Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica

Lahcen Jaafar and Jesús Zueco

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The structure and composition of the cell wall of yeast has so far been studied mainly in Saccharomyces cerevisiae. It is basically made up of three components: {beta}-glucans, chitin and mannose-containing glycoproteins, also called mannoproteins. Most covalently bound cell-wall mannoproteins belong to the so-called glycosylphosphatidylinositol cell-wall protein (GPI-CWP) family, cell-wall proteins that are bound through the remnant of a GPI residue to 1,6-{beta}-glucan. The non-conventional yeast Yarrowia lipolytica shares Generally Regarded As Safe (GRAS) status with S. cerevisiae, has some industrial applications and is increasingly being proposed as a host for the production of recombinant proteins and as a model in the study of dimorphism. However, very little information on cell-wall structure and composition is available for this organism. Here is described the isolation and characterization of YlCWP1, a homologue of the CWP1 gene from S. cerevisiae, which encodes a GPI-CWP, and the identification of its gene product. YlCWP1 encodes a 221 aa protein that contains a putative signal peptide and a putative GPI-attachment site. It shows 28·5 % overall identity with Cwp1 of S. cerevisiae and a hydropathy profile characteristic of GPI-CWPs. Disruption of YlCWP1, both in the wild-type and in an mnn9 glycosylation-deficient background, led to the identification of Ylcwp1 as a 60 kDa polypeptide present in cell-wall extracts. To the authors' knowledge, this is the first report of a GPI-CWP in Y. lipolytica, and it suggests that the cell-wall organization of Y. lipolytica is similar to that of S. cerevisiae.


Abbreviations: CWP, cell-wall protein; GPI, glycosylphosphatidylinositol; Pir, protein with internal repeats

The GenBank accession number for the YlCWP1 gene sequence reported in this article is AY084077.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The structure and composition of the cell wall of yeast has so far been studied mainly in Saccharomyces cerevisiae and to a lesser extent in Candida albicans. It is basically made up of three components: {beta}-glucans, chitin and mannose-containing glycoproteins, also called mannoproteins (Klis, 1994). The mannoproteins themselves can be divided into three groups according to the linkages that bind them to the structure of the cell wall: (i) non-covalently bound, (ii) covalently bound to the structural glucan and (iii) disulfide-bound to other proteins that are covalently bound to the structural glucan of the cell wall (De Nobel & Lipke, 1994; Kapteyn et al., 1999). Covalently bound mannoproteins are normally referred to as cell-wall proteins (CWPs) and fall into two different categories, Pir-CWPs and GPI-CWPs (Kapteyn et al., 1999). Pir-CWPs belong to the PIR (protein with internal repeats) gene family (Toh-e et al., 1993) and are supposed to be bound to 1,3-{beta}-glucan in the cell wall through a kind of linkage sensitive to mild alkali treatment, although some Pir-CWPs have been reported as disulfide-bound CWPs (Moukadiri et al., 1999; Moukadiri & Zueco, 2001). GPI-CWPs receive a glycosylphosphatidylinositol (GPI) anchor during their passage through the secretory pathway and, in addition, they become N-glycosylated and/or O-glycosylated (Orlean, 1997). The addition of the GPI anchor happens at the endoplasmic reticulum and substitutes a hydrophobic domain present at the carboxy terminus of CWPs (Caro et al., 1997). GPI-CWPs, once exported, become attached to 1,6-{beta}-glucan chains through a remnant of the GPI anchor (Lu et al., 1995; Kollár et al., 1997). The 1,6-{beta}-glucan side-chains are linked to 1,3-{beta}-glucan which may be in turn linked to chitin, in a basic structure that repeats itself to give the overall shape of the cell wall (Klis et al., 1997; Orlean, 1997; Lipke & Ovalle, 1998; Kapteyn et al., 1999). In addition, some GPI-CWPs, such as Cwp1, in conditions of low environmental pH, may also bind directly to 1,3-{beta}-glucan through an alkali-sensitive linkage, presumably in a Pir-CWP-like fashion (Kapteyn et al., 2001; Klis et al., 2002). This basic model of structure seems to apply also for C. albicans (Kapteyn et al., 2000).

