Unidad de Microbiología, Facultad de Farmacia, Universidad de Valencia, Avda Vicente Andrés Estelles s/n, 46100-Burjassot (Valencia), Spain
Correspondence
Jesús Zueco
jesus.zueco{at}uv.es
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ABSTRACT |
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The GenBank accession number for the YlCWP1 gene sequence reported in this article is AY084077.
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INTRODUCTION |
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Yarrowia lipolytica is one of the most extensively studied non-conventional yeasts and constitutes a good alternative model for the study of dimorphism (Barth & Gaillardin, 1997; Hurtado et al., 1999
, 2000
). However, relatively little is known about its cell-wall structure, especially at the level of CWPs. So far, only two CWPs, Ywp1 and Ylpir1, have been characterized (Ramon et al., 1996
; Jaafar et al., 2003a
). Ywp1 is reported to be specific to the mycelial cell wall and to be covalently linked to the cell-wall structure, although it does not contain the features characteristic of Pir- or GPI-CWPs, whilst Ylpir1 is the homologue of Pir4 of S. cerevisiae and can be extracted from the cell wall by reducing agents.
In this work, we describe the isolation and characterization of YlCWP1, a homologue of the CWP1 gene from S. cerevisiae that encodes a GPI-CWP (Van der Vaart et al., 1995), the isolation and characterization of ylcwp1
strains both in a wild-type and in a ylmnn9 background and the identification of the Ylcwp1 polypeptide encoded by YlCWP1.
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METHODS |
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Reagents.
Agar, yeast extract, peptone and yeast nitrogen base were purchased from Difco Laboratories; PMSF was from Roche; DNA restriction and modification enzymes were from Roche, New England Biolabs and Amersham Biosciences. The usual chemicals were purchased from Sigma and Panreac. Electrophoresis reagents were from Bio-Rad. Nitrocellulose membranes and the chemiluminescence ECL reagents for developing Western immunoblots were from Amersham. Goat anti-rabbit IgGperoxidase was from Bio-Rad.
Screening of gt11 expression libraries.
About 300 000 plaques containing inserts of a mean size of 1 kbp from a Y. lipolytica (yeast morphology) cDNA library in gt11 (provided by Eulogio Valentin, and obtained by Rosario Gil, Daniel Gozalbo and Eulogio Valentin, Unidad de Microbiología, Facultad De Farmacia, Universidad de Valencia) were screened with a polyclonal antibody that reacts with Pir-CWPs of S. cerevisiae (Moukadiri et al., 1999
). The screening of the library was done by the procedures described by Huyhn et al. (1985)
. The inserts of interest contained in the positive clones were recovered by PCR using the M13 forward and M13 reverse primers and subcloned in pGEMT-easy vectors (Promega).
Transformation of strains, DNA isolation and sequencing.
Basic DNA manipulation and transformation in E. coli was performed as described by Sambrook et al. (1989). Yeast transformation was carried out by the lithium acetate method (Ito et al., 1983
; Gietz & Sugino, 1988
). Plasmid DNA from E. coli was prepared using the Flexi-Prep kit (Pharmacia) and DNA fragments were purified from agarose gels using the Sephaglass Band-Prep kit, also from Pharmacia. Sequencing was performed using AmpliTaq polymerase with a Dye Terminator kit (Perkin Elmer) in an Applied Biosystems 373A automatic sequencer.
Isolation of genomic DNA.
The cells of an overnight 40 ml culture at 28 °C in YPD were harvested, washed in sterile distilled water and incubated for 2 h at 37 °C in 10 ml SEB buffer (0·9 M sorbitol, 0·1 M EDTA, 0·8 % -mercaptoethanol) containing 5 mg Zymolyase 20T (Seikagaku Kogyo Co.). Protoplast formation was monitored by phase-contrast microscopy. The protoplasts were harvested and resuspended in 3 ml TE buffer (10 mM Tris/HCl pH 7·5, 0·1 mM EDTA); 300 µl of 10 % SDS were added and the samples were incubated for 30 min at 65 °C. Then, 1 ml of 5 M potassium acetate was added and the samples were kept in ice for 1 h. The supernatant was recovered after centrifugation and DNA was precipitated by adding 0·1 vols of 3 M sodium acetate and 2·5 vols ethanol at -20 °C for at least 1 h. The DNA was recovered by centrifugation, resuspended in 3 ml TE, extracted with phenol/chloroform, precipitated again as above and resuspended in 500 µl TE buffer. DNA concentration was determined using a GeneQuantII spectrophotometer (Amersham-Pharmacia).
Southern analysis.
