DNA AND CELL BIOLOGY Volume 11, Number 8, 1992 Mary Ann Liebert, Inc., Publishers Pp. 621-626
Immunoelectron Microscopic Detection of the Hepatitis B Virus Major Surface Protein in Dilated Perinuclear Membranes of Yeast Cells RALPH
BIEMANS,*'Î
DENISE THINES.t BRIGITTE PETRE-PARENT.t MICHEL DE WILDE,* TINEKE RUTGERS,* and'TERESA CABEZÓN*
ABSTRACT The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.
INTRODUCTION infected by hepatitis B virus (HBV) and release, in addition to virions, subviral lipoprotein particles of 22 nm diameter, representing empty viral envelopes (HBsAg). These particles are constituted of the major viral surface protein (S) and two related minor viral proteins, the middle (M) and the large (L) surface protein (Tiollais et ai, 1985). Expression of the S protein of HBV in mammalian cells leads to secretion into the medium of S protein containing particles that closely resemble the 22-nm particles (Dubois et ai, 1980; Michel et ai, 1984; Patzer et ai, 1984). The S protein contains internal uncleaved signal sequences that direct portions of the molecule across the endoplasmic reticulum (ER) membrane. After assembly of monomers, particles are generated by budding into the lumen of the ER and subsequently transported through the Golgi apparatus and secreted (Patzer et ai, 1984, 1986; Simon et ai, 1988b). No secretion of particles takes place from Saccharomyces cerevisiae cells producing HBV surface proteins. However, lipoprotein particles are present in cellular extracts of such yeast cells (Valenzuela et ai, 1982; Harford et ai, 1983; Hitzeman et ai, 1983; Miyanohara et ai, 1983). It has
Human synthetize
liver cells
glutaraldehyde-fixed yeast-derived HBsAg particles. MATERIALS AND METHODS
Plasmid constructions, transformation, and strains The coding sequence for the S protein was ligated to DNA fragments containing the TDH3 (Harford et ai, 1987) or ARG3 (Cabezón et al., 1984) promoter region and
Beecham Biologicals, B-1330 Rixensart, Belgium. Catholic University of Louvain, B-1348 Louvain-La-Neuve, Belgium. address: Service for Applied Genetics, University of Brussels, B-1400 Nivelles, Belgium.
•Research
Department, SmithKline
^Laboratory of Cell Biology, ÎPresent
been suggested that the lipoprotein particles detected in cell lysates do not exist as such within the cells but are formed instead during cell lysis and extract preparation (Hitzeman et ai, 1983; Wampler et ai, 1985; Bitter et ai, 1988). The N-glycan which is present on about one-third of the S molecules synthesized in human liver cells is not added in 5. cerevisiae. This observation has led to the speculation tht either the S protein is not exposed to the lumenal side of the yeast ER where core glycosylation occurs or that the glycosylation site is not accessible due to misfolding of the protein or that the S protein does not enter the yeast secretory pathway. In this study, immunoelectron microscopy on thin sections of transformed yeast cells was used to analyze the subcellular localization of the S protein. Dilated ER structures were observed that reacted specifically with polyclonal antibodies raised against
621
622 ARG3 transcription termination region (Harford et ai, 1987) and inserted into a pBR322 derivative vector by standard recombinant DNA methodologies (Maniatis et ai, 1982). The expression cassette was transferred into a Escherichia co//-yeast shuttle vector composed of the yeast 2-micron DNA and the yeast LEU2 (Harford et ai, 1987) or LEU2D gene (Beggs, 1978) as marker of selection in yeast. The recombinant plasmids pRIT12363 (TDH3 promoter, LEU2 selection) (Harford et ai, 1987), pRIT12455 (TDH3 promoter, LEU2D selection), and pRIT13503 (ARG3 promoter, LEU2D selection) were obtained. These plasmids were used to transform S. cerevisiae strain DC5 (his3-ll, his3-15, leu2-3, leu2-112, canl-11, cir") (Harford and Peeters, 1987) to give transformants Y191, Y1592, and Y1593, respectively. The same plasmids were used to transform another S. cerevisiae strain TCY1 (leu2-3, leu2-112, pep4-3, cir0) (Hoylaerts et ai, 1986) and
BIEMANS ET AL.
transformants were named Y589, Y1535, and Y1616, spectively. Transformation with a plasmid lacking an pression cassette served as control strain (Y1017).
Preparation and analysis of yeast
reex-
extracts
Yeast extracts were analyzed for the presence of the S protein and of HBsAg particles by immunoblot and particle-associated antigenicity tests (AUSRIA-II, Abbott laboratories or Enzygnost-HBsAg micro test, Behring), respectively (Biemans et ai, 1991).
