Proc. Nati. Acad. Sci. USAVol. 78, No. 3, pp. 1746-1750, March 1981Cell Biology

Passage of an integral membrane protein, the vesicular stomatitisvirus glycoprotein, through the Golgi apparatus en route to theplasma membrane

(membrane biogenesis/intracellular glycoprotein transport/immunoelectron microscopy/cryo-ultramicrotomy)

JOHN E. BERGMANN, K. T. TOKUYASU, AND S. J. SINGERDepartment of Biology, University of California at San Diego, La Jolla, California 92093

Contributed by S. J. Singer, December 12, 1980

ABSTRACT The intracellular pathway of biogenesis of thevesicular stomatitis virus transmembrane glycoprotein was inves-tigated in situ by using indirect immunofluorescence of whole in-fected Chinese hamster ovary cells and immunoelectron micros-copy of ultrathin frozen sections of infected cells. Transport of theglycoprotein was synchronized by using the temperature-sensitivevirus mutant Orsay-45 and a temperature shift-down protocol.Sequential appearance of the glycoprotein in the rough endo-plasmic reticulum, Golgi apparatus, and plasmalemma was dem-onstrated. The potential of this system for further studies isdiscussed.

Essential to an understanding of membrane function is a bettercomprehension of the mechanisms of biogenesis and assemblyof the different membrane components. Transmembrane pro-teins are a particularly interesting class of membrane compo-nents because they are thought to be involved in such importantprocesses as transmembrane signaling and the transport of ionsand small molecules across membranes.

Although much has been learned about posttranslationalchanges in the proteins during their passage to the cell surface(or to the extracellular milieu), much less has been learnedabout the intracellular structures through which these proteinspass. Data from autoradiographic and cell fractionation exper-iments have suggested that the addition of most of the terminalsugars occurs in the smooth membrane systems making up theGolgi complex (1-4). As a result, it has been widely assumedthat all glycoproteins, including integral membrane glycopro-teins, pass through the Golgi complex on their way to the cellsurface. However, all the evidence to date has been indirect.Thus, when Farquhar recently discussed the evidence for Golgiapparatus involvement in the biogenesis of integral membraneproteins, she concluded that "passage (of integral proteins)through the Golgi is implied but not directly demonstrated, andit is clear that what is needed to settle the issue is the purifi-cation of a membrane protein from both Golgi and plasma-lemma membranes and demonstration of a kinetic relationshipbetween the two" (5). Unfortunately, such biochemical ap-proaches have been complicated by the difficulty of obtainingpure, well-defined subcellular fractions. In addition, cell dis-ruption often leads to the rearrangement of cellular constituentsbetween different cellular compartments (6). We therefore un-dertook to follow the intracellular pathway of such a protein inintact cells.

Recently, techniques involving the immunolabeling of ul-trathin frozen sections of cell or tissue specimens have beendeveloped in this laboratory that allow one to unambiguously

localize an antigen within a cell to a resolution of 200-300 A(7-10). We wished to use these techniques to map the intra-cellular pathway taken by an integral membrane protein fromits site of synthesis to its final destination. The protein we choseto study was the glycoprotein (G) of vesicular stomatitis virus(VSV). This system has already been extensively utilized as amodel for plasma membrane protein biogenesis. The G proteinis the only VSV-specified transmembrane protein. BecauseVSV induces the synthesis of only five proteins, it is thoughtthat VSV makes use of the host cell's machinery to translate,modify, and transport the G protein to the plasma membrane,from which it is incorporated into virions as they bud off fromthe cell surface. The G protein is synthesized on ribosomesbound to the rough endoplasmic reticulum (RER) (11), cotrans-lationally glycosylated and injected through the membrane ofthe RER (12, 13), and finally transferred via lighter densitymembranes to the plasma membrane (14). During this post-translational transport, the G protein becomes further modi-fied-it becomes attached to several molecules of fatty acid (15)and is further glycosylated (14, 16-18). In addition, our choiceof the G protein for these studies was based on several advan-tageous aspects of the VSV system. First, and foremost, manyviral mutants have been isolated with temperature-sensitivelesions in the G protein (19-21). (Other mutants with temper-ature-sensitive lesions in the other viral proteins have also beenisolated.) Second, during infection, the mutant or wild-type Gprotein can be readily introduced into a wide variety of hostcells. Third, intracellular localization is simplified because thefive viral proteins of VSV are synthesized in large quantities ininfected cells. Finally, the proteins can be purified from virionsfor antibody production or biochemical characterization.

