1 Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
2 Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, 657-8501, Kobe, Japan
3 Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, 657-8501, Kobe, Japan
Correspondence
Masayuki Azuma
azuma{at}bioa.eng.osaka-cu.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Many genes involved in 1,6--glucan biosynthesis have been identified based on resistance to K1 killer toxin, which kills yeast following binding to a 1,6-
-glucan-containing cell surface receptor (Al-Aidroos & Bussey, 1978
; Boone et al., 1990
; Brown et al., 1993
), and on hypersensitivity to Calcofluor white (Ram et al., 1994
; Lussier et al., 1997
). Their role in 1,6-
-glucan biosynthesis is supported by the fact that null mutations of these genes lead to a reduction in the level of 1,6-
-glucan. These gene products and their functional homologues are located along the secretory pathway, including the endoplasmic reticulum (ER), the Golgi apparatus, the cytoplasm and the cell surface, suggesting that 1,6-
-glucan is synthesized along the secretory pathway and completed at the cell surface (Shahinian & Bussey, 2000
). Among those gene products, Kre5p is particularly worth noting. The Kre5p protein is a soluble N-glycoprotein located in the ER (Meaden et al., 1990
; Levinson et al., 2002
), and mutation of the Kre5p gene greatly reduces formation of cell wall 1,6-
-glucan, leading to severe growth defects or lethality (Meaden et al., 1990
; Levinson et al., 2002
; Shahinian et al., 1998
; Azuma et al., 2002
). The biological activity of this protein is still unknown, but it is reported to have a limited but significant sequence similarity with UDP-glucose : glycoprotein glucosyltransferases (UGGT) from Drosophila melanogaster (Parker et al.; 1995
) and Schizosaccharomyces pombe (Fernandez et al., 1996
). Recently, we have found that deletion of BIG1 leads to an approximately 95 % reduction in cell wall 1,6-
-glucan (Page et al., 2003
). In addition, Big1p is an N-glycosylated integral membrane protein with a type I topology, it is located in the ER, and some phenotypes of a big1
mutant resemble those of a kre5
mutant. Although a Big1p homologue is also found in Candida albicans (Azuma et al., 2002
), its biochemical function has not yet been defined.
BIG1 was first described as a multicopy suppressor of the synthetic lethality of a rot1-1 rot2-1 double mutant. Mutations in ROT1 (reversal of TOR2) and ROT2 cause cell wall defects and suppress the loss of TOR2, an essential phosphatidylinositol-kinase-like protein kinase (Bickle et al., 1998). ROT2 was identified based on the similarity of its protein sequence with that of the
subunit of mammalian glucosidase II (Trombetta et al., 1996
). In addition, a rot2
mutant has a partial 1,6-
-glucan defect (Simons et al., 1998
). However, like Big1p, the functional role of Rot1p remains unknown. ROT1 is essential for growth, with rot1
ascospores germinating and arresting growth within one cell cycle (Bickle et al., 1998
). In the disruption consortium BY4741 strain series used in this work, ROT1 is also classified as an essential gene (Winzeler et al., 1999
).
Here, to examine whether the presence of Rot1p is necessary for formation of normal levels of cell wall 1,6--glucan, we begin an analysis of rot1
mutants.
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METHODS |
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FACS analysis.
Cells were pre-cultured at 30 °C in 4 ml YPDS for 1 day, then 0·2 ml of the culture was transferred to 4 ml YPDS and cells were cultured to exponential phase. Hydroxyurea (0·4 ml of 1·0 M solution) was added to the culture solution and incubated for 4 h. The cells were washed with YPDS three times to remove hydroxyurea, resuspended in 10 ml YPDS and cultured at 30 °C for 29 h. Cells were fixed in 70 % cold ethanol at 20 °C overnight, washed with 0·2 M Tris/HCl (pH 7·5) and treated with 1 mg RNase A ml1 (Sigma) at 37 °C overnight. Cells were washed with 0·2 M Tris/HCl (pH 7·5), and resuspended in 100 µl Na-PI solution (0·05 mg propidium iodide ml1, 1·0 mg sodium citrate ml1, 0·58 mg sodium chloride ml1); 10 µl of 2·0 mg propidium iodide ml1 was then added to the solution. Samples were allowed to stand at room temperature for 30 min and diluted with 900 µl 0·2 M Tris/HCl buffer and 10 µl of 2·0 mg propidium iodide ml1. After the cell suspensions had been briefly sonicated, samples were analysed using a Becton Dickinson FACSCalibur and Cell Quest. Flow cytometric analysis was performed using FACSCalibur (Becton Dickinson). Event rate was maintained at 300 cells s1 and data for 20 000 events were collected.