Electron microscopy Cells were fixed overnight with 3% formaldehyde/0.5% glutaraldehyde and embedded in epon. Thin sections were, after etching with NaI04, exposed to monoclonal antibod-
FIG. 1. Subcellular localization of the S protein. Electron micrographs of thin sections of control strain Y1017 (a), Y191 (b) (S gene, TDH3 promoter, LEU2 selection), Y1592 (c) (S gene, TDH3 promoter, LEU2D selection), and immunogold labeling of Y1593 (d) (S gene, ARG3 promoter, LEU2D selection) utilizing rabbit antibodies raised against formaldehyde/glutaraldehyde-fixed HBsAg as primary antibody. Dilation of the perinuclear membrane is indicated by arrows. Magnifications: a, 36,000 x; b, 40,000 x; c, 40,000 x; d, 80,000 x. Bar, 0.5 ¡im. N, Nucleus; V, vacuole; M, mitochondrion.
ELECTRON MICROSCOPY ANALYSIS OF HEPATITIS B ANTIGEN
623
ies (MAb) specific for HBV surface proteins (Mab RF1 and RF6 from H. Thomas, Royal Free Hospital, London or the Mab HBS1 from SmithKline Beecham Biologicals) and goat anti-mouse IgG coupled to 10-nm gold particles from Biocell (Cardiff-UK) or protein A-colloidal gold (10 nm). Rabbit antibodies to formaldehyde/glutaraldehydefixed HBsAg (anti-gfHBs) were produced by incubation of recombinant yeast-derived HBsAg particles (SmithKline Beecham Biologicals) with formaldehyde and glutaraldehyde, and injected as described by Patzer et al. (1986). Anti-yeast antibodies were removed from anti-gfHBs and preimmune serum by two cycles of adsorption on the cellular debris of broken yeast cells. Immunodecorated thin sections were counterstained with Pb-citrate and uranyl acetate. Detailed procedures are described by Jacobs et ai
(1989).
For the cryoelectron microscopy, cells were embedded in 10% gelatin, immersed in 2.3 M sucrose for 1 hr, mounted on the sample bolder of a Reichert FC-4, cryo-ultramicrotome, and frozen in liquid N2. Sections were cut at -100°C according to Tokuyasu (1984). The sections were collected with a platinum loop containing 2.3 M sucrose and deposited on grids covered with a formvar film. Im-
munodecoration
was
performed as described above. RESULTS
Localization of the S protein in dilated
FIG. 2. Immunoblot analysis of the S protein produced in yeast, a. Lanes 1 and 2, 40 and 20 ¡ig, respectively, of crude extract of Y1592 (S gene, TDH3 promoter, LEU2D selection); lanes 3 and 4, 40 and 20 ¡ig, respectively, of crude extract of Y191 (S gene, TDH3 promoter, LEU2 selection); lane 5, extract from Y1017 (control strain); lane 6, molecular size markers. Detection of the S protein was with the S-specific mAb HBS1. b. Lane 1, Molecular size markers; lane 2, extract from Y1017 (control strain); lane 3, extract from Y191 (S protein). Detection of the S protein was with the anti-gfHBs. In addition to the 24-kD S protein (lane 3), a slower-migrating yeast protein of about 33 kD was revealed nonspecifically (lanes 2 and 3).