This paper presents the results of our first studies of the in-tracellular pathway followed by the G protein during its passageto the cell surface. To conduct this study we made use of a tem-perature-sensitive mutant of VSV, Orsay-45 (0-45), which en-abled us to synchronize G protein transport. The rationale be-hind the experiment was as follows: At the restrictive tempera-ture, 39.70C, this mutant is able to infect cells, replicate itsgenome, and transcribe its mRNA. The infected cell is thereforeable to produce all of the viral proteins. However, the G proteinthat is made is defective and is not transported out of the RER.When these infected cells are shifted to 320C, much of the Gprotein that was made previously is unable to move, but the Gprotein made after the temperature shift is transported to thecell surface (20, 22). Using immunolabeling methods to study

Abbreviations: VSV, vesicular stomatitis virus; G protein, glycoproteinof VSV; M protein, membrane protein of VSV; RER, rough endo-plasmic reticulum; CHO cells, Chinese hamster ovary cells.

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Proc. NatL Acad. Sci. USA 78 (1981) 1747

the intracellular distribution of G protein at various times aftersuch a temperature shift, we were. able to follow the progressof the G protein from the RER to the cell surface and in par-ticular to observe directly the involvement of the Golgi appa-ratus in this pathway.

MATERIALS AND METHODSCell Culture and Virus .Infections. Chinese -hamster ovary

(CHO) cells were grown in suspension in Joklik's modified Ea-gle's medium supplemented with 7% fetal bovine serum, non-essential amino acids, penicillin (100 units/ml), streptomycin(100 units/ml), and Fungizone (250 ng/ml). For indirect im-munofluorescence experiments, cells in the same medium wereseeded onto no. 1 glass coverslips at a density of 104 cells percm2 and allowed to attach overnight.VSV (Indiana serotype) was grown by low multiplicity pas-

sage on monolayers of CHO cells. For antibody production,virus was purified by sedimentation and equilibrium densitybanding in a 15-50o (wt/vol) sucrose gradient.

Production and Purification of Antibodies. G protein wasisolated as described by Kelley et al. (23). Immunization of rab-bits was performed as described (24).G protein was also used in the preparation of an affinity col-

umn. Briefly, the G protein was bound to an agarose columncontaining immobilized lectin from Lens culinaris (Sigma) andsubsequently crosslinked to that column with 2% glutaralde-hyde. Affinity chromatography of antibody to the VSV G pro-tein was performed as described (25).

Immunofluorescence Experiments. CHO cells plated on no.1 coverslips in 35-mm tissue culture dishes were infected with3 X 107 plaque-forming units of 0-45. The coverslips weretransferred to test tubes containing 10 ml of medium at 39.70Cand a magnetic stirring bar. After the cells had incubated for3 hr at 39.70C, the tubes were transferred to the 320C waterbath. After the times indicated in the legend to Fig. 1 the tubeswere removed from the water bath, and the medium was pouredoff and replaced with 3% (wt/vol) formaldehyde in phosphate-buffered saline. Indirect immunofluorescence staining andmicroscopy were performed as described (26). The concentra-tion of IgG used in both the primary and the secondary stainingwas 10 ,ug/ml. The secondary label was a rhodamine conjugateof affinity-purified goat antibodies to rabbit IgG.

Cell Preparation for Electron Microscopy. CHO cells grownin suspension were infected at a multiplicity of 10 plaque-form-ing units per cell. The cells were incubated in test tubes con-taining stirring bars at 39.70C for 3 hr and transferred to 320Cfor the time indicated in Results. The cells were rapidly cooledby addition to shell frozen medium and collected by centrifu-gation. The resulting pellet was fixed in 3% formaldehyde/2%glutaraldehyde, embedded in agarose, and cut into smallblocks. The blocks were infused with sucrose, frozen, cryosec-tioned, immunolabeled, positively stained with uranyl acetate,and embedded in methylcellulose as described (7-9). The con-centration of immunolabeling reagents was always 10 ,g of IgGper ml of buffer. The secondary label was a conjugate of horsespleen ferritin and goat antibodies to rabbit IgG.

RESULTSImmunofluorescence Observations. In order to gain an

overall low-resolution view of the intracellular transport pro-cess, we began by studying the 0-45-infected cell with indirectimmunofluorescence staining and light microscopy. WhenCHO cells grown in monolayer culture and infected for 3 hr at39.7°C were fixed and immunolabeled, a diffuse pattern wasobserved (Fig. la). The only clearly defined structural featurethat was stained in the infected cells was the nuclear envelope.This pattern was consistent with our expectations that the Gprotein had remained confined to the RER at the nonpermissivetemperature. Mock-infected cells treated in the same mannershowed no detectable labeling under these conditions (notshown).When cells infected at 39. 7°C were shifted to 32°C for 5 min

prior to fixation, the staining pattern appeared essentially un-changed (Fig. lb). However, 8 min after the temperature shift,local concentrations of the G protein could be seen near thenucleus and staining of the nuclear envelope was reduced (Fig.lc). The accumulation of G protein in the perinuclear structuresbecame continually more pronounced 13 and 25 min after thetemperature shift (Fig. 1 d and e) but decreased 50 min afterthe shift (Fig. lf). These observations suggested that a waveof the G protein was being transported from the RER throughthe Golgi complex, and thence to the plasmalemma.