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{Delta} strains both in a wild-type and in a ylmnn9 background and the identification of the Ylcwp1 polypeptide encoded by YlCWP1.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and media.
Escherichia coli DH5{alpha} was used for the propagation of plasmids; it was grown in Luria broth supplemented with 100 µg ampicillin ml-1 when necessary. E. coli Y1090 was used in experiments involving {lambda}gt11. Strain PO1A (MATA leu2-270 ura3-202) of Y. lipolytica (C. Gaillardin, Laboratoire de Génétique des Micro-organismes, INRA-CNRS, Thiverval-Grignon, France) was used in all experiments involving Y. lipolytica. Yeast strains were grown in YPD (1 % yeast extract, 2 % Bacto Peptone, 2 % glucose) or synthetic minimal medium (MM; 0·7 % yeast nitrogen base without amino acids, 2 % glucose and amino acids as required).

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 IgG–peroxidase was from Bio-Rad.

Screening of {lambda}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 {lambda}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 % {beta}-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{Delta} 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 IgG–peroxidase 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).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of the YlCWP1 gene
In an attempt to isolate Pir-CWPs of Y. lipolytica, we proceeded to screen a {lambda}gt11-based gene library from Y. lipolytica using an antibody that reacts with Pir-CWPs from S. cerevisiae (Moukadiri et al., 1999). After the screening of some 300 000 plaques (mean insert 1 kbp), we obtained 13 positives, two of which were confirmed after secondary and tertiary screening. These were named L1 and L2, and the inserts contained in them were amplified by PCR, using the M13 forward and reverse universal primers, and sequenced. The two inserts, of 700 and 500 bp, respectively, were found to correspond to the same gene, and comparison of the amino acid sequence encoded by the ORF contained in the longer of the two inserts revealed homology with Cwp1 of S. cerevisiae, a GPI-CWP first reported by Van der Vaart et al. (1995). To characterize the chromosome region containing the insert contained in L1, Southern-blot analysis was performed using genomic DNA from Y. lipolytica PO1A and the 700 bp L1 insert as probe. The results from this analysis suggested the existence of a single copy of the gene and, at the same time, gave us an idea of the approximate restriction map of the chromosome region containing it. To isolate the complete gene, we prepared a mini-gene library by digestion of genomic DNA from Y. lipolytica PO1A with the restriction enzymes PstI and HindIII. The restriction fragments generated were separated by electrophoresis in a 0·7 % agarose gel; a slice of the gel supposed to contain the DNA fragments of around 3·3 kbp in size was cut out, the DNA fragments were eluted from the gel and ligated in pBlueScript II (Stratagene) vector, previously digested with PstI and HindIII, to give the mini-gene library. Screening of the mini-gene library, using the L1 insert as a probe, gave a positive clone with a 3·3 kbp insert that was confirmed by PCR to contain the L1 insert. The sequence of the complete gene was obtained using the oligonucleotides LNN1 (5'-TTGCCCCCTTTAACTGCAATG-3') and LNN2 (5'-TTGATGGTGAACTTGGCGCCG-3') which hybridize at the extremes of the known sequence of the L1 insert and which prime outwards from it, and further confirmed using the oligonucleotides LF (5'-TTACACCAGACTACCGAC-3') and LR (5'-TCCAGTCTCGTGATGTG-3') to give a double-strand reading in all the length of the 1456 bp fragment sequenced. Analysis of the sequence revealed the existence of an ORF of 666 bp in length that encoded a putative protein 221 aa long with homology to Cwp1 of S. cerevisiae (Van der Vaart et al., 1995). Accordingly, we named this gene YlCWP1.

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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Fig. 1. Sequence alignment of the predicted amino acid sequence of Cwp1 of Y. lipolytica (YlCwp1) with that of Cwp1 from S. cerevisiae. Percentage identity of YlCwp1 with Cwp1 is 28·51 %.

 


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Fig. 2. Kyte and Doolittle hydropathy profile of Cwp1 (a) and YlCwp1 (b).

