Departamentos de Microbiología y Ecología1, and Bioquímica y Biología Molecular2, Universitat de València, Avda Vicent Andrés Estellés s/n, 46100 Burjasssot (Valencia), Spain
Sección de Biología y Patología Celular, Centro de Investigación, Hospital la Fe3, Valencia, Spain
Author for correspondence: Daniel Gozalbo. Tel: +34 96 398 3026. Fax: +34 96 386 4299. e-mail: daniel.gozalbo{at}uv.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: insertion mutations, yeast, cell surface, glycolytic enzymes, immunodetection
Abbreviations: G3-P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IIF, indirect immunofluorescence; ßME, ß-mercaptoethanol; pAb anti-SC-GAPDH, polyclonal antibody against S. cerevisiae GAPDH
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As most C. albicans genes are functional when expressed in Saccharomyces cerevisiae, we thought it would be interesting to express the cloned C. albicans TDH1 gene, encoding GAPDH (Gil-Navarro et al., 1997 ), in S. cerevisiae cells, to determine whether the gene product is directed to the yeast cell wall in an active form. Unexpectedly, we found that untransformed wild-type intact cells of S. cerevisiae possess GAPDH activity. This observation prompted us to determine whether the S. cerevisiae GAPDH is also a cell wall protein.
Three unlinked GAPDH structural genes (TDH13) are present per haploid S. cerevisiae genome; they encode closely related, but not identical, polypeptides (McAlister & Holland, 1985a , b
). None of the structural genes is individually essential for cell viability; however, the presence of a functional TDH2 or TDH3 structural gene is required for cell viability. The three single mutants tdh1, tdh2 and tdh3, as well as the tdh1 tdh2 and tdh1 tdh3 double mutants, can grow using glucose as carbon source, albeit at lower rates (except the tdh1 mutant) than wild-type cells. No growth phenotype has been described for strains lacking only TDH1, and double mutant tdh2 tdh3 appears not to be viable, indicating that the TDH1 gene product cannot support growth and may carry out a function that is different from glycolysis (McAlister & Holland, 1985a
, b
). Synthesis of Tdh polypeptides is not coordinately regulated; Tdh1 is only synthesized when cells enter stationary phase or in heat-shocked cells, whereas synthesis of Tdh2 is repressed by heat shock (Boucherie et al., 1995
).
In the present study, using wild-type strains and single and double tdh mutants, we found that each of the three GAPDH polypeptides encoded by the TDH13 genes is associated with the cell wall of S. cerevisiae. This enlarges the new emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of polyclonal antibodies against S. cerevisiae GAPDH (pAb anti-SC-GAPDH).
Female New Zealand White rabbits were immunized with GAPDH from S. cerevisiae (Sigma) according to standard protocols (Harlow & Lane, 1988 ). Levels of anti-SC-GAPDH antibodies in sera were determined by ELISA.
Determination of GAPDH activity.
The assay for GAPDH activity was carried out according to the method originally described by Ferdinand (1964) , with some modifications. An assay for intact whole cells, previously used in C. albicans (Gil-Navarro et al., 1997
), was performed to determine the presence of an active GAPDH enzyme at the cell wall of S. cerevisiae cells. Triplicate samples of different amounts of exponentially growing cells were incubated with and without glyceraldehyde 3-phosphate (G3-P) (7 µl of a solution containing 49 mg substrate ml-1; Sigma) in the presence of NAD (100 µl of a 10 mM solution; Boehringer Mannheim) in assay buffer (40 mM triethanolamine, 50 mM Na2HPO4, 5 mM EDTA, 0·1 mM dithiothreitol; pH 8·6) to a final volume of 1 ml. After incubation of the reaction mixtures at 28 °C, cells were removed by centrifugation and the supernatants were analysed for the presence of NADH by recording the absorbance at 340 nm. Background absorbance measured in negative controls (without substrate) were subtracted from positive absorbance values. Activity is expressed as the concentration (µM) of NADH generated during the assay. The cytosolic activity was determined in 50 µl cytosol sample, and the NADH produced was measured after 10 min of incubation. Specific activity is expressed as nmol NADH min-1 mg-1. Enzyme activities were also determined (as indicated in Results) on cells pretreated with trypsin (250 µg ml-1) for 30 min at 37 °C, prior to the enzyme assay, and on cells preincubated with pAb anti-SC-GAPDH (1:10 dilution), for 30 min at 37 °C.
Indirect immunofluorescence (IIF) and flow cytometry analysis.
