- Email: [email protected]
Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface
FEMS Microbiology Letters 192 (2000) 243^248
www.fems-microbiology.org
Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface Yumi Shibasaki a , Naomi Kamasawa b , Seiji Shibasaki c , Wen Zou c , Toshiyuki Murai c , Mitsuyoshi Ueda c , Atsuo Tanaka c , Masako Osumi a;b; * a
Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan c Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
b
Received 11 May 2000 ; received in revised form 19 September 2000; accepted 19 September 2000
Abstract A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase^K-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cell surface engineering; K-Agglutinin ; Glycosyl phosphatidylinositol anchor; Fluorescence microscopy ; Immunoelectron microscopy ; Saccharomyces cerevisiae
1. Introduction We established a method for displaying certain proteins on the cell surface of Saccharomyces cerevisiae by connecting a C-terminal half of K-agglutinin to the protein which was aimed at displaying on the cell surface [1^4]. In a previous study, a starch-utilizing S. cerevisiae strain was constructed by displaying a glucoamylase^K-agglutinin fusion protein on the cell wall with retaining glucoamylase activity [1]. The gene of Rhizopus oryzae glucoamylase, cleaving K-1,4-linked and K-1,6-linked glucose, was fused with the 3P- half of K-agglutinin gene. K-Agglutinin is a cell wall mannoprotein involved in sexual adhesion, which has a signal sequence for attachment of a glycosyl phosphatidylinositol (GPI) anchor.
* Corresponding author. Tel. : +81 (3) 3943 3131 ext. 7260; Fax: +81 (3) 3942 6188; E-mail : [email protected]
Cell wall proteins which contain the GPI attachment signal are connected by GPI anchors in the endoplasmic reticulum (ER), and passed through the secretory pathway to the cell membrane. On the cell membrane, the GPI anchor is cleaved and the remnant is transferred to form a glycosidic linkage with the branched L-1,6-glucan [5^8]. C-terminal regions of these proteins have been proved to covalently bind heterologous proteins on the cell wall of S. cerevisiae [7,8]. Localization of native cell wall proteins anchored by GPI has been morphologically analyzed [9^11]. An K-agglutinin protein was visualized by immunoelectron microscopy on the ¢brillar mannan layer of the cell wall [9], and a £occulin protein was located in the ER, periplasmic region and external mannan layer [10]. Distribution of two cell wall proteins, Cwp1p and Cwp2p, on the cell surface was shown by tagging with green £uorescent protein [11]. Although the morphological studies on heterologous proteins constructed for anchoring to the cell wall
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 4 2 - 0
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
244
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
have been examined only by £uorescence microscopy, their transport pathway in the cell has not been identi¢ed. In this study, we further analyzed the distribution of glucoamylase^K-agglutinin fusion protein in relation to the cell cycle by indirect £uorescence microscopy, and its localization on the secretory pathway using a sec1 mutant strain by immunoelectron microscopy.
ing, a cellulose capillary tube was ¢lled with cell pellets, dipped into 1-hexadecene, and cut to a 2-mm length. Two or three pieces of capillaries were put into an aluminum platelet (BTBUO12126-T, Bal-Tech, Liechtenstein) ¢lled with 1-hexadecene, covered with another platelet, and frozen [16].
2. Materials and methods
Cryo¢xed cells were substituted at 380³C for 2 days, 320³C for 2 h, and 4³C for 2 h in absolute acetone as described [14]. Specimens were washed with absolute acetone and ethanol, embedded in a resin of LR white (London Resin, UK), and polymerized at 50³C for 24 h. Ultrathin sections of specimens were immuno-stained with an antibody against glucoamylase [1] diluted 1:1000, and a goat anti-rabbit IgG conjugated with 10 nm of colloidal gold (British Biocell International, UK) diluted 1:40 [17]. The sections were then stained with uranyl acetate.