 
Deletion of the YlCWP1 gene and characterization of ylcwp1{Delta} strains
Disruption ylcwp1{Delta} strains were created both in wild-type strain PO1A and in mnn9 (Jaafar et al., 2003b) backgrounds to determine the possible phenotypes associated with the disruption of the gene. The disruption was performed in both strains using the two-step ‘pop in/pop out’ method (Rothstein, 1991) with URA3 as marker. For this, the gene and part of its regulatory sequences were amplified with oligonucleotides LF and LR as a 1456 bp DNA fragment that was subcloned in the pGEMT-easy vector (Promega). This construction was then digested with NarI and re-ligated, with the loss of 435 bp of the coding sequence of the gene, and a SalI–SalI fragment containing the URA3 marker was subcloned in the single SalI site in the construction. Finally, the construction was rendered linear by digestion with HpaI to produce the disruption cassette that was then transformed in the PO1A and mnn9 strains. After monitoring the correct integration of the disruption cassette, a selected clone for each strain was plated onto plates containing 5'-fluoroorotic acid (5'-FOA), and clones derived from these plates and grown on YPD were monitored for the loss of the wild-type allele both by PCR and by Southern analysis. PCR was performed on DNA from colonies growing on YPD plates after the 5'-FOA selection step, using the oligonucleotides LF2 (5'-TTACACCAGACTACCGAC-3') and LR. The expected result was the amplification of either a 1340 bp band in the case of the wild-type allele or a 905 bp band in the case of the disrupted allele. As can be seen in Fig. 3(a), both clones, derived respectively from the PO1A and mnn9 strains, contained the disrupted YlCWP1 allele. These clones were also confirmed by Southern-blot analysis by digestion of DNA with EcoRV and HindIII, using the 1340 bp fragment generated by oligonucleotides LF2 and LR as probe. In this case, the expected result was either a band of around 3·065 kbp in the case of the disrupted allele or a band of 3·5 kbp in the case of the wild-type allele. As can be seen, both clones, based on the PO1A and mnn9 strains, respectively, were confirmed to contain the disrupted YlCWP1 allele (Fig. 3b). These results show that we had created ylcwp1{Delta} for both the wild-type PO1A and mnn9 strains of Y. lipolytica. However, a preliminary analysis failed to reveal any differences between the parental PO1A with the PO1A ylcwp1{Delta} and between the parental mnn9 strain and the double disruptant mnn9 ylcwp1{Delta}, with respect to morphology of the cells, growth rate or yeast-to-mycelium transition, at least in the conditions used. A more cell-wall-biased phenotypic analysis, using hypersensitivity to Calcofluor white and Congo red, did show a slight increase of the sensitivity to Congo red of the ylcwp1{Delta} strains with respect to their respective parental strains (Fig. 4), a result that suggests a role for Ylcwp1 in the cell wall.



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Fig. 3. Validation of the ylcwp1{Delta} deletion strains. (a) PCR using the LF2 and LR oligonucleotides to generate either a 1·34 kbp band (parental) or a 905 bp band (disrupted allele). Lanes: M, {lambda} DNA digested with EcoRI and HindIII; 1, ylcwp1{Delta} strain; 2, ylmnn9{Delta} ylcwp1{Delta} strain; 3, strain PO1A. (b) Southern analysis of the parental PO1A (lane 1), ylcwp1{Delta} (lane 2) and ylmnn9{Delta} ylcwp1{Delta} (lane 3) strains. Genomic DNA was digested with restriction enzymes EcoRV and HindIII, separated by agarose electrophoresis, transferred onto nylon membranes and hybridized with the 700 bp PCR-generated L1 insert. M corresponds to {lambda} DNA digested with EcoRI and HindIII. Expected size was 3·5 kbp (parental) or 3·1 kbp (disrupted allele).

 


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Fig. 4. Effect of YlCWP1 deletion on the sensitivity to Congo red of the parental (PO1A) and ylmnn9{Delta} strains. Cells of the parental and ylcwp1{Delta} strains (c and d) or the ylmnn9{Delta} and ylmnn9{Delta} ylcwp1{Delta} strains (a and b) were grown in YPD and 1/10 dilution series of 2 µl droplets of each strain was inoculated onto the surface of YPD plates containing 1 µg Congo red ml-1 (b) or 35 µg Congo red ml-1 (d). (a) and (c) correspond to YPD control plates.