Samples of genomic DNA (25 µg) were digested with restriction enzymes and the resulting fragments were separated by electrophoresis in 0·8 % agarose gels in TAE buffer (40 mM Tris/HCl pH 7·6, 5 mM sodium acetate, 1 mM EDTA). The agarose gels were then submerged in 0·25 M HCl for 15 min twice, in 0·5 M NaOH, 1·5 M NaCl for 30 min and, finally, in 0·5 M Tris/HCl pH 7, 1·5 M NaCl for a further 30 min. The DNA was then transferred onto a positively charged nylon membrane (Roche or Amersham Biosciences) by capillarity, and the membrane was baked at 120 °C for 30 min to ensure DNA immobilization. Pre-hybridization was performed in 5x SSC, 0·1 % N-laurylsarcosine, 0·02 % SDS, 1 % Blocking Reagent (Roche Prehybridization Solution) for 1 h at 42 °C. The blot was then hybridized with a digoxigenin (DIG)-labelled DNA probe, which had previously been prepared according to the protocols provided by the manufacturer (Roche), at a concentration of 20 ng ml-1 in Prehybridization Solution for at least 16 h at 42 °C. The membrane was then washed twice in 2x SSC, 0·1 % SDS for 5 min at room temperature, and twice more in 0·1x SSC, 0·1 % SDS at 68 °C. Detection of the hybridized probe was carried out according to the manufacturer's instructions for the DIG-DNA labelling and detection kit (Roche).
Phenotypic analysis of the ylcwp1 strains.
Calcofluor white and Congo red sensitivities were tested by streaking cells onto plates containing different concentrations of these substances. Samples (2 µl) of serial 1/10 dilutions of cells grown overnight in YPD and adjusted to OD660 8 were deposited onto the surfaces of YPD plates containing different concentrations of Calcofluor white or Congo red, and growth was monitored after 3 days.
Isolation of cell-wall mannoproteins.
Cell walls from Y. lipolytica were purified and extracted with Zymolyase 20T as follows. Cells in the early-exponential phase were harvested and washed twice in 10 mM Tris/HCl pH 7·4, 1 mM PMSF (buffer A). The harvested biomass was resuspended in buffer A in a proportion of 2 ml (g wet weight)-1; glass beads (0·45 mm in diameter) were added up to 50 % of the final volume, and the cells were broken by shaking four times for 30 s, with 1 min intervals, in a CO2 refrigerated MSK homogenizer (Braun Melsungen). Breakage was confirmed by phase-contrast microscopy and the walls were washed six to eight times in buffer A. Removal of non-covalently bound proteins was achieved by boiling the walls in buffer A containing 2 % SDS [10 ml (g walls, wet weight)-1] for 10 min, followed by six to eight washes in buffer A. The purified cell walls were then extracted in buffer A containing 500 µg Zymolyase 20T ml-1, using 10 ml (g walls, wet weight)-1, for 3 h at 30 °C in an orbital incubator at 200 r.p.m. The extract was separated from the cell walls by centrifugation and concentrated 20-fold using a Centriprep-10 concentration device (Amicon/Millipore).
SDS-polyacrylamide gels and Western-blot analysis.
Proteins were separated by SDS-PAGE according to the method of Laemmli (1970) in 10 or 12 % polyacrylamide gels. The proteins separated by SDS-PAGE were either stained with Coomassie brilliant blue or transferred onto Hybond-C nitrocellulose membranes as described by Towbin et al. (1979)
and Burnette (1981)
. Membranes were blocked overnight in Tris-buffered saline containing 0·05 % Tween 20 (TBST) and 5 % non-fat milk. The blocked membranes were washed three times in TBST and incubated for 1 h in TBST containing the antibody at a dilution of 1 : 5000. After three washes in TBST, the membranes were incubated for 20 min in TBST containing goat anti-rabbit IgGperoxidase at a dilution of 1 : 12 000 and washed in TBST. Finally, antibody binding was visualized on X-ray film using the ECL method (Amersham).
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RESULTS |
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Structural analysis of the amino acid sequence encoded by YlCWP1
Alignment of the amino acid sequence encoded by YlCWP1 with that of Cwp1p of S. cerevisiae (Fig. 1) shows 28·5 % overall identity and the presence of several common features. Ylcwp1 has a putative signal peptide with a possible peptidase site between positions 16 and 17, and a putative GPI-attachment site at the asparagine in position 200 that closely resembles the consensus GPI-attachment signal in S. cerevisiae (Nuoffer et al., 1993
; Van der Vaart et al., 1995
), defined by an asparagine followed by glycine and alanine (NAG or NGA) NGA in Ylcwp1 followed by a hydrophobic carboxy-terminal region. In this context, it is important to note that the Kyte and Doolittle hydropathy profiles of Cwp1 and Ylcwp1 are both characteristic of GPI-CWPs (Fig. 2
). Other common features are the high content of serine and alanine, 77 out of 239 aa in Cwp1 and 72 out of 221 aa in Ylcwp1, and the presence of the motif DGQIQA close to the carboxy terminus. This feature is shared by at least three GPI-CWPs in S. cerevisiae, Cwp1, Cwp2 and Srp1 (Van der Vaart et al., 1995
), and is also present in all four Pir-CWPs of S. cerevisiae (Toh-e et al., 1993
; Moukadiri et al., 1999
), being part of the internal repeats' that give them their name, but not in the single Pir-CWP characterized so far in Y. lipolytica (Jaafar et al., 2003a
). The presence of the DGQIQA feature would also explain why we have isolated YlCWP1, a GPI-CWP, using an antibody that reacts with Pir-CWPs of S. cerevisiae.
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DISCUSSION |
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However, the initial aim of our work was to isolate additional members of the Pir-CWP family of Y. lipolytica, following the characterization of Ylpir1, the first Pir-CWP characterized in Y. lipolytica (Jaafar et al., 2003a). For this, we screened
gt11-based Y. lipolytica expression libraries using a polyclonal antibody that reacts with Pir-CWPs of S. cerevisiae. The isolation of a clone corresponding to a GPI-CWP can only be explained by the presence of the DGQIQA motif, which is also part of the internal repeat' that gives its name to the Pir-CWPs and which can be repeated up to 11 times in the sequence of some of them. By contrast, this motif is not found in the only Pir-CWP characterized in Y. lipolytica (Jaafar et al., 2003a
).
Disruption of YlCWP1 was performed in two different strains, strain PO1A and a ylmnn9 strain (Jaafar et al., 2003b). The use of strains harbouring the mnn9 allele plus the disruption of a specific GPI-CWP-encoding gene has been used recently to highlight co-operative functions in the biogenesis and maintenance of the cell wall in S. cerevisiae (Horie & Isono, 2001
). Changes in the cell wall of the disruptant ylcwp1
and of the double disruptant ylcwp1
ylmnn9
were detected by testing their sensitivities to Calcofluor white and Congo red, substances that disturb the cell wall, aggravating the consequences of cell-wall defects (Elorza et al., 1983
; Kopecka & Gabriel, 1992
; Ram et al., 1994
). However, the results of this assay showed a slight increase in the sensitivity to Congo red only, both in the ylcwp1
and in the ylcwp1
ylmnn9
strains, compared to their respective parental strains. This result is in agreement with that reported for the cwp1 strain in S. cerevisiae where only a slight increase in sensitivity to Calcofluor white and Congo red was detected (Van der Vaart et al., 1995
), and confirms the possible role of Ylcwp1 in the cell wall of Y. lipolytica. Moreover, we have presumably identified Ylcwp1 as a 60 kDa band present in the Zymolyase 20T extract from cell walls of the ylmnn9
strain, by comparison with an identical extract obtained from the cell walls of the ylcwp1
ylmnn9
strain. The identification of this 60 kDa band as Ylcwp1 is supported by its absence in the corresponding ylcwp1
strain and, also, by having a similar size, 55 kDa in Cwp1 and 60 in Ylcwp1, and identical localization to Cwp1 in S. cerevisiae (Van der Vaart et al., 1995
). As is the case with Cwp1, there are no putative N-glycosylation sites in Ylcwp1; however, the discrepancy between the expected molecular mass, as deduced from the amino acid sequence, and that observed in SDS-PAGE could be accounted for by O-glycosylation.
Finally, Kapteyn et al. (2001) have reported that, in S. cerevisiae, at low environmental pH, Cwp1 becomes anchored through an alkali-labile linkage to 1,3-
-glucan, instead of, or in addition to, the GPI-derived linkage. In the case of the Ylcwp1 band we detected in the Zymolyase 20T extracts, although we presume it may correspond to GPI-anchored Ylcwp1, we cannot discard the possibility that it represents the protein directly bound to 1,3-
-glucan through an alkali-labile linkage, as described by Kapteyn et al. (2001)
for Cwp1 in S. cerevisiae.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Burnette, W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195203.[Medline]
Caro, L. H., Tettelin, H., Vossen, J. H., Ram, A. F., van den Ende, H. & Klis, F. M. (1997). In silico identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 14771489.[CrossRef][Medline]
De Nobel, H. & Lipke, P. N. (1994). Is there a role for GPIs in yeast cell-wall assembly? Trends Cell Biol 4, 4246.[CrossRef]
Elorza, M. V., Rico, H. & Sentandreu, R. (1983). Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. J Gen Microbiol 129, 15771582.[Medline]
Gietz, R. D. & Sugino, A. (1988). New yeast-Escherichia coli shuttle vector constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527534.[CrossRef][Medline]
Horie, T. & Isono, K. (2001). Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae. Yeast 18, 14931503.[CrossRef][Medline]
Hurtado, C. A. & Rachubinski, R. A. (1999). MHY1 encodes a C2H2-type zinc finger protein that promotes dimorphic transition in the yeast Yarrowia lipolytica. J Bacteriol 181, 30513057.