2.1. Strains S. cerevisiae strains MT8-1 (MATa ade his3 leu2 trp1 ura3) [12] and a sec1 mutant, RSY45 (MATK K sec1-1 his4 ura3 trp1 leu2) [13], were used as host cells. A sec1 mutant accumulates vesicles during incubation at the restrictive temperature, 37³C [13]. A multicopy plasmid pGA11 and an integrative plasmid pIGA11 were constructed as described previously [1,3]. 2.2. Media and cultivation MT8-1 cells integrated with plasmid pIGA11 at the URA3 locus were grown in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) and MT8-1 cells harboring plasmid pGA11 were grown in modi¢ed Burkholder's medium [1] at 30³C to the exponential phase. RSY45 cells harboring plasmid pGA11 were grown in SD medium (0.67% yeast nitrogen base without amino acid (Difco) but with 0.002% adenine sulfate, 0.002% L-histidine^HCl, 0.003% L-leucine, 0.002% uracil and 2% glucose) at 24³C to the exponential phase, and then shifted up to the restrictive temperature, 37³C, for 3 h to accumulate vesicles. 2.3. Immuno£uorescence microscopy Indirect immuno£uorescence microscopy was performed as reported previously[1]. Fixed cells were incubated with an antibody against R. oryzae glucoamylase [1] diluted 1:100 for 1.5 h. After washing, goat anti-rabbit IgG conjugated with £uorescein-5-isothiocyanate (Cappel, ICN Pharmaceuticals, USA) was reacted at the dilution of 1:300 for 1 h. Cells were observed by a £uorescence microscope (BX 50, Olympus, Japan) and a confocal laser scanning microscope (TCS NT, Leica, Germany). Serial optical images of cells were superimposed and processed by the method described previously [14]. 2.4. Cryo¢xation Cryo¢xation was performed by rapid freezing with a KF80 machine (Leica, Austria) and by high-pressure freezing with an HPM 010 machine (Bal-Tech, Liechtenstein). For rapid-freezing, cell pellets were frozen by the sandwich method [15] using liquid propane. For high pressure freez-
FEMSLE 9661 26-10-00
2.5. Immunoelectron microscopy
2.6. Conventional electron microscopy Cryo¢xed cells were substituted in absolute acetone with 2% osmium tetroxide, and embedded in a Quetol 812 mixture (Nissin EM, Japan) [15]. Ultrathin sections were stained with uranyl acetate and lead citrate. 3. Results and discussion 3.1. Observation of glucoamylase^K-agglutinin fusion protein localized on the surface of MT8-1 cells by £uorescence microscopy Fluorescence localized speci¢cally on the cell surface was detected by £uorescence microscopy. In photographs taken at di¡erent focus-phases, £uorescence from the glucoamylase^K-agglutinin fusion protein was seen to be composed of many small particles (Fig. 1a1;2 ). Fluorescent particles were distributed unevenly, resulting in heavy and light £uorescent dots on the cell surface. This phenomenon was clearly con¢rmed by the confocal laser scanning microscopic observation (Fig. 1b,c). The superimposed images showed that the outline of a cell was not continuous (Fig. 1b). Another image also had uneven £uorescence (Fig. 1c1 ), and the processed image was emphasized by di¡erent colors corresponding to the £uorescence intensity (Fig. 1c2 ). Although cytochemical evaluation was done on the basis of a state of uniform expression of the fusion protein, immuno£uorescence microscopy showed that the amount of the expressed protein was distributed unequally on the cell surface. The distribution of £uorescence was analyzed according to the cell cycle in cells inserted with pIGA11 (Fig. 2). Cells grown to the exponential phase were classi¢ed into four growth stages by budding cell sizes, which were dis-
Cyaan Magenta Geel Zwart
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
245
Fig. 1. Immuno£uorescent labeling with anti-glucoamylase antibody of MT8-1 cells harboring pGA11 or integrated with pIGA11. Upper panel: Fluorescence micrographs were taken at di¡erent focus-phases, equatorial (a1 ), and upper phase (a2 ). Matching Nomarsky di¡erential interference micrograph (a3 ). U2250. Lower panel: Confocal laser scanning microscopic images of MT8-1 cells harboring pGA11 (b) or integrated with pIGA11 (c). Superimposed images of optical sections (b,c1 ), and a processed image (c2 ) of which £uorescent intensity is shown in arti¢cial colors.
Fig. 2. Immuno£uorescent images on budding process of MT8-1 cells integrated with pIGA11. 2250U.