endoplasmic reticulum
Immunoelectron microscopy on thin sections of yeast cells producing the S protein was carried out to determine the localization and the assembly of intracellular HBsAg particles. Ultrastructural examination of cell sections demonstrated that expression of the S protein provoked dilation of the perinuclear membrane (Fig. lb). About 8090% of cell sections examined showed this phenomenon, whereas such dilations were not observed in cells of the control strain Y1017 (Fig. la). Immunodecoration of the epon-embedded sections was used to determine whether the S protein was localized in these dilated membranes. No labeling was observed with the MAbs HBS1 or RF6, both directed against a sequential epitope, nor with the Mab RF1 against a conformational epitope of HBsASg. These results suggested that the epitopes of the S protein were not available in glutaraldehyde-treated cells or that the expression level was too low to permit a visualization of the protein by immunodecoration. Increase of the expression of the S protein was obtained by inserting its expression cassette in a E. co//-yeast shuttle vector harboring the LEU2D gene as a selective marker. Indeed, immunoblot analysis and antigenicity tests on crude extracts showed that under LEU2D selection the expression of the S protein reached a level two-fold (ARG3 promoter) or 4- to 7fold (TDH3 promoter) higher than with the LEU2 gene as selection in yeast (Fig. 2a). However, specific labeling of cells utilizing the anti-S antibodies mentioned above was not observed (Fig. lc). Because this lack of reactivity might be due to sample preparation for electron microscopy, i.e., the fixation procedure, antibodies against for-
maldehyde/glutaraldehyde-fixed HBsAg (anti-gfHBs) were
elicited in rabbits. The anti-gfHBs recognized the S protein in immunoblot (Fig. 2b, lane 3). Immunoelectron microscopy with the anti-gfHBs revealed labeling of the space formed by the dilation of the perinuclear membrane (Fig. Id). Only background levels of label were observed in the cytoplasm, plasma membrane, or mitochondria. No labeling was observed in the control strain Y1017. Some sections gave the appearance that the dilated areas were engulfed in the nucleus, indicating that the dilated structures are endoplasmic reticulum (ER) extensions of the perinuclear space, which follow the indentations of the nucleus. In cells that overexpressed the S protein, some gold grains were observed in the periplasmic space, suggesting that HBsAg may be secreted across the plasma membrane into the periplasm. No labeling was obtained with the preimmune serum. Results were similar in two yeast strains with different genetic backgrounds, strains DC5 and TCY1. In spite of higher magnifications of the dilated areas, positive identification of particles was difficult owing to the lack of any further ultrastructural detail. To improve the preservation and visualization of membranes, ultrathin cryosectioning techniques were used. The dilated ER of cells producing the S protein was well immunodecorated with the anti-gfHBs (Fig. 3b, c). The dilated structures were rich in lipids and seemed to be filled with particular or tubular structures similar of particles observed in the ER of Chinese hamster ovary (CHO) cells producing the S protein (Patzer et ai, 1986). In spite of the improved structural preservation of membranes revealing ultrastructural de-
624
FIG. 3.
BIEMANS ET AL.
Subcellular localization of the S protein. Immunogold labeling with the anti-gfHBs as primary antibody on thin cryosections of yeast cells, a. Control strain Y1017. b. Y1593 (S protein), c. High magnification of a thin section of Y1593. Magnifications: a, 18,000x; b, 18,000x; c, 83,000x. Bar, 0.5 iim.
ELECTRON MICROSCOPY ANALYSIS OF HEPATITIS B ANTIGEN
tails, discrete particles remained elusive possibly due to low contrast of the lipoprotein particles against the lipid background. However, the specific and general immunodecoration of the dilated ER area provided for the first time the indication that in S. cerevisiae the S protein enters the secretory pathway and accumulates in the ER lumen. DISCUSSION In summary, the results described here suggest that, as in mammalian cells, particle formation does occur in S. cerevisiae and takes place in the ER. The majority of the S protein was found in dilated ER structures, suggesting that further transport along the secretion pathway is blocked. Similar results were found in other expression systems. In Xenopus oocytes only 2.5% of the S protein was secreted. The majority of the HBsAg remained as an integral membrane protein and was never fully transferred into the ER lumen (Simon et ai, 1988a). The S protein synthesized in insect cells was assembled into lipoprotein particles but was not secreted efficiently (Lanford et ai, 1989). In CHO cells, HBsAg particles were shown within large dilated areas of the ER (Patzer et ai, 1986). Transport to the Golgi apparatus appeared to be the rate-limiting step in secretion. The transport of HBsAg particles might be affected by the absence of oligomeric subunits cross-linked by interchain disulfide bonds. Oligomeric assembly represents an essential step in the intracellular transport of certain hetero-oligomeric membrane and secretory proteins (Hurtley and Helenius, 1989). Indeed, trimerization of vesicular stomatitis virus G protein and of influenza virus hemagglutinin glycoproteins are essential for their export from the ER (Rose and Doms, 1988). Thus, the secretion of the HBsAg might depend on the presence of interchain disulfide bonds. Indeed, mutations of certain cysteine residues prevents the secretion of the S protein from mammalian cells (C. Mangold, personal communication). Intracellular particle formation was not analyzed, however. In S. cerevisiae, the HBsAg particles were hypothesized to be formed during the process of isolation by formation of disulfide bonds in vitro (Wampler et ai, 1985). Alternatively, the absence of an AMinked oligosaccharide on the yeast-derived S protein might provoke aberrant formation of disulfide bonds and thus the formation of aggregates that accumulate in the ER, as shown for a nonglycosylated G protein of the vesicular stomatitis virus expressed in mammalian cells (Machamer and Rose, 1988). These aggregates might disassemble during the extraction process. The aggregates and particles that are assembled in the ER might be difficult to transport to the Golgi complex owing to size constraints. If the transport is mediated by transport vesicles, there may be a limit to the size of particles that can be accommodated inside the vesicles. Immunodecoration of thin sections of cells overproducing the S protein revealed some gold grains in the periplasmic space, suggesting that to a limited extent HBsAg may be secreted across the plasma membrane into the periplasm. Recently, it was demonstrated that the middle protein of
625
HBV expressed in Hansenula polymorpha can be secreted outside the plasma membrane into the periplasm and further excreted into the culture medium when the cell wall is permeabilized with glucanase (Shen et ai, 1989). We have recently executed immunogold labeling experiments on cells from H. polymorpha that produce the S protein (Janowicz et ai, 1991). A dilation and specific labeling of the perinuclear membrane was also observed in this host cell (R. Biemans and D. Thines, unpublished). No labeling with the anti-gfHBs was observed in the periplasmic space or in the cell wall, suggesting that transport of particles along the secretory pathway is blocked as observed in S. cerevisiae.