Cells from exponentially growing cultures were washed twice with phosphate-buffered saline (PBS), and resuspended at 5x106 cells in 50 µl pAb anti-SC-GAPDH, diluted 1:10 in PBS. After 1 h at 37 °C, cells were washed with PBS and incubated under the same conditions in FITC-conjugated goat anti-rabbit IgG (Pierce, 1:20 dilution) in PBS. After washing with PBS, cells were examined for epifluorescence with a Nikon Eclipse E800 microscope. In flow cytometry analysis, after the immunofluorescence assay, cells were fixed in 1% paraformaldehyde in PBS, and analysed on an EPICS Elite Cell Sorter (Coulter Electronics), as previously described (Peñalver et al., 1996 ). Control experiments were performed by omitting incubation of cells with the pAb anti-SC-GAPDH.
Immunoelectron microscopy.
Immunoelectron microscopy detection of GAPDH protein in S. cerevisiae S173-29A wild-type cells with the pAb anti-SC-GAPDH was performed by the postembedding method, as previously described (Gozalbo et al., 1998 ).
Preparation of cell extracts.
Protein and glycoprotein components of the walls were released from intact cells by treatment with ß-mercaptoethanol (ßME) as previously described (Casanova & Chaffin, 1991 ), with minor modifications. Briefly, cells were collected and resuspended in sterile distilled water containing 1% (v/v) ßME, and incubated for 30 min at 37 °C with shaking. After treatment, the cells were sedimented, and the supernatant fluid was recovered, filtered, and concentrated by freeze-drying (ßME extract). The ßME-extracted cells were washed with ice-cold PBS, and broken by vortexing with glass beads. After addition of cold PBS, unbroken cells and particulate matter were removed from the supernatant (cytosol) by centrifugation.
Western immunoblotting.
Proteins were separated by SDS-PAGE (Laemmli, 1970 ) and electrophoretically transferred to PVDF membranes, using a Trans-Blot cell (Bio-Rad), according to the manufacturers instructions. Immunodetection was performed using the pAb anti-SC-GAPDH diluted 1:2000, and peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, diluted 1:2000); the reactive bands were developed with hydrogen peroxide and 4-chloro-1-naphthol as the chromogenic reagent (Gil-Navarro et al., 1997
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
These results indicate that at least the polypeptides encoded by TDH2 and TDH3 are associated with the cell wall in an active form, whereas no conclusion can be deduced regarding TDH1 due to the lack of a tdh2 tdh3 double mutant.
Immunodetection of GAPDH protein at the surface of S. cerevisiae cells
pAb anti-SC-GAPDH was used in an IIF assay to assess the presence of the protein on the surface of intact cells. Fluorescence was detected in most of the cells from both parental wild-type strains and all five tdh mutants by microscopic observation (Fig. 2) and flow cytometry analysis (not shown), indicating that GAPDH polypeptides are exposed on their surface. Labelling intensity was heterogeneously distributed among the cell population, and showed a patchy binding distribution, as previously described in C. albicans blastoconidial cells (Gil-Navarro et al., 1997
; Gozalbo et al., 1998
). Fluorescence was not observed when the cells were reacted with the second FITC-labelled marker antibody alone, indicating that the reactivity found was dependent on the previous interaction of the specific antibody (pAb anti-SC-GAPDH) with cells. This confirms the presence of Tdh2 and Tdh3 at the yeast cell surface.
|
|
The ßME extracts from exponentially growing cells were analysed by SDS-PAGE and Western blotting using the pAb anti-SC-GAPDH as a probe. The antibody reacted with the control protein (GAPDH from S. cerevisiae, Sigma) and immunodetected a polypeptide(s) with the same apparent molecular mass as the control protein (36 kDa) (Fig. 4a). The presence of soluble GAPDH from S. cerevisiae (1 mg ml-1, Sigma) in the pAb preparation used to develop the Western blot inhibited immunodetection of the 36 kDa polypeptide, which further supports the specificity of the reaction. In cells expressing one TDH gene (tdh1 tdh2 and tdh1 tdh3 double mutants) only a single polypeptide was detected, corresponding to Tdh3 and Tdh2, respectively; Tdh2 migrated slightly faster than Tdh3. This was confirmed by the detection of two polypeptides in tdh1 mutant cells. In parental strains these two polypeptides were also detected, and in tdh2 and tdh3 single mutants only one polypeptide was detected. These data show that Tdh1 is not present in these extracts. This pattern of reactive polypeptides was also found when the immunoblotting was performed with cytosol samples (Fig. 4b
), thus indicating that Tdh2 and Tdh3 cytosolic polypeptides are also present in the cell wall, whereas Tdh1 is not detected. In agreement with previous reports, the Tdh3 polypeptide represents the major Tdh polypeptide in exponentially growing cells, whereas Tdh1 is not found, as it is synthesized only as cells enter stationary phase (Boucherie et al., 1995
; McAlister & Holland, 1985b