Electron Microscope Observations. Definitive identifica-tion of the fluorescent structures required high-resolution im-

FIG. 1. Immunofluorescence micrographs ofCHO cells infected with 0-45 VSV. CHO cells were infected and incubated for 3 hr at 39.7°C, shiftedto 32°C and incubated for various times as indicated below, fixed, made permeable, and stained forG protein. Prior to fixation, the times of incubationat 32°C were: 0 min (a),-5 min (b), 8 min (c), 13 min (d), 25 min (e), and 50 min (f). Ring staining of the nucleus is indicatedby arrowheads. Surfacelabel is marked by the long arrow in f. The bars indicate 10 ,um.

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FIG. 2. Immunoelectron micrograph of CHO cell infected with 0-45 at the nonpermissive temperature. Ultrathin frozen sections of infectedCHO cells were immunolabeled with rabbit anti-G protein antibody followed by ferritin-conjugated goat antibodies directed against rabbit IgG. TheGolgi apparatus (G), nucleus (N), and cytosol are not significantly labeled. The nuclear envelope (NE) and the RER are labeled to a greater extent.To aid in the identification of ferritin particles in the entire field, every ferritin in the circumscribed area was marked with an arrowhead. The barindicates 100 nm.

munoferritin staining and electron microscopy. Accordingly, asuspension of CHO cells was infected with:0-45 and incubatedfor 3 hr at 39.70C. The infected cells were then shifted to 320C,incubated for various times, cooled rapidly to 00C, and fixed.The fixed cells were sectioned, immunolabeled, and furtherprepared for observation in the electron microscope as outlinedin Materials and Methods. Fig. 2 shows a representative mi-crograph of cells infected with 0-45 at 39.7C but not shiftedto 32TC (the zero time point). No structures were heavily la-beled. In particular, the levels of labeling of the Golgi apparatusand plasmalemma were much lower than in cells infected withwild-type virus (not shown but see below). More label was as-sociated with the RER and nuclear envelope, although the levelof labeling of these structures was also low. Uninfected controlcells sectioned and immunolabeled in the same manner as thecells shown in Figs. 2-4 were far more lightly labeled; areasencompassing the entirety of Fig. 2 typically had no ferritinparticles (not shown).

By 11 min after the shift to 32TC (Fig; 3a), labeling of theGolgi apparatus was very intense; and the label was alwaysfound uniformly distributed across the entire stack of saccules.Label covered both the tightly apposed central portions and thedilated rims of the saccules. Within the same cells there wasno increase in the level of labeling of the plasma membrane overthe zero time point. Fig. 3b shows the plasma membrane fromthe same cell as Fig. 3a; and Fig. 3c indicates the parts of thecell that were magnified in Fig. 3a and b.

By 25 min after the temperature shift, plasma membranelabeling was significantly increased (Fig. 4). The label was oftenfound in clusters or associated with budding virus. We con-cluded that the G protein moved from. the RER to the Golgiapparatus prior to the plasma membrane.

DISCUSSIONIn this study we examined the intracellular location of an in-tegral membrane glycoprotein (G) at a level of resolution thathad been unattainable by using autoradiography. Particular at-

tention was paid to the potential involvement, of the Golgi ap-paratus in the intracellular pathway of integral membrane pro-tein biogenesis. Utilizing a temperature-sensitive G protein, wewere able to synchronize its passage to the cell surface and thusunambiguously map stages in the intracellular pathway that itfollowed.The VSV mutant used in these studies synthesizes a defective

G protein at the nonpermissive temperature. Previous findings(20, 22) that the G protein of this mutant is largely restrictedto the RER at the nonpermissive temperature were confirmed(Fig. 2). After release of the temperature block, G proteinmoved rapidly to the Golgi apparatus. Within 11 min, G proteinwas found uniformly distributed over the entire stack.of sac-cules (Fig. 3). As was seen in the indirect immunofluorescenceexperiments, the labeling over the Golgi apparatus at this timewas much heavier than the labeling over the endoplasmic re-ticulum, suggesting that the G protein had been greatly-con-centrated as it was transported to the Golgi-apparatus. Becausethe G protein had not yet reached the plasma membrane, it wasclear that the Golgi- apparatus was a definite part of the intra-cellular pathway taken by G on its way to the cell surface. Inthese experiments it was not possible that the G protein in theGolgi apparatus had arrived there via a recycling of the plasmamembrane. PreVious investigations of the intracellular pathwayfollowed by integral membrane proteins were hampered by aninability simultaneously to-synchronize the release of the pro-tein of interest from the RER and to recognize it unambiguouslywithin the cell (5, 27-30). Thus we have directly demonstratedthat- the saccules of the Golgi complex participate in the intra-cellular. transport of an integral membrane protein bound forthe plasma membrane.