Phenotypes of rot1 cells.
Assays for K1 killer toxin sensitivity were carried out as previously described (Bussey, 1991). Yeast strains were grown on YPD+0·6 M sorbitol at 30 °C for 18 h. Cells were suspended at approximately 1x107 cells ml1 in 100 µl of a sterilized solution of 1·0 M sorbitol, and 5 µl of this suspension was added to 5 ml medium (1 % Difco yeast extract, 2 % peptone, 1 % agar, 0·001 % methylene blue, 0·6 M sorbitol and 1x Halvorson medium buffered at pH 4·7) kept at 45 °C. The medium was quickly poured into Petri dishes (60x15 mm). After the agar had gelled and attained room temperature, 5 µl K1 killer toxin (1000x stock diluted 1 : 10) was spotted onto the centre of the medium. The plate was incubated at 18 °C overnight, followed by 2448 h at 30 °C, after which the death zone was measured and photographed.
Drug sensitivity was determined by spotting diluted yeast cultures onto agar media containing various drugs (Ram et al., 1994; Lussier et al., 1997
). Cells were cultured in liquid YPD+1·0 M sorbitol overnight at 30 °C. The cell density was adjusted to 0·5 (optical density at 600 nm), and 2 µl drops of a set of 1 : 10 serial dilutions were spotted onto agar plates. We used YPD+1·0 M sorbitol agar medium containing the following drug concentrations: hygromycin B, 1100 µg ml1; or SDS, 0·00050·005 %. Cells were cultured at 30 °C for 72 h, and the growth was then observed.
Alkali-soluble 1,6--glucan assay.
Yeast strains were pre-grown on YPD+0·6 M sorbitol plates for 2 or 3 days, and then cells were transferred to YPD+0·6 M sorbitol liquid medium (25 ml) with a toothpick and cultured overnight. The cells were harvested by centrifugation for 10 min at 1860 g, washed with 1·0 M sorbitol (10 ml), and resuspended in 500 µl water. Glass beads were added to the cell suspension, and the mixtures were vortexed five times for 30 s, with intervals (at least 5 min) on ice, and lysates removed from the beads. The protein levels in lysates were determined using the Bradford assay. Lysates containing 8 µg total cell wall protein were brought up to 50 µl with water, and 50 µl NaOH (1·5 M) was added to each, followed by incubation for 1 h at 75 °C. After removal of the alkali-insoluble components by centrifugation (5 min, 11 000 g), a 1 : 2 serial dilution of the alkali-soluble fractions was spotted on Hybrid-C nitrocellulose membrane (Amersham). The immunoblotting was carried out in TBST (10 mM Tris/HCl, pH 8·0/150 mM NaCl/0·05 % Tween 20) containing 5·0 % non-fat dried milk powder using a 1 : 2000 dilution of the affinity-purified rabbit anti-1,6--glucan primary antibody (Kollar et al., 1997
) and a 1 : 2000 dilution of horseradish peroxidasegoat anti-rabbit secondary antibody (Amersham). The glucan signal on the membrane was visualized following development with a chemiluminescence detection kit.
Alkali-insoluble -glucan assay.
Alkali-insoluble -glucan levels were determined as described by Kapteyn et al. (1999)
. Cells were cultured in 50 ml flasks containing 10 ml YPD+1·0 M sorbitol, harvested by centrifugation (10 min, 2600 g) and washed with 1·0 M sorbitol (25 ml). The cells were broken with glass beads on ice, and the cell wall fraction was collected by centrifugation (15 min, 2600 g). Alkali-insoluble
-glucans were extracted three times in 3 % (w/v) NaOH at 75 °C for 1 h. The pellet was washed twice in 0·1 M Tris/HCl (pH 7·5), washed in 0·01 M Tris/HCl (pH 7·5), resuspended in 0·01 M Tris/HCl (pH 7·5) containing 1,3-
-glucanase (Zymolyase 100T, 1·0 mg ml1) and incubated at 37 °C overnight. After dialysis, the 1,6-
-glucan was collected and quantified. The total alkali-insoluble glucan was measured as the hexose content before dialysis. The alkali-insoluble 1,3-
-glucan level was calculated by subtraction of the 1,6-
-glucan content from total glucan.
Fluorescence microscopy.
Chitin was visualized by Calcofluor white staining. Cells grown on solid medium (YPD+1·0 M sorbitol) for 48 h were suspended in 1·0 M sorbitol, stained with Calcofluor white M2R (Sigma) at 0·1 mg ml1, washed with 1·0 M sorbitol, and observed with a fluorophotomicroscope (Olympus BX50-34FLAD/PM30). The images were recorded with a Coolsnap camera (Nippon Roper). Mannan was visualized by staining with concanavalin Afluorescein isothiocyanate (ConAFITC, Sigma). Cells grown on YPD+0·6 M sorbitol for 48 h were fixed with 3·7 % formaldehyde for 30 min and washed with PBS. ConAFITC (0·1 mg) was added to the cell suspension (1·0 ml). The suspension was incubated at room temperature for 10 min, washed with PBS, and observed.
Observation of GFPFlo1p.
To evaluate cell surface proteins in rot1 cells, the fluorescence of a fusion protein of GFP and the C-terminal part of Flo1p (a GPI-anchored protein located in cell wall) (Bony et al., 1997
) was analysed. Cells with pMUF-GFP, bearing the gene encoding the fusion protein, were cultured in YNB (without uracil)+0·6 M sorbitol containing 50 mM HEPES (pH 7·2) at 30 °C. Cells were washed, resuspended in the same medium and observed with a fluorophotomicroscope. The cultures were also centrifuged, and the fluorescence intensities of the supernatant and the pellet (cells) resuspended in a volume of the same medium equal to the culture were measured (excitation, 488 nm; emission, 510 nm) with a Shimadzu RF-5000 fluorometer.
SEM and TEM images.
Cells were cultured on YPDS plates at 30 °C for 2 days. Specimens were fixed by the freeze-substitution method with minor modifications (Baba & Osumi, 1987). The cells were mounted on the copper meshes to form a thin layer and plunged into liquid propane cooled with liquid N2. Frozen cells were transferred to anhydrous acetone containing 2·5 % OsO4, cooled in a solid CO2/acetone bath, and kept in the bath for 48 h. The solution including cells was incubated at 20 °C for 2 h, at 4 °C for 2 h, and subsequently at room temperature for 2 h. After washing the cells with anhydrous acetone three times, cells for SEM were incubated in anhydrous acetone/isoamyl acetate (1 : 1, v/v) for 20 min, and then in 100 % isoamyl acetate for 20 min. The cells were dried with a critical point dryer (Hitachi), coated with a gold layer and observed with a Hitachi Natural SEM S-3500N scanning electron microscope. Cells for TEM were embedded using ERL4206-Quentol 653 (Nissin EM). Ultrathin sections were prepared with an ULTRACUT N microtome (Reichert-Nisei); they were stained with uranyl acetate and lead citrate and viewed with a Hitachi H-7000 electron microscope.
Genetic interaction.
Crossing of rot1 and big1
cells was carried out. The rot1
and big1
cells were mixed on a YPDS plate, incubated at 30 °C for 6 h, transferred to a YNBS plate (not containing Met and Lys) to select only heterozygous diploid cells, and cultured at 30 °C for 4 or 5 days. The heterozygous diploid cells that grew on the selective medium were transferred to a GNA plate, cultured for 1 day, transferred to a SPO plate to make spores, and incubated at room temperature for 7 or 9 days. These cells were treated with 0·5 mg Zymolyase 100T ml1. The spores in individual asci were dissected onto YPDS plates and incubated at 30 °C for 6 days. Genotypes of resulting spores were determined by growth on media containing G418 and by PCR analysis using primers KanB, ScROT1-A and ScBIG1-A (Table 2
).