).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regenerating protoplasts of S. cerevisiae have been described to secrete three glycolytic proteins: enolase (Eno2), fructose biphosphate aldolase (Fba1) and GAPDH (Tdh23) (Pardo et al., 1999 ). A genetic approach has been used to demonstrate that Eno2 and Fba1, although lacking a conventional N-terminal signal sequence, are able to be secreted into the periplasmic space (Pardo et al., 1999
). Furthermore, heterologous overexpression in S. cerevisiae of the Kluyveromyces marxianus GAP1 gene, encoding a GAPDH protein, results in the accumulation of GAPDH in the cell wall of yeast cells and confers a flocculent phenotype (Falcao Moreira et al., 2000
), providing additional evidence of the ability of S. cerevisiae to target GAPDH protein, lacking N-terminal signal peptide, to the cell wall. The presence of members of the Hsp70 family (Ssa1 and Ssa2) in the cell wall of S. cerevisiae has been described (López-Ribot & Chaffin, 1996
), in addition to its well known cytosolic location; also members of the Hsp70 family (Ssa1, Ssa2, as well as Ssb1 and Ssb2) are secreted by regenerating protoplasts of S. cerevisiae (Pardo et al., 1999
). Interestingly, some cytosolic proteins such as enolase, members of the Hsp70 family, and phosphoglycerate kinase, have been described to be bona fide components of the C. albicans cell wall (Alloush et al., 1997
; Angiolella et al., 1996
; Chaffin et al., 1998
; López-Ribot et al., 1996
; Martínez et al., 1998
). Hence, the presence of cytosolic proteins, including glycolytic enzymes, as autochthonous microbial cell wall components, appears to be a more general phenomenon than was previously imagined (Alloush et al., 1997
; Angiolella et al., 1996
; Chaffin et al., 1998
; Fernandes et al., 1992
; Gil-Navarro et al., 1997
; López-Ribot et al., 1996
; López-Ribot & Chaffin, 1996
; Modun & Williams, 1999
; Pancholi & Fischetti, 1992
, 1998
; Winram & Lottenberg, 1996
). All these observations favour the idea of the involvement of a nonconventional secretion mechanism for these cell-wall-associated proteins. However, the alternative possibility that they originate, at least in part, by cell leakage and release of cytosolic proteins from a small amount of damaged cells cannot be discarded, particularly in the case of Tdh polypeptides, which are abundant proteins that at acidic pH may bind to negatively charged molecules at the yeast cell surface.
As S. cerevisiae GAPDH is encoded by three structural genes (TDH13), we wondered whether the cell-wall-associated protein corresponded to one or more gene products. We therefore searched for the presence of cell-wall-associated GAPDH in single tdh mutant cells and double (tdh1 tdh2 and tdh1 tdh3) mutants. Overall our results indicate that all three polypeptides encoded by TDH13 are associated with the cell wall. Tdh2 and Tdh3 are found at the cell surface of exponentially growing cells, as well as in the cytosol. The TDH1 gene, when expressed in stationary-phase cells, directs the synthesis of a GAPDH polypeptide that is located both in the cytosol and in the cell wall.
The absence of correlation between cytosolic activity and cell-wall-associated activity in tdh mutants may reflect that: (i) different TDH genes may possess distinct ability to direct the protein to the cell wall, (ii) the catalytic activity of individual isozymes may vary between the cytosol and the cell wall due to possible modification of polypeptides during their association with the cell wall, (iii) activity may depend on the assembly of the cell wall polypeptides into active GAPDH, a process which may differ from that of the cytosol.
Our results incorporate the GAPDH of S. cerevisiae into the newly emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity, although the precise role of the cell-wall-associated GAPDH in S. cerevisiae physiology remains to be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angiolella, L., Facchin, M., Stringaro, A., Maras, B., Simonetti, N. & Cassone, A.(1996). Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J Infect Dis 173, 684-690.[Medline]
Boucherie, H., Bataille, N., Fitch, I. T., Perrot, M. & Tuite, M. F.(1995). Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells. FEMS Microbiol Lett 125, 127-134.[Medline]
Casanova, M. & Chaffin, W. L.(1991). Cell wall glycoproteins of Candida albicans as released by different methods. J Gen Microbiol 137, 1045-1051.[Medline]
Chaffin, W. L., López-Ribot, J. L., Casanova, M., Gozalbo, D. & Martinez, J. P.(1998). Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev 62, 130-180.
Charrier-Ferrara, S., Caillol, D. & Goudot-Crozel, V.(1992). Complete sequence of the Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase gene encoding a major surface antigen. Mol Biochem Parasitol 56, 339-344.[Medline]
Falcao Moreira, R., Fernandes, P. A. & Moradas-Ferreira, P.(1998). Kluyveromyces marxianus flocculence and growth at high temperature is dependent on the presence of the protein p37. Microbiology 144, 681-688.