ACKNOWLEDGMENTS We thank A. Bollen for helpful discussions. This work supported in part by the Walloon Region (STN-1109). D. Thines is Senior Research Associate at the Fonds National de la Recherche Scientifique (Belgium). was
REFERENCES BEGGS, J.D. (1978). Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104-109. BIEMANS, R., THINES, D., RUTGERS, T., DE WILDE, M., and CABEZÓN, T. (1991). The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement. DNA Cell Biol. 10, 191-200. BITTER, G.A., EGAN, K.M., BURNETTE, W.N., SAMAL, B., FIESCHKO, J.C., PETERSON, D.L., DOWNING, M.R., WYPYCH, J., and LANGLEY, K.E. (1988). Hepatitis B vaccine produced in yeast. J. Med. Virol. 25, 123-140. CABEZÓN, T., DE WILDE, M., HERION, P., LORIAU, R., and BOLLEN, A. (1984). Expression of human al-antitrypsine cDNA in the yeast Saccharomyces cerevisiae. Proc. Nati. Acad. Sei. USA 81, 6594-6598. DUBOIS, M.F., POURCEL, C, ROUSSET, S., CHANY, C, and TIOLLAIS, P. (1980). Excretion of hepatitis B surface antigen particles from mouse cells transformed with cloned viral DNA. Proc. Nati. Acad. Sei. USA 77, 4549-4553. HARFORD, M.N., and PEETERS, M. (1987). Curing of endogenous 2 micron DNA in yeast by recombinant vectors. Curr. Genet. 11, 315-319. HARFORD, N., CABEZÓN, T., CRABEEL, M., SIMOEN, E., RUTGERS, A., and DE WILDE, M. (1983). Expression of hepatitis B surface antigen in yeast. Develop. Biol. Standard 54, 125-130. HARFORD, N., CABEZÓN, T., COLAU, B., DELISSE A.-M., RUTGERS, T., and DE WILDE, M. (1987). Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen. Postgrad. Med. J. 63, 65-70. HITZEMAN, R.A., CHEN, C.Y., HAGIE, F.E., PATZER, E.J., LIU, C.-C, ESTELL, D.A., MILLER, J.V., YAFFE, A., KLEID, D.G., LEVINSON, A.D., and OPPERMANN, H. (1983). Expression of hepatitis B virus surface antigen in yeast. Nucleic Acids Res. 11, 2745-2763. HOYLAERTS, M., WEYENS, A., BOLLEN, A., HARFORD, N., and CABEZÓN, T. (1986). High-level production and iso-
BIEMANS ET AL.
626 lation of human recombinant al-proteinase inhibition in yeast. FEBS Lett. 204, 83-87. HURTLEY, S.M., and HELENIUS, A. (1989). Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 277-307.
JACOBS, E., GHEYSEN, D., THINES, D., FRANCOTTE, M., and DE WILDE, M. (1989). The HIV-1 gag precursor Pr55sas
synthesized in yeast is myristoylated and membrane. Gene 79, 71-81.
targeted to the plasma
JANOWICZ, Z.A., MELBER, K., MERCKELBACH, A., JACOBS, E., HARFORD, N., COMBERBACH, M., and HOLLENBERG, C.P. (1991). Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 7, 431-443. LANFORD, R.E., LUCKOW, V., KENNEDY, R.C., DREESMAN, G.R., NOTVALL, L., and SUMMERS, M.D. (1989). Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system. J. Virol. 63, 1549-1557. MACHAMER, CE., and ROSE, J.K. (1988). Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J. Biol. Chem. 263, 5955-5960.