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RESULTS AND DISCUSSION |
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The PSORT and Sosui programs predicted the presence of a transmembrane domain between residues 238 and 256 in Rot1p. Although a signal peptide was not found using either of these two programs, another program, SignalP, predicted an N-terminal signal peptide. Rot1p also has a consensus sequence for GPI-modified proteins, suggesting that it remains attached to the plasma membrane (Caro et al., 1997, De Groot et al., 2003
). On the other hand, Huh et al. (2003)
predicted that Rot1p may be localized in the ER, based on analyses of a collection of yeast strains expressing Rot1p tagged with GFP at the C-terminus (Huh et al., 2003
). However, further work is required to determine the subcellular location of Rot1p more precisely.
According to a BLASTP search and the C. albicans genome sequence at the Stanford University Genome Center (http://genome-www.stanford.edu/), Rot1p has homologues in Sz. pombe and C. albicans. Because these homologues have high identities with Rot1p, we refer to them here as SzRot1p (43 % identity) and CaRot1p (53 % identity), respectively. The presence of a transmembrane domain near the C-terminus and an N-terminal signal peptide was predicted in both SzRot1p and CaRot1p using PSORT and Sosui.
Isolation of a haploid rot1 mutant
The rot1-1 mutant has an altered content of chitin (188 % of wild-type), suggesting the participation of the ROT1 gene product in cell wall synthesis (Bickle et al., 1998). To understand its role, we performed a phenotypic analysis of ROT1 by isolating a haploid rot1
mutant. The heterozygous rot1
/ROT1 mutant was sporulated, and spores were dissected on YPD containing 0·6 M sorbitol. After 6 days of culture, the rot1
mutant produced small colonies on this medium; it was unable to produce colonies on YPD without sorbitol. A representative tetrad from the rot1
/ROT1 heterozygous diploid is shown in Fig. 1
. The colony size was similar or slightly smaller than the big1
colonies (data not shown). To test if the haploid mutants obtained were indeed disruptants with an insertion of the G418 resistance gene, cells were picked and transferred to medium containing G418. The mutants grew, although poorly, on YPD containing sorbitol and G418. The poor growth of the mutants was complemented by transformation with the plasmid pRS426-ROT1 (data not shown). Confirmation of the disruption of ROT1 was also obtained by PCR analysis.
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Bickle et al. (1998) described that rot1-null spores germinated and arrested growth within one cell cycle as medium- or large-budded cells. To examine cell cycle progression, the DNA content of rot1
cells was quantified by FACS. After arresting the cells in S phase with hydroxyurea, cell progression was restarted by removing the hydroxyurea. In wild-type cells there was a progression of (
G2/M
G1) over a 4 h period (Fig. 2
a). In both rot1
and big1
mutants (Fig. 2b and 2c, 0 h), the cell cycle was not completely arrested in S phase by hydroxyurea. This may be due to slow growth of the mutants. However, after removal of the hydroxyurea most of the cells arrested in G2/M for at least 4 h. These results suggested that rot1
cells are delayed at the G2/M phase.
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Recently, a system to display proteins on the cell surface of S. cerevisiae was developed using the prepro--factor leader region and the C-terminal GPI-anchor attachment signal sequences of Flo1p, a native cell wall protein (Sato et al., 2002
). To examine the effect of the 1,6-
-glucan defect on cell surface proteins, wild-type, big1
, cwh41
and rot1
cells were transformed with pMUF-GFP (bearing the gene encoding the GFPFlo1p fusion protein) and observed by fluorescence microscopy. We found that the fluorescence of GFP in big1
, cwh41
, and rot1
cells was similar to that in the wild-type (Fig. 5c
).
Because of the role of 1,6--glucan in anchoring mannoproteins to the cell wall, we expected that disruption of Big1p or Rot1p would cause a release of the GFPFlo1 fusion protein into the medium. We therefore examined the intensity of GFP fluorescence in the culture supernatant of the rot1
, big1
, cwh41
, and wild-type cells (Fig. 6
). After 72 h of culture, the fluorescence intensities of the supernatants from rot1
and big1
cells were approximately fivefold stronger than those in wild-type and the cwh41
cells. These results indicate that mannoproteins in rot1
and big1
cells fail to be correctly linked to the cell wall and are released to the medium.