Hurtado, C. A., Beckerich, J. M., Gaillardin, C. & Rachubinski, R. A. (2000). A rac homolog is required for induction of hyphal growth in the dimorphic yeast Yarrowia lipolytica. J Bacteriol 182, 23762386.
Huyhn, T. V., Young, R. A. & Davis, R. W. (1985). Constructing and screening of cDNA libraries in gt10 and
gt11. In DNA Cloning: a Practical Approach, vol. 1, pp. 4978. Edited by D. M. Glover. Oxford: IRL Press.
Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153, 163168.[Medline]
Jaafar, L., Moukadiri, I. & Zueco, J. (2003a). Characterization of a disulfide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica. Yeast 20, 417426.[CrossRef][Medline]
Jaafar, L., León, M. & Zueco, J. (2003b). Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 strain. Yeast 20, 633644.[CrossRef][Medline]
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 1426, 373383.[Medline]
Kapteyn, J. C., Hoyer, L. L., Hecht, J. E., Muller, W. H., Andel, A., Verkleij, A. J., Makarow, M., Van Den Ende, H. & Klis, F. M. (2000). The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 35, 601611.[CrossRef][Medline]
Kapteyn, J. C., ter Riet, B., Vink, E., Blad, S., De Nobel, H., Van den Ende, H. & Klis, F. M. (2001). Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall. Mol Microbiol 39, 469479.[CrossRef][Medline]
Klis, F. M. (1994). Review: cell wall assembly in yeast. Yeast 10, 851869.[Medline]
Klis, F. M., Caro, L. H., Vossen, J. H., Kapteyn, J. C., Ram, A. F., Montijn, R. C., Van Berkel, M. A. & Van den Ende, H. (1997). Identification and characterization of a major building block in the cell wall of Saccharomyces cerevisiae. Biochem Soc Trans 25, 856860.[Medline]
Klis, F. M., Mol, P., Hellingwerf, K. & Brul, S. (2002). Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26, 239256.[CrossRef][Medline]
Kollár, 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. Beta(16)-glucan interconnects mannoprotein, beta(1
)3-glucan, and chitin. J Biol Chem 272, 1776217775.
Kopecka, M. & Gabriel, M. (1992). The influence of Congo red on the cell wall and (13)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae. Arch Microbiol 158, 115126.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lipke, P. N. & Ovalle, R. (1998). Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180, 37353740.
Lu, C. F., Montijn, R. C., Brown, J. L., Klis, F. M., Kurjan, J., Bussey, H. & Lipke, P. N. (1995). Glycosyl phosphatidylinositol-dependent crosslinking of -agglutinin and
1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol 128, 333340.[Abstract]
Moukadiri, I. & Zueco, J. (2001). Evidence for the attachment of Hsp150/Pir2 to the cell wall of Saccharomyces cerevisiae through disulfide bridges. FEMS Yeast Res 1, 241245.[CrossRef][Medline]
Moukadiri, I., Jaafar, L. & Zueco, J. (1999). Identification of two mannoproteins released from cell walls of a Saccharomyces cerevisiae mnn1 mnn9 double mutant by reducing agents. J Bacteriol 181, 47414745.
Nuoffer, C., Horvath, A. & Riezman, H. (1993). Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein. J Biol Chem 268, 1055810563.
Orlean, P. (1997). Biogenesis of yeast wall and surface components. In The Molecular and Cellular Biology of the Yeast Saccharomyces, vol. 3, Cell Cycle and Cell Biology, pp. 229262. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ram, A. F. J., 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]
Ramon, A. M., Gil, R., Burgal, M., Sentandreu, R. & Valentin, E. (1996). A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica. Yeast 12, 15351548.[CrossRef][Medline]
Rothstein, R. (1991). Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194, 281301.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Toh-e, A., Yasunaga, S., Nisogi, H., Tanaka, K., Oguchi, T. & Matsui, Y. (1993). Three yeast genes, PIR1, PIR2 and PIR3, containing internal tandem repeats are related to each other, and PIR1 and PIR2 are required for tolerance to heat shock. Yeast 9, 481494.[Medline]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 43504354.[Abstract]
Van der Vaart, J. M., Caro, L. H., Chai Man, J. W., Klis, F. M. & Verrips, C. T. (1995). Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177, 31043110.[Abstract]
Received 17 April 2003;
revised 15 October 2003;
accepted 20 October 2003.
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