 cells, whentreated with CCCP. In contrast, we find transport of Gprotein through this compartment in BHK cells, underconditions where ATP levels are as low as 40 % of control.Our data, both for G protein (this paper) and for Ig(Argon et al. 1989) do indeed correlate the overallinhibition of exocytosis by CCCP with the depletion ofATP. In the case of G protein, however, this inhibition ismanifested primarily at the second ATP-sensitive stage,

640 J. K. Burkhardt and Y. Argon

transport from the Golgi to the cell surface.It must be noted that as a protonophore, CCCP can

affect cellular metabolism in several ways. In addition todepleting cellular ATP pools, it raises the pH of acidiccompartments (Poole & Okhuma, 1981). In fact, it isdifficult to definitively separate the effects of ATPdepletion and intracellular pH, since ATP-dependentproton pumping is required for maintenance of low pH,and selective proton permeability is required for oxidat-ive phosphorylation. The low pH of compartments suchas endosomes and lysosomes is known to be required fornormal protein movement (Gonzalez-Noriegaet al. 1980;Mellman et al. 1986). The /raws-Golgi is also acidic(Anderson & Pathak, 1985), but the role of this acidity inprotein transport has not been determined. Thus, thedistal CCCP block may result directly from the alkaliniz-ation of this compartment. This could, in fact, explainthe differences in sensitivity of G protein to CCCP at thetwo transitional transport steps.

The presence of a CCCP-sensitive stage very late intransport has already been described for two otherproteins, Semliki Forest virus glycoprotein and IgD(Kaarianen et al. 1980; Argon & Milstein, 1984). In bothcases, however, the presence of another, earlier, CCCP-sensitive stage complicates the analysis of the late trans-port compartment. The apparent absence or leakiness ofthe early CCCP-sensitive stage in the system describedhere makes it possible to characterize the late transitionalcompartment. In the accompanying paper (Burkhardt etal. 1989), we have used both immunofluorescence andimmunoelectron microscopy to determine where G pro-tein accumulates when arrested at the late stage. We showthat CCCP disrupts the ultrastructure of a distal Golgicompartment, and present evidence that the arrested Gprotein accumulates in this compartment.

We thank Dr D. Lyles for his generous supply of monoclonalantibodies, Dr J. Keene for advice and for gifts of VSV stocks,and Dr A. Balber for advice and for help with FACS analysis.We are indebted to Drs J. Leeds and M. Hershfield for theirhelp with ATP determinations. Our thanks also to A. Stock-dale, D. Wiest and Drs P. Cresswell, J. Keene, M. Snider, C.Lapham and J. Dul for critical reading of this manuscript. Thiswork is in partial fulfilment of the requirements for the Ph.Ddegree of Duke University (J.K.B.). It was supported by NIHgrants AI-08817 and AI-23282. J.K.B. was supported by NIHtraining grants GM-07184 and CA-09058.

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{Received 27 October 1988 - Accepted 6 January 1989)

642 J. K. Burkhardt and Y. Argon