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
246
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
tinguished from the ratio of the major axis between mother and daughter cells. About 50 cells in each stage were observed, and their £uorescent patterns and percentages are shown in Fig. 3. In unbudded cells, £uorescence is discontinuously localized on the cell surface (Fig. 2-1), or the areas with intense £uorescence were restricted to being opposite each other on the cell surface (Fig. 2-2). Small buds which were one-third or less the size of the mother cells had hard £uorescent intensity in most cells
(Fig. 2-3). When the buds enlarged to more than one-third but less than two-thirds of the mother cells, two £uorescent patterns were observed: in one only the buds had hard £uorescence intensity (Fig. 2-4), and in the other the entire cell surface of mother and bud was covered with £uorescent dots (Fig. 2-5). When buds grew to more than two-third of the mother cells, £uorescence was detected all over the cell surface of mother and bud (Fig. 2-6), and was intense at the neck region (Fig. 2-7,U). Fluorescence of this fusion protein especially localized on small buds and the neck region; these areas are known to be those where cell wall materials are newly synthesized. It is possible that insertion of the fusion protein into the cell wall is carried out together with other native cell wall materials. The direction of insertion of new materials of a cell wall has been reported to be changed from apex to isotropy in a cell cycle [18,19]. In very small buds, insertion occurred only on the bud surface, but isotropic insertion took place overall both buds and mother cells when buds reached two-third the size of the mother cells. Although Schreuder et al. [20] showed that a fusion protein which had the signal of the C-terminal half of K-agglutinin was labeled uniformly in buds of various sizes, our results suggested that heterologous proteins were ¢rst inserted in buds, and then dispersed over the entire cell surface, as in other native cell wall materials. 3.2. Behavior of the glucoamylase K-agglutinin fusion protein in S. cerevisiae sec1 cells
Fig. 3. Illustration of classi¢ed £uorescent pattern in budding process. *The numbers correspond to cell images in Fig. 2. Cells were classi¢ed in four stages by the ratio of cell size between a bud (a) and a mother cell (b); unbudded (a = 0), less than one-third (a/b 6 0.33), more than one-third but less than two-third (0.33Aa/b 6 0.66), more than twothirds (0.66Aa/b). Small buds were strongly labeled in 70% of the cells. Fluorescence then dispersed through the cell wall and accumulated at the neck region.
FEMSLE 9661 26-10-00
We used an immunoelectron microscopic technique to observe the intracellular localization of the glucoamylase^ K-agglutinin fusion protein. In cryo¢xed MT 8-1 cells, their intracellular ultrastructures, nucleus (N), vacuoles (V), and mitochondria (M) were observed, but there were only a few ERs and vesicles (Ves.) (Fig. 4a). Gold particles, indicating the glucoamylase^K-agglutinin fusion protein, were located on both the outside and inside of the cell wall (Fig. 4b). A few gold particles were seen in cytoplasm (Fig. 4b). It seemed di¤cult to determine the intracellular localization of the fusion protein in MT8-1 cells, because there were few secretory organelles under normal growth conditions. We therefore used a sec1 mutant, RSY45, as the host for pGA11, in which secretory vesicles would accumulate at the restrictive temperature, 37³C [13]. If the heterologous proteins pass through a secretory pathway, they had to remain in accumulated vesicles of this mutant cell. After cultivation at 37³C for 3 h, many accumulated vesicles were observed in the sec1 cell (Fig. 5a). These vesicles had di¡erent electron densities, and probably depended on the substances inside. An expanded membranous structure, perhaps ER, was often observed in the sec1 cells harboring pGA11, in which the glucoamylase^K-agglutinin fusion protein was expressed (Fig. 5b). A few gold particles indicated the fusion protein localized on the ac-
Cyaan Magenta Geel Zwart
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
247
Fig. 4. Electron micrographs of cryo¢xed MT8-1 cells harboring pGA11. Ultrastructure (a), and immunogold labeling with anti-glucoamylase antibody (b). Gold particles (U) indicate glucoamylase^K-agglutinin fusion protein. 30 000U.
cumulated vesicles (Fig. 5c), but most of the particles were in ERs and the nuclear envelope (Fig. 5d). Bony et al. observed the existence of an abundant, expanded ER holding the expressed Flo1p, a cell wall protein involved in £occulation [10]. They argued that the phenomenon frequently occurred by the overexpression
of Flo1p which has a GPI anchor attachment signal for targeting to the cell wall. GPI anchors were proved to be connected with the protein in the rough ER in S. cerevisiae [21]. We observed that most of the gold particles with the fusion protein were stored in ERs; this may be due to overexpression of the protein which has a GPI anchor
Fig. 5. Electron and immunoelectron micrographs of cryo¢xed sec1 cells. Ultrastructure of the sec1 host cell (a), and the same cells harboring pGA11 (b). Immunogold labeling with anti-glucoamylase antibody on cells harboring pGA11 (c,d). 30 000U.
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
248
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
attachment signal. In cwh6/gpi3 cells, which are de¢cient in GPI anchor biosynthesis, limited availability of GPI anchors might cause accumulation of GPI-dependent proteins in the ER [22]. In our study, the GPI anchor was synthesized at the normal level, but overexpression of a GPI anchor-dependent fusion protein might cause a relative shortage of the GPI anchor, resulting in accumulation of the fusion protein in the ER. In cells in which the intact glucoamylase was secreted, gold particles were located on secretory vesicles and in ERs (data not shown). This also supported the possibility of a shortage of GPI anchors due to overproduction of the fusion protein. This evidence indicates that the highly e¤cient GPI anchoring of heterologous proteins is required for displaying on the cell surface of S. cerevisiae. Acknowledgements The authors thank Randy Scheckman of the University of California, Berkeley, CA, USA, for providing the S. cerevisiae sec1 strain. References [1] Murai, T., Ueda, M., Yamamura, M., Atomi, H., Shibasaki, Y., Kamasawa, N., Osumi, M., Amachi, T. and Tanaka, A. (1997) Construction of a starch-utilizing yeast by cell surface engineering. Appl. Environ. Microbiol. 63, 1362^1366. [2] Murai, T., Ueda, M., Atomi, H., Shibasaki, Y., Kamasawa, N., Osumi, M., Kawaguchi, T., Arai, M. and Tanaka, A. (1997) Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 48, 499^503. [3] Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T. and Tanaka, A. (1999) Development of an arming yeast strain for e¤cient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol. 51, 65^70. [4] Ueda, M., Murai, T., Shibasaki, Y., Kamasawa, N., Osumi, M. and Tanaka, A. (1998) Molecular breeding of polysaccharide-utilizing yeast cells by cell surface engineering. Ann. N. Y. Acad. Sci. 864, 528^537. [5] Lipke, P.N. and Ovalle, R. (1998) Cell wall architecture in yeast: new structure and new challenges. J. Bacteriol. 180, 3735^3740. [6] Kapteyn, J.C., van den Ende, H. and Klis, F.M. (1999) The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim. Biophys. Acta 1426, 373^383.
FEMSLE 9661 26-10-00
[7] Schreuder, M.P., Mooren, A.T.A., Toschka, H.Y., Verrips, C.T. and Klis, F.M. (1996) Immobilizing proteins on the surface of yeast cells. Trends Biotechnol. 14, 115^120. [8] van der Vaart, J.M., te Biesebeke, R., Chapman, J.W., Toschka, H.Y., Klis, F.M. and Verrips, C.T. (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous protein. Appl. Environ. Microbiol. 63, 615^ 620. [9] Cappellaro, C., Baldermann, C., Rachel, R. and Tanner, W. (1994) Mating type-speci¢c cell-cell recognition of Saccharomyces cerevisiae : cell wall attachment and active sites of a- and K-agglutinin. EMBO J. 13, 4737^4744. [10] Bony, M., Sempoux, D.T., Barre, P. and Blondin, B. (1997) Localization and cell surface anchoring of the Saccharomyces cerevisiae £occulation protein Flo1p. J. Bacteriol. 179, 4929^4936. [11] Ram, A.F.J., van den Ende, H. and Klis, F.M. (1998) Green £uorescent protein^cell wall fusion proteins are covalently incorporated into the cell wall of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 162, 249^255. [12] Tajima, T., Nogi, Y. and Fukasawa, T. (1985) Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1, 67^77. [13] Novick, P. and Schekman, R. (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 76, 1858^1862. [14] Kamasawa, N., Yoshida, T., Ueda, M., Tanaka, A. and Osumi, M. (1999) Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells. J. Electron Microsc. 48, 173^176. [15] Baba, M. and Osumi, M. (1987) Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae yeast cells. J. Electron Microsc. Tech. 5, 249^261. [16] Hohenberg, H., Mannweiler, K. and Muller, M. (1994) High-pressure freezing of plant cell suspension in cellulose capillary tubes. J. Microsc. 175, 34^43. [17] Kamasawa, N., Naito, N., Kurihara, T., Kamada, Y., Ueda, M., Tanaka, A. and Osumi, M. (1992) Immunoelectron microscopic localization of thiolase, L-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis. Cell Struct. Funct. 17, 203^207. [18] Lew, D.J. and Reed, S. (1995) Cell cycle control of morphogenesis in budding yeast. Curr. Opin. Genet. Dev. 5, 17^23. [19] Finger, F.P. and Novic, P. (1998) Spatial regulation of exocytosis : lesson from yeast. J. Cell Biol. 142, 609^612. [20] Schreuder, M.P., Brekelmans, S., van den Ende, H. and Klis, F.M. (1993) Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast 9, 399^409. [21] Conzelmann, A., Reizman, H., Desponds, C. and Bron, C. (1988) A major 125-kDa membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 7, 2233^2240. [22] Vossen, J.H., Muller, W.H., Lipke, P.N. and Klis, F.M. (1997) Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins. J. Bacteriol. 179, 2202^2209.