 
Identification of Ylcwp1 in Zymolyase 20T extracts from the cell walls of the mnn9 strain of Y. lipolytica
Finally, based on the described localization of Cwp1 in extracts obtained from cell walls of S. cerevisiae (Van der Vaart et al., 1995; Kapteyn et al., 2000), we proceeded to identify Ylcwp1 in similar extracts from cell walls of the mnn9 strain of Y. lipolytica. This strain was chosen because of the lower degree of glycosylation of its glycoproteins, which makes the identification of individual bands in electrophoresis easier. With this aim, we isolated cell walls from ylmnn9{Delta} and ylmnn9{Delta} ylcwp1{Delta} strains from Y. lipolytica, and treated them sequentially with SDS, to eliminate non-covalently bound proteins, and with Zymolyase 20T, to release GPI-CWPs. The analysis of the electrophoretic pattern of the bands in the Zymolyase 20T extract was performed by Western immunoblot, using antibodies raised against purified cell walls of Y. lipolytica. The comparison of the pattern of bands of the extracts from the cell walls of the ylmnn9{Delta} and ylmnn9{Delta} ylcwp1{Delta} strains shows a single and relatively clear difference that consists of the presence of a band of some 60 kDa in the extract of the ylmnn9{Delta} strain, a band that is not present in the extract from the double ylmnn9{Delta} ylcwp1{Delta} disruptant strain (Fig. 5). Accordingly, we assumed this band to correspond to Ylcwp1.



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Fig. 5. Western-immunoblot analysis of proteins extracted by Zymolyase 20T from the purified cell walls of the ylmnn9{Delta} (lane 1) and ylmnn9{Delta} ylcwp1{Delta} (lane 2) strains of Y. lipolytica. Cell-wall extracts were submitted to SDS-PAGE in 10 % acrylamide gels, transferred onto nitrocellulose membranes and incubated with a polyclonal antibody raised against purified cell walls of the yeast form of Y. lipolytica. The arrow points to the band presumably corresponding to Ylcwp1.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work, we present the isolation and characterization of the YlCWP1 gene of Y. lipolytica, presumably encoding a GPI-CWP, and the study of the phenotypic effects of the disruption of this gene, both in the parental strain PO1A and in a ylmnn9{Delta} strain. This gene encodes a 221 aa protein that is homologous to Cwp1, a GPI-CWP of S. cerevisiae (Van der Vaart et al., 1995). Ylcwp1, similarly to Cwp1, contains a putative signal peptide and a putative GPI-attachment signal, consisting of an NGA sequence, compatible with the NGA or NAG sequences found in S. cerevisiae GPI-CWPs (Nuoffer et al., 1993; Van der Vaart et al., 1995), followed by a stretch of hydrophobic amino acids that constitutes the amino terminus of the protein, giving an overall hydropathy profile characteristic of a GPI-CWP. Other common features are the presence of a DGQIQA motif in both the sequence of Cwp1 and Ylcwp1, a motif that is also shared by Srp1 and Cwp2, other GPI-CWPs of S. cerevisiae (Van der Vaart et al., 1995), and the high content of serine and alanine in both Cwp1 and Ylcwp1. Taken together, these data strongly suggest that YlCWP1 encodes a GPI-CWP of Y. lipolytica, and clearly point to the existence of a family of GPI-CWPs in Y. lipolytica that shares the main features of the GPI-CWPs of S. cerevisiae.

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 {lambda}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{Delta} and of the double disruptant ylcwp1{Delta} ylmnn9{Delta} 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{Delta} and in the ylcwp1{Delta} ylmnn9{Delta} 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{Delta} strain, by comparison with an identical extract obtained from the cell walls of the ylcwp1{Delta} ylmnn9{Delta} strain. The identification of this 60 kDa band as Ylcwp1 is supported by its absence in the corresponding ylcwp1{Delta} 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-{beta}-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-{beta}-glucan through an alkali-labile linkage, as described by Kapteyn et al. (2001) for Cwp1 in S. cerevisiae.


   ACKNOWLEDGEMENTS
 
We thank Eulogio Valentin, Daniel Gozalbo and Rosario Gil (Unidad Departamental de Microbiología, Facultad de Farmacia, Universidad de Valencia) for the gift of the {lambda}gt11 cDNA library, and Maria Iranzo (Unidad Departamental de Microbiología, Facultad de Farmacia, Universidad de Valencia) for the gift of the antibody raised against the purified cell walls of Y. lipolytica. This work was supported by grant BMC2001-2761 from the Ministerio de Ciencia y Tecnología (Spain).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Barth, G. & Gaillardin, C. (1997). Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19, 219–237.[CrossRef][Medline]

Burnette, W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.[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, 1477–1489.[CrossRef][Medline]

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Received 17 April 2003; revised 15 October 2003; accepted 20 October 2003.



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