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Considering the role of 1,6--glucan in the cell wall, it was expected that big1
and rot1
mutants would have defects in anchoring cell surface mannoproteins. However, microscopic analyses using ConAFITC and GFPFlo1p showed no differences relative to the wild-type cells. In contrast, when a strain with defects in GPI anchor synthesis (mcd4 mutant) was observed as a negative control, there was little ConAFITC and GFPFlo1p fluorescence (unpublished results). In addition, the quantity of cell wall proteins obtained from rot1
cells was similar to or slightly more than that from the wild-type when the two cell types were cultivated to the same culture volume (data not shown). These results suggest that, even with this large defect in 1,6-
-glucan level, mannoproteins were transported to the cell surface and anchored to 1,3-
-glucan and chitin. In rot1
cells expressing pMUF-GFP, the fluorescence intensity in the supernatant was 77 % of that in the cells, suggesting that much of the GFPFlo1p was released to the medium. Furthermore, electron microscopic observations indicate that the cell walls of rot1
and big1
mutants are very rough, that these strains do not have the outer layer with a high density of mannoproteins, and that the outer boundary of the cell wall is irregular. Therefore, in these mutants, the mannoproteins do not localize in the outer layer at a high density, and, instead, may be spread around the whole cell wall.
Rot1p appears to be a membrane protein required for normal levels of the cell wall 1,6--glucan, and has homologues with high identity in Sz. pombe and C. albicans. Although in both SzRot1p and CaRot1p the presence of a transmembrane domain is predicted, the functional role of those proteins has not yet been defined. Analysis of Rot1p function should contribute to an understanding of fungal 1,6-
-glucan biosynthesis. Rot1p is also potentially interesting as a target for antifungal drugs because the protein is essential for growth and has a homologue in the pathogenic yeast C. albicans.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Azuma, M., Levinson, J. N., Page, N. & Bussey, H. (2002). Saccharomyces cerevisiae Big1p, a putative endoplasmic reticulum membrane protein required for normal levels of cell wall -1,6-glucan. Yeast 19, 783793.[CrossRef][Medline]
Baba, M. & Osumi, M. (1987). Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae yeast cells. J Electron Microsc Techn 5, 249261.
Bickle, M., Delley, P. A., Schmidt, A. & Hall, M. N. (1998). Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J 17, 22352245.
Bony, M., Thines-Sempoux, D., Barre, P. & Blondin, B. (1997). Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179, 49294936.[Abstract]
Boone, C., Sommer, S. S., Hensel, A. & Bussey, H. (1990). Yeast KRE genes provide evidence for a pathway of cell wall -glucan assembly. J Cell Biol 110, 18331843.[Abstract]
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. & Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115132.[CrossRef][Medline]
Brown, J. L., Kossaczka, Z., Jiang, B. & Bussey, H. (1993). A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1,6)--glucan synthesis. Genetics 133, 837849.