MANIATIS, T., FRITSCH, E.F., and SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). MICHEL, M.-L., PONTISSO, P., SOBCZAK, E., MALPIECE, Y., STREEK, R., and TIOLLAIS, P. (1984). Synthesis in animal cells of hepatitis B surface antigen particle carrying a receptor for polymerized human serum albumin. Proc. Nati. Acad. Sei. USA 81, 7708-7712. MIYANOHARA, A., TOH-E, A., NOZAKI, C, HAMADA, F., OHTOMO, N., and MATSUBARA, K. (1983). Expression of hepatitis B surface antigen gene in yeast. Proc. Nati. Acad. Sei. USA 80, 1-5. PATZER, E.J., NAKAMURA, G.R., and YAFFE, A. (1984). Intracellular transport and secretion of hepatitis virus surface antigen in mammalian cells. J. Virol. 51, 346-353. PATZER, E.J., NAKAMURA, G.R., SIMONSEN, C.C.,
LEVINSON, A.D., and BRANDS, R. (1986). Intracellular
as-
and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum. J. Virol. 58, 884-892. ROSE, J.K., and DOMS, R.W. (1988). Regulation of protein export from the endoplasmic reticulum. Annu. Rev. Cell Biol. 4, 257-288. SHEN, S.-H., BASTIEN, L., NGUYEN, T., FUNG, M., and SLILATY, S.N. (1989). Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha. Gene 84, 303-309. SIMON, K., LINGAPPA, V.R., and GANEM, D. (1988a). A block to the intracellular transport and assembly of hepatitis B surface antigen polypeptides in Xenopus oocytes. Virology 166, 76-81. SIMON, K., LINGAPPA, V.R., and GANEM, D. (1988b). Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor. J. Cell Biol. 107, 2163-2168. TIOLLAIS, P., POURCEL, C, and DEJEAN, A. (1985). The hepatitis B virus. Nature 317, 489-495. TOKUYASU, K.T. (1984). Immunocryoultramicrotomy. pp71-82. In Immunolabeling for Electron Microscopy. J.M. Polak and I.M. Varndell, eds. (Elsevier, New York). VALENZUELA, P., MEDINA, A., RUTTER, W., AMMERER, G., and HALL, B.D. (1982). Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 298, 347-350.
sembly
WAMPLER, D.E., LEHMAN, E.D., BOGER, J., McALEER, W.J., and SCHONICK, E.M. (1985). Multiple chemical forms of hepatitis B surface antigen produced in yeast. Proc. Nati. Acad. Sei. USA 82, 6830-6834. Address reprint requests to: Dr. Teresa Cabezón SmithKline Beecham Biologicals Research Department rue de l'Institut 89 B-1330 Rixensart, Belgium Received for publication January 4, 1992; in revised form May 25, 1992; accepted June 4, 1992.
Immunoelectron Microscopic Detection of the Hepatitis B Virus Major Surface Protein in Dilated Perinuclear Membranes of Yeast Cells RALPH
BIEMANS,*'Î
DENISE THINES.t BRIGITTE PETRE-PARENT.t MICHEL DE WILDE,* TINEKE RUTGERS,* and'TERESA CABEZÓN*
ABSTRACT The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.
INTRODUCTION infected by hepatitis B virus (HBV) and release, in addition to virions, subviral lipoprotein particles of 22 nm diameter, representing empty viral envelopes (HBsAg). These particles are constituted of the major viral surface protein (S) and two related minor viral proteins, the middle (M) and the large (L) surface protein (Tiollais et ai, 1985). Expression of the S protein of HBV in mammalian cells leads to secretion into the medium of S protein containing particles that closely resemble the 22-nm particles (Dubois et ai, 1980; Michel et ai, 1984; Patzer et ai, 1984). The S protein contains internal uncleaved signal sequences that direct portions of the molecule across the endoplasmic reticulum (ER) membrane. After assembly of monomers, particles are generated by budding into the lumen of the ER and subsequently transported through the Golgi apparatus and secreted (Patzer et ai, 1984, 1986; Simon et ai, 1988b). No secretion of particles takes place from Saccharomyces cerevisiae cells producing HBV surface proteins. However, lipoprotein particles are present in cellular extracts of such yeast cells (Valenzuela et ai, 1982; Harford et ai, 1983; Hitzeman et ai, 1983; Miyanohara et ai, 1983). It has
Human synthetize
liver cells
glutaraldehyde-fixed yeast-derived HBsAg particles. MATERIALS AND METHODS
Plasmid constructions, transformation, and strains The coding sequence for the S protein was ligated to DNA fragments containing the TDH3 (Harford et ai, 1987) or ARG3 (Cabezón et al., 1984) promoter region and
Beecham Biologicals, B-1330 Rixensart, Belgium. Catholic University of Louvain, B-1348 Louvain-La-Neuve, Belgium. address: Service for Applied Genetics, University of Brussels, B-1400 Nivelles, Belgium.