Cyaan Magenta Geel Zwart
www.fems-microbiology.org
Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface Yumi Shibasaki a , Naomi Kamasawa b , Seiji Shibasaki c , Wen Zou c , Toshiyuki Murai c , Mitsuyoshi Ueda c , Atsuo Tanaka c , Masako Osumi a;b; * a
Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan c Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
b
Received 11 May 2000 ; received in revised form 19 September 2000; accepted 19 September 2000
Abstract A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase^K-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cell surface engineering; K-Agglutinin ; Glycosyl phosphatidylinositol anchor; Fluorescence microscopy ; Immunoelectron microscopy ; Saccharomyces cerevisiae
1. Introduction We established a method for displaying certain proteins on the cell surface of Saccharomyces cerevisiae by connecting a C-terminal half of K-agglutinin to the protein which was aimed at displaying on the cell surface [1^4]. In a previous study, a starch-utilizing S. cerevisiae strain was constructed by displaying a glucoamylase^K-agglutinin fusion protein on the cell wall with retaining glucoamylase activity [1]. The gene of Rhizopus oryzae glucoamylase, cleaving K-1,4-linked and K-1,6-linked glucose, was fused with the 3P- half of K-agglutinin gene. K-Agglutinin is a cell wall mannoprotein involved in sexual adhesion, which has a signal sequence for attachment of a glycosyl phosphatidylinositol (GPI) anchor.
* Corresponding author. Tel. : +81 (3) 3943 3131 ext. 7260; Fax: +81 (3) 3942 6188; E-mail : [email protected]
Cell wall proteins which contain the GPI attachment signal are connected by GPI anchors in the endoplasmic reticulum (ER), and passed through the secretory pathway to the cell membrane. On the cell membrane, the GPI anchor is cleaved and the remnant is transferred to form a glycosidic linkage with the branched L-1,6-glucan [5^8]. C-terminal regions of these proteins have been proved to covalently bind heterologous proteins on the cell wall of S. cerevisiae [7,8]. Localization of native cell wall proteins anchored by GPI has been morphologically analyzed [9^11]. An K-agglutinin protein was visualized by immunoelectron microscopy on the ¢brillar mannan layer of the cell wall [9], and a £occulin protein was located in the ER, periplasmic region and external mannan layer [10]. Distribution of two cell wall proteins, Cwp1p and Cwp2p, on the cell surface was shown by tagging with green £uorescent protein [11]. Although the morphological studies on heterologous proteins constructed for anchoring to the cell wall
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 4 2 - 0
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
244
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
have been examined only by £uorescence microscopy, their transport pathway in the cell has not been identi¢ed. In this study, we further analyzed the distribution of glucoamylase^K-agglutinin fusion protein in relation to the cell cycle by indirect £uorescence microscopy, and its localization on the secretory pathway using a sec1 mutant strain by immunoelectron microscopy.
ing, a cellulose capillary tube was ¢lled with cell pellets, dipped into 1-hexadecene, and cut to a 2-mm length. Two or three pieces of capillaries were put into an aluminum platelet (BTBUO12126-T, Bal-Tech, Liechtenstein) ¢lled with 1-hexadecene, covered with another platelet, and frozen [16].
2. Materials and methods
Cryo¢xed cells were substituted at 380³C for 2 days, 320³C for 2 h, and 4³C for 2 h in absolute acetone as described [14]. Specimens were washed with absolute acetone and ethanol, embedded in a resin of LR white (London Resin, UK), and polymerized at 50³C for 24 h. Ultrathin sections of specimens were immuno-stained with an antibody against glucoamylase [1] diluted 1:1000, and a goat anti-rabbit IgG conjugated with 10 nm of colloidal gold (British Biocell International, UK) diluted 1:40 [17]. The sections were then stained with uranyl acetate.
2.1. Strains S. cerevisiae strains MT8-1 (MATa ade his3 leu2 trp1 ura3) [12] and a sec1 mutant, RSY45 (MATK K sec1-1 his4 ura3 trp1 leu2) [13], were used as host cells. A sec1 mutant accumulates vesicles during incubation at the restrictive temperature, 37³C [13]. A multicopy plasmid pGA11 and an integrative plasmid pIGA11 were constructed as described previously [1,3]. 2.2. Media and cultivation MT8-1 cells integrated with plasmid pIGA11 at the URA3 locus were grown in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) and MT8-1 cells harboring plasmid pGA11 were grown in modi¢ed Burkholder's medium [1] at 30³C to the exponential phase. RSY45 cells harboring plasmid pGA11 were grown in SD medium (0.67% yeast nitrogen base without amino acid (Difco) but with 0.002% adenine sulfate, 0.002% L-histidine^HCl, 0.003% L-leucine, 0.002% uracil and 2% glucose) at 24³C to the exponential phase, and then shifted up to the restrictive temperature, 37³C, for 3 h to accumulate vesicles. 2.3. Immuno£uorescence microscopy Indirect immuno£uorescence microscopy was performed as reported previously[1]. Fixed cells were incubated with an antibody against R. oryzae glucoamylase [1] diluted 1:100 for 1.5 h. After washing, goat anti-rabbit IgG conjugated with £uorescein-5-isothiocyanate (Cappel, ICN Pharmaceuticals, USA) was reacted at the dilution of 1:300 for 1 h. Cells were observed by a £uorescence microscope (BX 50, Olympus, Japan) and a confocal laser scanning microscope (TCS NT, Leica, Germany). Serial optical images of cells were superimposed and processed by the method described previously [14]. 2.4. Cryo¢xation Cryo¢xation was performed by rapid freezing with a KF80 machine (Leica, Austria) and by high-pressure freezing with an HPM 010 machine (Bal-Tech, Liechtenstein). For rapid-freezing, cell pellets were frozen by the sandwich method [15] using liquid propane. For high pressure freez-
FEMSLE 9661 26-10-00
2.5. Immunoelectron microscopy
2.6. Conventional electron microscopy Cryo¢xed cells were substituted in absolute acetone with 2% osmium tetroxide, and embedded in a Quetol 812 mixture (Nissin EM, Japan) [15]. Ultrathin sections were stained with uranyl acetate and lead citrate. 3. Results and discussion 3.1. Observation of glucoamylase^K-agglutinin fusion protein localized on the surface of MT8-1 cells by £uorescence microscopy Fluorescence localized speci¢cally on the cell surface was detected by £uorescence microscopy. In photographs taken at di¡erent focus-phases, £uorescence from the glucoamylase^K-agglutinin fusion protein was seen to be composed of many small particles (Fig. 1a1;2 ). Fluorescent particles were distributed unevenly, resulting in heavy and light £uorescent dots on the cell surface. This phenomenon was clearly con¢rmed by the confocal laser scanning microscopic observation (Fig. 1b,c). The superimposed images showed that the outline of a cell was not continuous (Fig. 1b). Another image also had uneven £uorescence (Fig. 1c1 ), and the processed image was emphasized by di¡erent colors corresponding to the £uorescence intensity (Fig. 1c2 ). Although cytochemical evaluation was done on the basis of a state of uniform expression of the fusion protein, immuno£uorescence microscopy showed that the amount of the expressed protein was distributed unequally on the cell surface. The distribution of £uorescence was analyzed according to the cell cycle in cells inserted with pIGA11 (Fig. 2). Cells grown to the exponential phase were classi¢ed into four growth stages by budding cell sizes, which were dis-
Cyaan Magenta Geel Zwart
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
245
Fig. 1. Immuno£uorescent labeling with anti-glucoamylase antibody of MT8-1 cells harboring pGA11 or integrated with pIGA11. Upper panel: Fluorescence micrographs were taken at di¡erent focus-phases, equatorial (a1 ), and upper phase (a2 ). Matching Nomarsky di¡erential interference micrograph (a3 ). U2250. Lower panel: Confocal laser scanning microscopic images of MT8-1 cells harboring pGA11 (b) or integrated with pIGA11 (c). Superimposed images of optical sections (b,c1 ), and a processed image (c2 ) of which £uorescent intensity is shown in arti¢cial colors.
Fig. 2. Immuno£uorescent images on budding process of MT8-1 cells integrated with pIGA11. 2250U.
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
246
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
tinguished from the ratio of the major axis between mother and daughter cells. About 50 cells in each stage were observed, and their £uorescent patterns and percentages are shown in Fig. 3. In unbudded cells, £uorescence is discontinuously localized on the cell surface (Fig. 2-1), or the areas with intense £uorescence were restricted to being opposite each other on the cell surface (Fig. 2-2). Small buds which were one-third or less the size of the mother cells had hard £uorescent intensity in most cells
(Fig. 2-3). When the buds enlarged to more than one-third but less than two-thirds of the mother cells, two £uorescent patterns were observed: in one only the buds had hard £uorescence intensity (Fig. 2-4), and in the other the entire cell surface of mother and bud was covered with £uorescent dots (Fig. 2-5). When buds grew to more than two-third of the mother cells, £uorescence was detected all over the cell surface of mother and bud (Fig. 2-6), and was intense at the neck region (Fig. 2-7,U). Fluorescence of this fusion protein especially localized on small buds and the neck region; these areas are known to be those where cell wall materials are newly synthesized. It is possible that insertion of the fusion protein into the cell wall is carried out together with other native cell wall materials. The direction of insertion of new materials of a cell wall has been reported to be changed from apex to isotropy in a cell cycle [18,19]. In very small buds, insertion occurred only on the bud surface, but isotropic insertion took place overall both buds and mother cells when buds reached two-third the size of the mother cells. Although Schreuder et al. [20] showed that a fusion protein which had the signal of the C-terminal half of K-agglutinin was labeled uniformly in buds of various sizes, our results suggested that heterologous proteins were ¢rst inserted in buds, and then dispersed over the entire cell surface, as in other native cell wall materials. 3.2. Behavior of the glucoamylase K-agglutinin fusion protein in S. cerevisiae sec1 cells
Fig. 3. Illustration of classi¢ed £uorescent pattern in budding process. *The numbers correspond to cell images in Fig. 2. Cells were classi¢ed in four stages by the ratio of cell size between a bud (a) and a mother cell (b); unbudded (a = 0), less than one-third (a/b 6 0.33), more than one-third but less than two-third (0.33Aa/b 6 0.66), more than twothirds (0.66Aa/b). Small buds were strongly labeled in 70% of the cells. Fluorescence then dispersed through the cell wall and accumulated at the neck region.
FEMSLE 9661 26-10-00
We used an immunoelectron microscopic technique to observe the intracellular localization of the glucoamylase^ K-agglutinin fusion protein. In cryo¢xed MT 8-1 cells, their intracellular ultrastructures, nucleus (N), vacuoles (V), and mitochondria (M) were observed, but there were only a few ERs and vesicles (Ves.) (Fig. 4a). Gold particles, indicating the glucoamylase^K-agglutinin fusion protein, were located on both the outside and inside of the cell wall (Fig. 4b). A few gold particles were seen in cytoplasm (Fig. 4b). It seemed di¤cult to determine the intracellular localization of the fusion protein in MT8-1 cells, because there were few secretory organelles under normal growth conditions. We therefore used a sec1 mutant, RSY45, as the host for pGA11, in which secretory vesicles would accumulate at the restrictive temperature, 37³C [13]. If the heterologous proteins pass through a secretory pathway, they had to remain in accumulated vesicles of this mutant cell. After cultivation at 37³C for 3 h, many accumulated vesicles were observed in the sec1 cell (Fig. 5a). These vesicles had di¡erent electron densities, and probably depended on the substances inside. An expanded membranous structure, perhaps ER, was often observed in the sec1 cells harboring pGA11, in which the glucoamylase^K-agglutinin fusion protein was expressed (Fig. 5b). A few gold particles indicated the fusion protein localized on the ac-
Cyaan Magenta Geel Zwart
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
247
Fig. 4. Electron micrographs of cryo¢xed MT8-1 cells harboring pGA11. Ultrastructure (a), and immunogold labeling with anti-glucoamylase antibody (b). Gold particles (U) indicate glucoamylase^K-agglutinin fusion protein. 30 000U.
cumulated vesicles (Fig. 5c), but most of the particles were in ERs and the nuclear envelope (Fig. 5d). Bony et al. observed the existence of an abundant, expanded ER holding the expressed Flo1p, a cell wall protein involved in £occulation [10]. They argued that the phenomenon frequently occurred by the overexpression
of Flo1p which has a GPI anchor attachment signal for targeting to the cell wall. GPI anchors were proved to be connected with the protein in the rough ER in S. cerevisiae [21]. We observed that most of the gold particles with the fusion protein were stored in ERs; this may be due to overexpression of the protein which has a GPI anchor
Fig. 5. Electron and immunoelectron micrographs of cryo¢xed sec1 cells. Ultrastructure of the sec1 host cell (a), and the same cells harboring pGA11 (b). Immunogold labeling with anti-glucoamylase antibody on cells harboring pGA11 (c,d). 30 000U.
FEMSLE 9661 26-10-00
Cyaan Magenta Geel Zwart
248
Y. Shibasaki et al. / FEMS Microbiology Letters 192 (2000) 243^248
attachment signal. In cwh6/gpi3 cells, which are de¢cient in GPI anchor biosynthesis, limited availability of GPI anchors might cause accumulation of GPI-dependent proteins in the ER [22]. In our study, the GPI anchor was synthesized at the normal level, but overexpression of a GPI anchor-dependent fusion protein might cause a relative shortage of the GPI anchor, resulting in accumulation of the fusion protein in the ER. In cells in which the intact glucoamylase was secreted, gold particles were located on secretory vesicles and in ERs (data not shown). This also supported the possibility of a shortage of GPI anchors due to overproduction of the fusion protein. This evidence indicates that the highly e¤cient GPI anchoring of heterologous proteins is required for displaying on the cell surface of S. cerevisiae. Acknowledgements The authors thank Randy Scheckman of the University of California, Berkeley, CA, USA, for providing the S. cerevisiae sec1 strain. References [1] Murai, T., Ueda, M., Yamamura, M., Atomi, H., Shibasaki, Y., Kamasawa, N., Osumi, M., Amachi, T. and Tanaka, A. (1997) Construction of a starch-utilizing yeast by cell surface engineering. Appl. Environ. Microbiol. 63, 1362^1366. [2] Murai, T., Ueda, M., Atomi, H., Shibasaki, Y., Kamasawa, N., Osumi, M., Kawaguchi, T., Arai, M. and Tanaka, A. (1997) Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 48, 499^503. [3] Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T. and Tanaka, A. (1999) Development of an arming yeast strain for e¤cient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol. 51, 65^70. [4] Ueda, M., Murai, T., Shibasaki, Y., Kamasawa, N., Osumi, M. and Tanaka, A. (1998) Molecular breeding of polysaccharide-utilizing yeast cells by cell surface engineering. Ann. N. Y. Acad. Sci. 864, 528^537. [5] Lipke, P.N. and Ovalle, R. (1998) Cell wall architecture in yeast: new structure and new challenges. J. Bacteriol. 180, 3735^3740. [6] Kapteyn, J.C., van den Ende, H. and Klis, F.M. (1999) The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim. Biophys. Acta 1426, 373^383.
FEMSLE 9661 26-10-00
[7] Schreuder, M.P., Mooren, A.T.A., Toschka, H.Y., Verrips, C.T. and Klis, F.M. (1996) Immobilizing proteins on the surface of yeast cells. Trends Biotechnol. 14, 115^120. [8] van der Vaart, J.M., te Biesebeke, R., Chapman, J.W., Toschka, H.Y., Klis, F.M. and Verrips, C.T. (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous protein. Appl. Environ. Microbiol. 63, 615^ 620. [9] Cappellaro, C., Baldermann, C., Rachel, R. and Tanner, W. (1994) Mating type-speci¢c cell-cell recognition of Saccharomyces cerevisiae : cell wall attachment and active sites of a- and K-agglutinin. EMBO J. 13, 4737^4744. [10] Bony, M., Sempoux, D.T., Barre, P. and Blondin, B. (1997) Localization and cell surface anchoring of the Saccharomyces cerevisiae £occulation protein Flo1p. J. Bacteriol. 179, 4929^4936. [11] Ram, A.F.J., van den Ende, H. and Klis, F.M. (1998) Green £uorescent protein^cell wall fusion proteins are covalently incorporated into the cell wall of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 162, 249^255. [12] Tajima, T., Nogi, Y. and Fukasawa, T. (1985) Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1, 67^77. [13] Novick, P. and Schekman, R. (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 76, 1858^1862. [14] Kamasawa, N., Yoshida, T., Ueda, M., Tanaka, A. and Osumi, M. (1999) Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells. J. Electron Microsc. 48, 173^176. [15] Baba, M. and Osumi, M. (1987) Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae yeast cells. J. Electron Microsc. Tech. 5, 249^261. [16] Hohenberg, H., Mannweiler, K. and Muller, M. (1994) High-pressure freezing of plant cell suspension in cellulose capillary tubes. J. Microsc. 175, 34^43. [17] Kamasawa, N., Naito, N., Kurihara, T., Kamada, Y., Ueda, M., Tanaka, A. and Osumi, M. (1992) Immunoelectron microscopic localization of thiolase, L-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis. Cell Struct. Funct. 17, 203^207. [18] Lew, D.J. and Reed, S. (1995) Cell cycle control of morphogenesis in budding yeast. Curr. Opin. Genet. Dev. 5, 17^23. [19] Finger, F.P. and Novic, P. (1998) Spatial regulation of exocytosis : lesson from yeast. J. Cell Biol. 142, 609^612. [20] Schreuder, M.P., Brekelmans, S., van den Ende, H. and Klis, F.M. (1993) Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast 9, 399^409. [21] Conzelmann, A., Reizman, H., Desponds, C. and Bron, C. (1988) A major 125-kDa membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 7, 2233^2240. [22] Vossen, J.H., Muller, W.H., Lipke, P.N. and Klis, F.M. (1997) Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins. J. Bacteriol. 179, 2202^2209.
Cyaan Magenta Geel Zwart