As mentioned above, migration of the G protein to:the Golgiapparatus was quite rapid, and the G protein was seen to beuniformly distributed throughout the Golgi apparatus, A gen-erally uniform distribution was also seen at later times after thetemperature shift-and in wild-type-infected cells. However, atearlier times after the temperature shift, the nucleus-proximal

Proc. Natl. Acad. Sci. USA 78 (1981)

Proc. Natl. Acad. Sci. USA 78 (1981) 1749

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FIG. 3. Immunoelectron micrographs of CHO cell infected with 0-45 for 3 hr and shifted to 320C for 11 min. Infected CHO cells were sectionedand immunolabeled as in Fig. 2. (a) The nucleus (N) and cytosol are not labeled. The nuclear envelope (NE) is more lightly labeled than in Fig. 2.The Golgi apparatus (G) is now heavily labeled. Ferritin is found both over the tightly apposed central regions of the saccules and over their dilatedrims (arrowheads). (b) The plasma membrane (PM) is not labeled at this time. (c) Overview of the cell indicating the regions shown at high mag-nification in a and b. The bars in a and b indicate 100 nm.

face of the Golgi apparatus was preferentially labeled (unpub-lished observations). Presumably, at such early times the G

protein had just entered the Golgi apparatus and had not yetreached its steady-state distribution. The uniform distribution

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FIG. 4. Immunoelectron micrograph of CHO cell infected with 0-

45 for 3 hr and shifted to 32TC for 25 min. Ferritin labeling patternindicates that G protein has reached the cell surface by this time. Gprotein is organized into "patches" and budding virus. The bar indi-cates 100 nm.

of the G protein seen at 11 min after the temperature shift isquite remarkable when one considers the diverse roles attrib-uted to the Golgi complex (see ref. 5 and 31 for critical reviewsof our knowledge of Golgi complex function). For example, itappears to be involved in the intracellular concentration andtransport of secretory proteins (32-35), the transport of lyso-somal proteins (31), and the recycling of membrane components

from the plasmalemma (5, 31). Further, there have already beena number of indications that the Golgi complex might be struc-turally differentiated, including the observed differences in themorphological appearance of the membranes of the differentsaccules and the nonuniformity of the staining of the Golgi com-plex as seen with a variety of cytochemical labeling protocolsboth in situ and on isolated Golgi fractions (5, 31). It remainsto be seen whether the uniform distribution. of the G proteinis a characteristic of integral membrane proteins passingthrough the Golgi apparatus or is an indication of virus-inducedperturbations in Golgi structure.

Finally, the results presented in this paper demonstrate thefeasibility of this system for the further study of the intracellularpathway followed by both integral and peripheral, membraneproteins. By examining 0-45-infected cells at times earlier than11 min after the temperature shift to 32TC, it may be possibleto learn more about morphological elements in the pathway fol-lowed by the G protein on its way to the Golgi apparatus. Bystudying other VSV mutants thought to be blocked in, the Golgiapparatus, it may be possible to obtain information at the struc-

tural level about the pathway G takes from the Golgi apparatus

to the cell surface. By using techniques for double immunola-beling of ultrathin frozen sections for the electron microscope(36), this information could be correlated with biochemical-stud-ies such as those by Rothman and his colleagues (37, 38), whichimplicate clathrin-coated vesicles in G protein transport. In ad-dition to the integral membrane G protein, VSV specifies a

peripheral membrane protein (M). After G protein has reachedthe cell surface, the M protein functions to associate the G pro-

tein with the soluble nucleocapsid of the virus and is thus re-

quired for viral budding (22, 39). Although the M protein isknown to be translated on ribosomes that are not bound to theRER (11), its subsequent intracellular location is not known. Byusing anti-M and anti-G antibodies and double immunolabelingtechniques it should be possible to examine in situ which mem-branes the M protein becomes associated with and whether theG protein is either necessary or sufficient for that association.

We are grateful to Mrs. Margie Adams, Mrs. Michele Wilhite, andMr. Michael McCaffery for excellent technical assistance.. J.E.B. is afellow of the Jane Coffin Childs Memorial Fund for Medical Research.S.J. S. is an American Cancer Society Research Professor. These studieswere supported by U.S. Public Health Service Grant GM-15971.

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Proc. Nad Acad. Sci.. USA 7& (I981)