•Research
Department, SmithKline
^Laboratory of Cell Biology, ÎPresent
been suggested that the lipoprotein particles detected in cell lysates do not exist as such within the cells but are formed instead during cell lysis and extract preparation (Hitzeman et ai, 1983; Wampler et ai, 1985; Bitter et ai, 1988). The N-glycan which is present on about one-third of the S molecules synthesized in human liver cells is not added in 5. cerevisiae. This observation has led to the speculation tht either the S protein is not exposed to the lumenal side of the yeast ER where core glycosylation occurs or that the glycosylation site is not accessible due to misfolding of the protein or that the S protein does not enter the yeast secretory pathway. In this study, immunoelectron microscopy on thin sections of transformed yeast cells was used to analyze the subcellular localization of the S protein. Dilated ER structures were observed that reacted specifically with polyclonal antibodies raised against
621
622 ARG3 transcription termination region (Harford et ai, 1987) and inserted into a pBR322 derivative vector by standard recombinant DNA methodologies (Maniatis et ai, 1982). The expression cassette was transferred into a Escherichia co//-yeast shuttle vector composed of the yeast 2-micron DNA and the yeast LEU2 (Harford et ai, 1987) or LEU2D gene (Beggs, 1978) as marker of selection in yeast. The recombinant plasmids pRIT12363 (TDH3 promoter, LEU2 selection) (Harford et ai, 1987), pRIT12455 (TDH3 promoter, LEU2D selection), and pRIT13503 (ARG3 promoter, LEU2D selection) were obtained. These plasmids were used to transform S. cerevisiae strain DC5 (his3-ll, his3-15, leu2-3, leu2-112, canl-11, cir") (Harford and Peeters, 1987) to give transformants Y191, Y1592, and Y1593, respectively. The same plasmids were used to transform another S. cerevisiae strain TCY1 (leu2-3, leu2-112, pep4-3, cir0) (Hoylaerts et ai, 1986) and
BIEMANS ET AL.
transformants were named Y589, Y1535, and Y1616, spectively. Transformation with a plasmid lacking an pression cassette served as control strain (Y1017).
Preparation and analysis of yeast
reex-
extracts
Yeast extracts were analyzed for the presence of the S protein and of HBsAg particles by immunoblot and particle-associated antigenicity tests (AUSRIA-II, Abbott laboratories or Enzygnost-HBsAg micro test, Behring), respectively (Biemans et ai, 1991).
Electron microscopy Cells were fixed overnight with 3% formaldehyde/0.5% glutaraldehyde and embedded in epon. Thin sections were, after etching with NaI04, exposed to monoclonal antibod-
FIG. 1. Subcellular localization of the S protein. Electron micrographs of thin sections of control strain Y1017 (a), Y191 (b) (S gene, TDH3 promoter, LEU2 selection), Y1592 (c) (S gene, TDH3 promoter, LEU2D selection), and immunogold labeling of Y1593 (d) (S gene, ARG3 promoter, LEU2D selection) utilizing rabbit antibodies raised against formaldehyde/glutaraldehyde-fixed HBsAg as primary antibody. Dilation of the perinuclear membrane is indicated by arrows. Magnifications: a, 36,000 x; b, 40,000 x; c, 40,000 x; d, 80,000 x. Bar, 0.5 ¡im. N, Nucleus; V, vacuole; M, mitochondrion.
ELECTRON MICROSCOPY ANALYSIS OF HEPATITIS B ANTIGEN
623
ies (MAb) specific for HBV surface proteins (Mab RF1 and RF6 from H. Thomas, Royal Free Hospital, London or the Mab HBS1 from SmithKline Beecham Biologicals) and goat anti-mouse IgG coupled to 10-nm gold particles from Biocell (Cardiff-UK) or protein A-colloidal gold (10 nm). Rabbit antibodies to formaldehyde/glutaraldehydefixed HBsAg (anti-gfHBs) were produced by incubation of recombinant yeast-derived HBsAg particles (SmithKline Beecham Biologicals) with formaldehyde and glutaraldehyde, and injected as described by Patzer et al. (1986). Anti-yeast antibodies were removed from anti-gfHBs and preimmune serum by two cycles of adsorption on the cellular debris of broken yeast cells. Immunodecorated thin sections were counterstained with Pb-citrate and uranyl acetate. Detailed procedures are described by Jacobs et ai
(1989).
For the cryoelectron microscopy, cells were embedded in 10% gelatin, immersed in 2.3 M sucrose for 1 hr, mounted on the sample bolder of a Reichert FC-4, cryo-ultramicrotome, and frozen in liquid N2. Sections were cut at -100°C according to Tokuyasu (1984). The sections were collected with a platinum loop containing 2.3 M sucrose and deposited on grids covered with a formvar film. Im-
munodecoration
was
performed as described above. RESULTS
Localization of the S protein in dilated
FIG. 2. Immunoblot analysis of the S protein produced in yeast, a. Lanes 1 and 2, 40 and 20 ¡ig, respectively, of crude extract of Y1592 (S gene, TDH3 promoter, LEU2D selection); lanes 3 and 4, 40 and 20 ¡ig, respectively, of crude extract of Y191 (S gene, TDH3 promoter, LEU2 selection); lane 5, extract from Y1017 (control strain); lane 6, molecular size markers. Detection of the S protein was with the S-specific mAb HBS1. b. Lane 1, Molecular size markers; lane 2, extract from Y1017 (control strain); lane 3, extract from Y191 (S protein). Detection of the S protein was with the anti-gfHBs. In addition to the 24-kD S protein (lane 3), a slower-migrating yeast protein of about 33 kD was revealed nonspecifically (lanes 2 and 3).
endoplasmic reticulum
Immunoelectron microscopy on thin sections of yeast cells producing the S protein was carried out to determine the localization and the assembly of intracellular HBsAg particles. Ultrastructural examination of cell sections demonstrated that expression of the S protein provoked dilation of the perinuclear membrane (Fig. lb). About 8090% of cell sections examined showed this phenomenon, whereas such dilations were not observed in cells of the control strain Y1017 (Fig. la). Immunodecoration of the epon-embedded sections was used to determine whether the S protein was localized in these dilated membranes. No labeling was observed with the MAbs HBS1 or RF6, both directed against a sequential epitope, nor with the Mab RF1 against a conformational epitope of HBsASg. These results suggested that the epitopes of the S protein were not available in glutaraldehyde-treated cells or that the expression level was too low to permit a visualization of the protein by immunodecoration. Increase of the expression of the S protein was obtained by inserting its expression cassette in a E. co//-yeast shuttle vector harboring the LEU2D gene as a selective marker. Indeed, immunoblot analysis and antigenicity tests on crude extracts showed that under LEU2D selection the expression of the S protein reached a level two-fold (ARG3 promoter) or 4- to 7fold (TDH3 promoter) higher than with the LEU2 gene as selection in yeast (Fig. 2a). However, specific labeling of cells utilizing the anti-S antibodies mentioned above was not observed (Fig. lc). Because this lack of reactivity might be due to sample preparation for electron microscopy, i.e., the fixation procedure, antibodies against for-
maldehyde/glutaraldehyde-fixed HBsAg (anti-gfHBs) were
elicited in rabbits. The anti-gfHBs recognized the S protein in immunoblot (Fig. 2b, lane 3). Immunoelectron microscopy with the anti-gfHBs revealed labeling of the space formed by the dilation of the perinuclear membrane (Fig. Id). Only background levels of label were observed in the cytoplasm, plasma membrane, or mitochondria. No labeling was observed in the control strain Y1017. Some sections gave the appearance that the dilated areas were engulfed in the nucleus, indicating that the dilated structures are endoplasmic reticulum (ER) extensions of the perinuclear space, which follow the indentations of the nucleus. In cells that overexpressed the S protein, some gold grains were observed in the periplasmic space, suggesting that HBsAg may be secreted across the plasma membrane into the periplasm. No labeling was obtained with the preimmune serum. Results were similar in two yeast strains with different genetic backgrounds, strains DC5 and TCY1. In spite of higher magnifications of the dilated areas, positive identification of particles was difficult owing to the lack of any further ultrastructural detail. To improve the preservation and visualization of membranes, ultrathin cryosectioning techniques were used. The dilated ER of cells producing the S protein was well immunodecorated with the anti-gfHBs (Fig. 3b, c). The dilated structures were rich in lipids and seemed to be filled with particular or tubular structures similar of particles observed in the ER of Chinese hamster ovary (CHO) cells producing the S protein (Patzer et ai, 1986). In spite of the improved structural preservation of membranes revealing ultrastructural de-
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FIG. 3.
BIEMANS ET AL.
Subcellular localization of the S protein. Immunogold labeling with the anti-gfHBs as primary antibody on thin cryosections of yeast cells, a. Control strain Y1017. b. Y1593 (S protein), c. High magnification of a thin section of Y1593. Magnifications: a, 18,000x; b, 18,000x; c, 83,000x. Bar, 0.5 iim.
ELECTRON MICROSCOPY ANALYSIS OF HEPATITIS B ANTIGEN
tails, discrete particles remained elusive possibly due to low contrast of the lipoprotein particles against the lipid background. However, the specific and general immunodecoration of the dilated ER area provided for the first time the indication that in S. cerevisiae the S protein enters the secretory pathway and accumulates in the ER lumen. DISCUSSION In summary, the results described here suggest that, as in mammalian cells, particle formation does occur in S. cerevisiae and takes place in the ER. The majority of the S protein was found in dilated ER structures, suggesting that further transport along the secretion pathway is blocked. Similar results were found in other expression systems. In Xenopus oocytes only 2.5% of the S protein was secreted. The majority of the HBsAg remained as an integral membrane protein and was never fully transferred into the ER lumen (Simon et ai, 1988a). The S protein synthesized in insect cells was assembled into lipoprotein particles but was not secreted efficiently (Lanford et ai, 1989). In CHO cells, HBsAg particles were shown within large dilated areas of the ER (Patzer et ai, 1986). Transport to the Golgi apparatus appeared to be the rate-limiting step in secretion. The transport of HBsAg particles might be affected by the absence of oligomeric subunits cross-linked by interchain disulfide bonds. Oligomeric assembly represents an essential step in the intracellular transport of certain hetero-oligomeric membrane and secretory proteins (Hurtley and Helenius, 1989). Indeed, trimerization of vesicular stomatitis virus G protein and of influenza virus hemagglutinin glycoproteins are essential for their export from the ER (Rose and Doms, 1988). Thus, the secretion of the HBsAg might depend on the presence of interchain disulfide bonds. Indeed, mutations of certain cysteine residues prevents the secretion of the S protein from mammalian cells (C. Mangold, personal communication). Intracellular particle formation was not analyzed, however. In S. cerevisiae, the HBsAg particles were hypothesized to be formed during the process of isolation by formation of disulfide bonds in vitro (Wampler et ai, 1985). Alternatively, the absence of an AMinked oligosaccharide on the yeast-derived S protein might provoke aberrant formation of disulfide bonds and thus the formation of aggregates that accumulate in the ER, as shown for a nonglycosylated G protein of the vesicular stomatitis virus expressed in mammalian cells (Machamer and Rose, 1988). These aggregates might disassemble during the extraction process. The aggregates and particles that are assembled in the ER might be difficult to transport to the Golgi complex owing to size constraints. If the transport is mediated by transport vesicles, there may be a limit to the size of particles that can be accommodated inside the vesicles. Immunodecoration of thin sections of cells overproducing the S protein revealed some gold grains in the periplasmic space, suggesting that to a limited extent HBsAg may be secreted across the plasma membrane into the periplasm. Recently, it was demonstrated that the middle protein of
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HBV expressed in Hansenula polymorpha can be secreted outside the plasma membrane into the periplasm and further excreted into the culture medium when the cell wall is permeabilized with glucanase (Shen et ai, 1989). We have recently executed immunogold labeling experiments on cells from H. polymorpha that produce the S protein (Janowicz et ai, 1991). A dilation and specific labeling of the perinuclear membrane was also observed in this host cell (R. Biemans and D. Thines, unpublished). No labeling with the anti-gfHBs was observed in the periplasmic space or in the cell wall, suggesting that transport of particles along the secretory pathway is blocked as observed in S. cerevisiae.
ACKNOWLEDGMENTS We thank A. Bollen for helpful discussions. This work supported in part by the Walloon Region (STN-1109). D. Thines is Senior Research Associate at the Fonds National de la Recherche Scientifique (Belgium). was
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WAMPLER, D.E., LEHMAN, E.D., BOGER, J., McALEER, W.J., and SCHONICK, E.M. (1985). Multiple chemical forms of hepatitis B surface antigen produced in yeast. Proc. Nati. Acad. Sei. USA 82, 6830-6834. Address reprint requests to: Dr. Teresa Cabezón SmithKline Beecham Biologicals Research Department rue de l'Institut 89 B-1330 Rixensart, Belgium Received for publication January 4, 1992; in revised form May 25, 1992; accepted June 4, 1992.