Bussey, H. (1991). K1 killer toxin, a pore-forming protein from yeast. Mol Microbiol 5, 23392343.[Medline]
Bussey, H., Sacks, W., Galley, D. & Saville, D. (1982). Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function. Mol Cell Biol 2, 346354.[Medline]
Caro, L. H., Tettelin, H., Vossen, J. H., Ram, A. F., van den Ende, H. & Klis, F. M. (1997). In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 14771489.[CrossRef][Medline]
Chen, D. C., Yang, B. C. & Kuo, T. T. (1992). One-step transformation of yeast in stationary phase. Curr Genet 21, 8384.[Medline]
Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. & Hieter, P. (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119122.[CrossRef][Medline]
Cid, V. J., Duran, A., Del Rey, F., Snyder, M. P., Nombela, C. & Sanchez, M. (1995). Molecular basis of cell integrity and morphogenesis in S. cerevisiae. Microbiol Rev 59, 345386.[Medline]
De Groot, P. W., Hellingwerf, K. J. & Klis, F. M. (2003). Genome-wide identification of fungal GPI proteins. Yeast 20, 781796.[CrossRef][Medline]
Fernandez, F., Jannatipour, M., Hellman, U., Rokeach, L. A. & Parodi, A. J. (1996). A new stress protein: synthesis of Schizosaccharomyces pombe UDP-Glc : glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability. EMBO J 15, 705713.[Abstract]
Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. (1995). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355360.[Medline]
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S. & O'Shea, E. K. (2003). Global analysis of protein localization in budding yeast. Nature 425, 686691.[CrossRef][Medline]
Jiang, B., Sheraton, J., Ram, A. F. J., Dijkgraaf, G. J., Klis, F. M. & Bussey, H. (1996). CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly. J Bacteriol 178, 11621171.[Abstract]
Kanai, T., Atomi, H., Umemura, K., Ueno, H., Teranishi, Y., Ueda, M. & Tanaka, A. (1996). A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis. Appl Microbiol Biotechnol 44, 759765.[CrossRef][Medline]
Kapteyn, J. C., Montijn, R. C., Vink, E., De La Cruz, J., Llobell, A., Douwes, J. E., Shimoi, H., Lipke, P. N. & Klis, F. M. (1996). Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 6, 337345.[Abstract]
Kapteyn, J. C., Van Den Ende, H. & Klis, F. M. (1999). The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 2, 373383.
Kollar, R., Reinhold, B. B., Petrakova, E., Yeh, H. J., Ashwell, G., Drgonova, J., Kapteyn, J. C., Klis, F. M. & Cabib, E. (1997). Architecture of the yeast cell wall. (1,6)-glucan interconnects mannoprotein,
(1,3)-glucan, and chitin. J Biol Chem 272, 1776217775.
Levinson, J. N., Shahinian, S., Sdicu, A. M., Tessier, D. C. & Bussey, H. (2002). Functional, comparative and cell biological analysis of Saccharomyces cerevisiae Kre5p. Yeast 19, 12431259.[CrossRef][Medline]
Lussier, M., White, A. M., Sheraton, J. & 17 other authors (1997). Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147, 435450.
Meaden, P., Hill, K., Wagner, J., Slipetz, D., Sommer, S. S. & Bussey, H. (1990). The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1,6)--D-glucan synthesis and normal cell growth. Mol Cell Biol 10, 30133019.[Medline]
Orlean, P. (1997). Biogenesis of yeast wall and surface components. In The Molecular Biology of the Yeast Saccharomyces, vol. 3, pp. 229362. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Page, N., Gerard-Vincent, M., Menard, P. & 9 other authors (2003). A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin. Genetics 163, 875894.
Parker, C. G., Fessler, L. I., Nelson, R. E. & Fessler, J. H. (1995). Drosophila UDP-glucose : glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins. EMBO J 14, 12941303.[Abstract]
Ram, A. F., Wolters, A., Ten Hoopen, R. & Klis, F. M. (1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 10191030.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sato, N., Matsumoto, T., Ueda, M., Tanaka, A., Fukuda, H. & Kondo, A. (2002). Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates. Appl Microbiol Biotechnol 60, 469474.[CrossRef][Medline]
Shahinian, S. & Bussey, H. (2000). -1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol 35, 477489.[CrossRef][Medline]
Shahinian, S., Dijkgraaf, G. J., Sdicu, A. M., Thomas, D. Y., Jakob, C. A., Aebi, M. & Bussey, H. (1998). Involvement of protein N-glycosyl chain glucosylation and processing in the biosynthesis of cell wall -1,6-glucan of Saccharomyces cerevisiae. Genetics 149, 843856.
Sherman, F., Fink, G. & Hicks, J. B. (1982). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sikorski, R. S. & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 1927.
Simons, J. F., Ebersold, M. & Helenius, A. (1998). Cell wall 1,6--glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p. EMBO J 17, 396405.
Trombetta, E. S., Simons, J. F. & Helenius, A. (1996). Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit. J Biol Chem 271, 2750927516.
Winzeler, E. A., Shoemaker, D. D., Astromoff, A. & 49 other authors (1999). Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901906.
Received 30 April 2004;
revised 24 July 2004;
accepted 29 July 2004.
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