Falcao Moreira, R., Ferreira-Da-Silva, F., Fernandes, P. A. & Moradas-Ferreira, P.(2000). Flocculation of Saccharomyces cerevisiae is induced by transformation with the GAP1 gene from Kluyveromyces marxianus. Yeast 16, 231-240.[Medline]
Ferdinand, W.(1964). The isolation and specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Biochem J 92, 578.[Medline]
Fernandes, P. A., Keen, J. N., Findlay, J. B. C. & Moradas-Ferreira, P.(1992). A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of flocculent Kluyveromyces marxianus. Biochim Biophys Acta 1159, 67-73.[Medline]
Gil, M. L., Villamón, E., Monteagudo, C., Gozalbo, D. & Martínez, J. P.(1999). Clinical strains of Candida albicans express the surface antigen glyceraldehyde-3-phosphate dehydrogenase in vitro and in infected tissues. FEMS Immunol Med Microbiol 23, 229-234.[Medline]
Gil-Navarro, I., Gil, M. L., Casanova, M., Martínez, J. P. & Gozalbo, D.(1997). The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen. J Bacteriol 179, 4992-4999.[Abstract]
Goudot-Crozel, V., Caillol, D., Djabali, M. & Dessein, A. J.(1989). The major parasite surface antigen associated with human resistence to schistosomiasis is a 37 kDa glyceraldehyde-3-phosphate dehydrogenase. J Exp Med 170, 2065-2080.[Abstract]
Gozalbo, D., Gil-Navarro, I., Azorín, I., Renau-Piqueras, J., Martínez, J. P. & Gil, M. L.(1998). The cell wall associated glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is also a fibronectin and laminin binding protein. Infect Immun 66, 2052-2059.
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Laemmli, U. K.(1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
López-Ribot, J. L. & Chaffin, W. L.(1996). Members of the hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 178, 4724-4726.[Abstract]
López-Ribot, J. L., Alloush, H. M., Masten, B. J. & Chaffin, W. L.(1996). Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family. Infect Immun 64, 3333-3340.[Abstract]
McAlister, L. & Holland, M. J.(1985a). Isolation and characterization of yeast strains carrying mutations in the glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 260, 15013-15018.
McAlister, L. & Holland, M. J.(1985b). Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 260, 15019-15027.
Martínez, J. P., Gil, M. L., López-Ribot, J. L. & Chaffin, W. L.(1998). Serologic response to cell wall mannoproteins and proteins of Candida albicans. Clin Microbiol Rev 11, 121-141.
Modun, B. & Williams, P.(1999). The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosphate dehydrogenase. Infect Immun 67, 1086-1092.
Pancholi, V. & Fischetti, V. A.(1992). A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate dehydrogenase with multiple binding activity. J Exp Med 176, 415-426.[Abstract]
Pancholi, V. & Fischetti, V. A.(1993). Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme. Proc Natl Acad Sci USA 90, 8154-8158.
Pancholi, V. & Fischetti, V. A.(1997). Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells. J Exp Med 186, 1633-1643.
Pancholi, V. & Fischetti, V. A.(1998). -Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273, 14503-14515.
Pardo, M., Monteoliva, L., Pla, J., Sanchez, M., Gil, C. & Nombela, C.(1999). Two-dimensional analysis of proteins secreted by Saccharomyces cerevisiae regenerating protoplasts: a novel approach to study the cell wall. Yeast 15, 459-472.[Medline]
Peñalver, M. C., OConnor, J. E., Martínez, J. P. & Gil, M. L.(1996). Binding of human fibronectin to Aspergillus fumigatus conidia. Infect Immun 64, 1146-1153.[Abstract]
Sirover, M. A.(1997). Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology. J Cell Biochem 66, 133-140.[Medline]
Sirover, M. A.(1999). New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta 1432, 159-184.[Medline]
Villamón, E., Gozalbo, D., Martínez, J. P. & Gil, M. L.(1999). Purification of a biologically active recombinant glyceraldehyde-3-phosphate dehydrogenase from Candida albicans. FEMS Microbiol Lett 179, 61-65.[Medline]
Winram, S. B. & Lottenberg, R.(1996). The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase. Microbiology 142, 2311-2320.[Abstract]
Winram, S. B. & Lottenberg, R.(1998). Site-directed mutagenesis of streptococcal plasmin receptor protein (Plr) identifies the C-terminal Lys334 as essential for plasmin binding, but mutation of the plr gene does not reduce plasmin binding to group A streptococci. Microbiology 144, 2025-2035.[Abstract]
Received 27 July 2000;
revised 9 October 2000;
accepted 18 October 2000.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |