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Fungal Genetics and Biology 22, 77–83 (1997) Article No. FG971000

Expression of YWP1, a Gene That Encodes a Specific Yarrowia lipolytica Mycelial Cell Wall Protein, in Saccharomyces cerevisiae

Ana M. Ramo ´ n,* Eulogio Valentı´n,* Sergi Maicas,† and Rafael Sentandreu*,1 *Seccio´ de Microbiologı´a, Facultat de Farmacia, Universitat de Vale`ncia, Avgda. Vicent Andre´s Estelle´s, s/n, 46100 Burjassot, Vale`ncia, Spain; and †Department de Microbiologia i Ecologia, Facultat de Biologia, Universitat de Vale`ncia, C/Dr. Moliner, 50, 46100 Burjassot, Vale`ncia, Spain

Accepted for publication June 25, 1997

Ramo´n, A. M., Valentı´n, E., Maicas, S., and Sentandreu, R. 1997. Expression of YWP1, a gene that encodes a specific Yarrowia lipolytica mycelial cell wall protein, in Saccharomyces cerevisiae. 22, 77–83. The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids. Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 58 noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S. cerevisiae promoters. A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants. Ywp1 processing and incorporation to the cell wall were similar in both Y. lipolytica and S. cerevisiae but not in its final localization in the cell wall. In Y. lipolytica Ywp1 is covalently bound to the cell wall (it is released only by Zymolyase digestion), whereas in S. cerevisiae it was not (it was released by boiling SDS solutions). These results suggest that the sequences involved in recognition, anchoring of a protein to the cell wall, or the catalytic activities implicated are different, at least for Ywp1, in Y. lipolytica and S. cerevisiae. Another possibility is that the

1 To whom correspondence should be addressed. Fax: 34 6 386 46 82. E-mail: rafael.sentandreu@uv.es. 2 Abbreviations used: SDS, sodium dodecyl sulfate; SDS-PAGE, SDS– polyacrylamide gel electrophoresis.

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target for attachment of Ywp1 is missing or cryptic in the cell wall of S. cerevisiae. r 1997 Academic Press

Index Descriptors: YWP1; Ywp1; expression; cell wall; SDS-released; Saccharomyces cerevisiae. The fungal cell wall is a type of extracellular matrix made up of an association of different components, mainly polysaccharides and proteins. Chitin and b-glucans are considered responsible for the morphology and the strength of the cell walls of different fungal species. Experimental evidence exists that glucans and chitin are covalently bound (Kollar et al., 1995; Sonnenberg et al., 1983; Surarit et al., 1988; Mol and Wessels, 1987; Wessels and Sietsma, 1983). The proteins present in the walls, mainly mannoproteins, can be divided into three classes: sodium dodecyl sulfate (SDS)2-released proteins, which are generally low molecular weight (Valentı´n et al., 1984) and glucanase- and chitinase-released proteins, both of which are polydispersed proteins of high molecular weight (Marcilla et al., 1991; Pastor et al., 1984). Glucanase-released proteins are likely to be covalently linked to glucan (van Berkel et al., 1994; van Rinsum et al., 1991). Yarrowia lipolytica is a dimorphic organism that grows as a yeast-like or mycelial form depending on the environmental factors (Rodriguez and Dominguez, 1984). In previous studies we have identified a cell wall protein specific for the mycelial form of Y. lipolytica Ywp1, which is covalently bound to glucan (Ramo´n et al., 1996). The YWP1 gene has been cloned and sequenced, and from its

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predicted amino acid sequence it was deduced that the N-terminal half is very rich in threonine and valine and the C-terminal half is very rich in serine and proline (Ramo´n et al., 1996). In Saccharomyces cerevisiae the C-terminal half of the a-agglutinin, a glucanase-released mannoprotein (Hauser and Tanner, 1989), is also very rich in serine and threonine, this region being considered responsible, after GPI modification, for anchoring this molecule in the cell wall (Leidich et al., 1995; Schreuder et al., 1993; Vossen et al., 1997). To study whether the signals (mechanisms) involved in the secretion and incorporation of proteins into the cell walls were common in different fungal species, the YWP1 gene was cloned under the control of different promoters and used to transform S. cerevisiae. We show in this paper that Ywp1 is incorporated in the S. cerevisiae cell wall but in a manner different from that in Y. lipolytica. In this species Ywp1 is a glucanase-released protein, whereas in S. cerevisiae it is a SDS-released protein, indicating that YWP1 is not covalently bound to the S. cerevisiae cell wall. Our results could suggest that the signals for anchoring a protein to the cell wall or the catalytic activities involved are different in both Y. lipolytica and S. cerevisiae.

transformations were carried out according to Hanahan (1983). Plasmid DNA was extracted from E. coli by the alkaline–lysis procedure of Birnboim and Doly (1979); for large-scale preparation, plasmid DNA was obtained using the Flexi Prep kit (Pharmacia) according to the manufacturer’s instructions. S. cerevisiae strains were transformed according to Ito et al. (1978). Leucine or uracil prototrophy of the transformants was found to be conferred by the presence of the plasmids since auxotroph colonies were recovered after growing on rich medium. Plasmids were rescued from S. cerevisiae by E. coli transformation with yeast DNA, extracted according to Hoffman and Winston (1987).

Northern Analysis Yeast total RNA extraction from exponentially growing cultures of transformants, RNA denaturation, gel electrophoresis, and transfer to nylon membranes were carried out as previously described (Ramo´n et al., 1996). DNA probe was labeled by random primed incorporation of digoxigenin-labeled deoxiuridine-triphosphate (DIG-labeled DNA) using the DIG DNA Labeling kit (Boehringer Mannheim) according to the manufacturer’s instructions.

MATERIALS AND METHODS Cell Wall Purification Strains and Media Escherichia coli strain DH5a F80d, lacD DM15, 2 recA, endA1, gyrA96, thi1, hsdR17, (r2 k , mk ), supE44, relA1, deoR, D(lacZYA-argF)U169] was grown in LB medium (1% peptone, 0.5% yeast extract, 0.5% NaCl) supplemented with ampicillin (50 mg/liter) when required. S. cerevisiae strains OL1 (Mata, leu2-3, leu2-112, his3-11, his3-15, ura3-251, ura3-337) (Boy-Marcotte and Jacquet, 1982) and AN1.7D (Mata, leu2-3, leu2-112, trp1, ura3-52, sec18-1ts) (Nieto et al., 1993) were used. Yeast cells were selected and grown in synthetic minimal YNB medium containing 0.7% yeast nitrogen base without amino acids (YNB, Difco) and 2% of either glucose (MM medium) or galactose (MMG medium) plus 50 mg/liter of the required amino acids or bases according to the auxotrophic markers. In some experiments, where indicated, tunicamycin (10 mg/liter) was added. [F2,

Purified cell walls were obtained as previously described (Pastor et al., 1984; Valentı´n et al., 1984) except that intact cells were broken with glass beads by shaking in a vortex mixer (eight periods of 1 min each). Cell breakage, practically completed, was assessed by examination of the preparations in a phase-contrast microscope.

Solubilization of Wall Proteins and Preparation of Membraneous and Cytosolic Fractions Conditions for solubilization of cell wall proteins with SDS or Zymolyase 20T have been described (Pastor et al., 1984; Valentı´n et al., 1984). Spent media, membraneous and cytosolic fractions were obtained as previously described for Candida albicans (Marcilla et al., 1993).

Plasmids and Transformation

Western Analysis

Plasmids listed in Table 1 were constructed using standard techniques (Sambrook et al., 1989). E. coli

The different fractions (spent media, cytosolic, membraneous, sodium dodecyl sulfate (SDS)2-released and Zymoly-

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Expression of YWP1 by S. cerevisiae

ase-released materials) obtained from S. cerevisiae transformants were run on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970) using 12% (w/v) acrylamide gels. Blotting to Hybond-C nitrocellulose filters (Amersham) and immunodetection with rabbit antibodies against GST-YWP1 fusion protein (Ywp1pAbs) were carried out as previously described (Ramo´n et al., 1996; Towbin et al., 1979).

TABLE 1 Plasmids Used Designation YEplac181 YEplac181ACT

pEMBLyex4

RESULTS Construction and Selection of Expression Plasmids We cloned and sequenced the YWP1 gene, which encodes a protein with an apparent molecular mass of 70 kDa that is expressed only in mycelial cells of Y. lipolytica. This protein is covalently bound to the glucan of the cell wall since it is not released from the cell wall by SDS extraction but by Zymolyase digestion (Ramo´n et al., 1996). One of our initial purposes was to express YWP1 in S. cerevisiae and analyze whether processing and localization in the cell wall of the encoded protein were similar to that in Y. lipolytica. For this purpose we first cloned YWP1 cDNA from pYAE33 in two different multicopy plasmids: pEMBLyex4 with the GAL/CYC galactose-regulated promoter and YEplac181ACT, which carries the ACT actin constitutive promoter, resulting in pYAE34CG and pYAE34ACT, respectively (Table 1). When S. cerevisiae OL1 was transformed with pYAE34CG and grown in MMG medium, a transcript of approximately 1.2 kb was detected by Northern analysis using DIG-labeled YWP1 cDNA as a probe (Fig. 1). However, two transcripts greater than 2.5 kb were discovered when pYAE34ACT was used (Fig. 1). The difference observed in the size of both transcripts could be due to the fact that YWP1 cDNA does not contain the consensus sequences for transcriptional termination, and although in pYAE34CG the plasmid has the ‘‘termination’’ sequences of the CYC1 gene, this does not occur in pYAE34ACT and hence a longer transcript appeared. We also constructed two plasmids, pYAE35BB and pYAE35ES (Table 1), containing the Y. lipolytica YWP1 gene including the 58 noncoding promoter sequences (806 and 1406 bp, respectively) in order to determine whether the YWP1 gene was expressed by S. cerevisiae under the control of its own promoter. Nevertheless the YWP1 gene was not expressed (data not shown), indicating that factors

pYAE33

pYAE33-1

pYAE34ACT

pYAE33EC

pExCell pYAE34CG

pYAE35BB

pYAE35ES

Description LEU2 and 2-µm plasmid from S. cerevisiae Actin promoter from S. cerevisiae at the SmaI/ BamHI site of YEplac181 URA3, LEU2, 2-µm plasmid, GAL/CYC promoter from S. cerevisiae Y. lipolytica YWP1 gene cDNA at the EcoRV of pGEM Y. lipolytica 4.8-kb Sau3AI fragment containing YWP1 gene at the BamHI site of pINA240 0.95-kb PstI/SphI fragment from pYAE33 at the PstI/ SphI site of YEplac181ACT 0.95-kb EcoRI fragment from pYAE33 at the EcoRI site of pExCell AmpR 0.95-kb XhoI/BamHI fragment from pYAE33EC at the SalI/BamHI site of pEMBLyex4 2.15-kb BamHI fragment from pYAE33-1 at the BamHI site of YEplac181 3.20-kb EcoRI/SacI fragment from pYAE33-1 at the EcoRI/SacI site of YEplac181

Source or Reference Gietz and Sugino (1988) Randez-Gilet al. (1995) Cesareni and Murray (1987) Ramo´n et al. (1996)

Ramo´n et al. (1996)

This work

This work

Pharmacia This work

This work

This work

that regulate YWP1 gene expression in mycelial cells of Y. lipolytica are not present in S. cerevisiae or they are inactive under the growth conditions. Because pYAE35BB and pYAE35ES were not active in YWP1 gene expression, pYAE34CG and pYAE34ACT were used.

Ywp1 Is Incorporated in the Cell Wall of S. cerevisiae Initially the ability of the pYAE34CG and pYAE34ACT clones to express the YWP1 gene in MM and MMG media was assayed. No differences were observed in the extent of growth of different clones in either medium (data not shown). To analyze whether Ywp1 was incorporated into the cell wall of S. cerevisiae, cells of S. cerevisiae OL1 transformed

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with pYAE34CG or pYAE34ACT were grown in MMG or MM medium, respectively. Cells were harvested and the spent media collected, the cells broken, and the cytoplasmic and membrane preparations collected. Isolated cell walls were then treated with SDS or Zymolyase. All samples were analyzed by SDS-PAGE followed by immunoblot detection using Ywp1pAbs (Ramo´n et al., 1996). As a control, SDS and Zymolyase cell wall extracts from S. cerevisiae OL1 transformed with pEMBLyex4 or YEplac181ACT were included. A main band with an apparent molecular mass of 70 kDa together with a band of 81 kDa was detected in the material released by SDS from the cell walls (Fig. 2, lanes 2 and 4). No reactive bands were observed, either in the controls and Zymolyasereleased cell wall materials or in the cytoplasmic, membrane preparation or spent media (Fig. 2, lanes 5 to 8). Since Ywp1 from Y. lipolytica has an apparent molecular mass of 70 kDa and Ywp1pAbs recognized in transformed S. cerevisiae cell wall a band of apparent 70 kDa molecular mass too, we assumed that this molecular species is Ywp1. While in Y. lipolytica Ywp1 is covalently bound to the glucan cell wall (being a Zymolyase-released molecule), it is not in S. cerevisiae (it is a SDS-released molecule). This fact could indicate that recognition or anchoring sequences of Ywp1 for binding to glucan or the enzymatic activities

Ramo´n et al.

FIG. 2. Western blot analysis of the spent media and different cell wall extracts from S. cerevisiae transformants. 20 µg of proteins was run in SDS-PAGE, transferred to nitrocellulose filters, and incubated with Ywp1pAbs. (A) SDS-released cell wall material from pEMBLyex4 (lane 1), pYAE34CG (lane 2), YEplac181ACT (lane 3), and pYAE34ACT (lane 4) transformants. (B) Zymolyase-released material, cytoplasmic and membrane fractions, and medium supernatants from the same transformants as in (A) (lanes 5 to 8). ST, molecular weight markers. Proteins with an apparent molecular mass of 70 kDa (Ywp1) are marked with arrowheads.

implicated are different in Y. lipolytica and S. cerevisiae. Another possibility is that Ywp1 cannot be covalently attached because the receptors in the cell wall are missing or cryptic. The fact that an additional band of apparent 81 kDa molecular mass was recognized by Ywp1pAbs could be explained by some kind of covalent interaction with other molecules, although the precise nature of the phenomenon is at present unknown. Because the results using pYAE34CG or pYAE34ACT constructs were similar we decided to use pYAE34CG for further studies.

The Processing of Ywp1 Is Similar in S. cerevisiae and Y. lipolytica FIG. 1. Northern blot analysis of Y. lipolytica and S. cerevisiae transformants. 25 µg of different total RNA samples was electrophoresed through an agarose–formaldehyde gel and transferred to nylon membranes. The blotted membranes were hybridized with a DIG-YWP1 cDNA labeled probe. The positions of the rRNAs (18S and 28S) are indicated. (A) Total RNAs from the Y. lipolytica yeast-like form (lane 1) and mycelial form (lane 2). (B) Total RNAs from pEMBLyex4 (lane 3) and pYAE34CG (lane 4) S. cerevisiae transformants. (C) Total RNAs from YEplac181ACT (lane 5) and pYAE34ACT (lane 6) S. cerevisiae transformants.

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Ywp1 is an integral cell wall protein of the mycelial form of Y. lipolytica that has, in its original native form, an N-terminal region with characteristics of a signal peptide and a possible N-glycosylation signal although it is not N-glycosylated (Ramo´n et al., 1996). To find out whether these signals were recognized by S. cerevisiae, and hence Ywp1 processed as in Y. lipolytica, a S. cerevisiae sec18 mutant was transformed with pYAE34CG. The clones expressing the YWP1 gene were detected using DIG-


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labeled YWP1 cDNA as a probe after Northern blot analysis. As expected a transcript of 1.2 kb was detected (Fig. 3A). Cells of the selected clone expressing the YWP1 gene were grown at room temperature for 12 h and then shifted to 37°C for 30 min in order to express the sec mutation. Later, membraneous and cytosolic fractions were obtained and analyzed by SDS-PAGE followed by immunoblotting with Ywp1pAbs. As a control an SDS cell wall extract from the mutant transformed was included. A main band of 70 kDa was detected in the membraneous fraction and in the SDS cell wall extract, but not in the cytosolic one (Fig. 3B), indicating that the signal peptide of Ywp1 was recognized by the machinery that mediates cotranslational translocation into the endoplasmic reticulum in S. cerevisiae. Two minor bands of apparent 81 and 86 kDa molecular masses were detected in the membraneous fraction and SDS cell wall extract. The nature of these two bands remains unknown but some kind of covalent interaction with S. cerevisiae proteins could be contemplated. To study whether Ywp1 was N-glycosylated by S. cerevisiae, a culture of the S. cerevisiae sec18 mutant transformed with pYAE34CG was treated with tunicamycin for 30 min at room temperature and then grown at 37°C for an additional 30 min. The membraneous fraction was then obtained and processed as above. As shown in Fig. 4A

FIG. 4. (A) Immunodetection by Western blot of Ywp1 in the membraneous fraction of S. cerevisiae AN1.7D transformed with pYAE34CG and treated (1) or not (2) with 10 µg/ml of tunicamycin. (B) The same filter stripped and incubated with concanavalin A conjugated with peroxidase.

there was no modification in the apparent molecular mass of the bands recognized by Ywp1pAbs, indicating that Ywp1 was not N-glycosylated in S. cerevisiae. To verify that tunicamycin treatment was correct, the same filter was stripped and incubated with concanavalin-A conjugated to peroxidase. Different band patterns were detected in cultures treated with the antibiotic and in the control (Fig. 4B), indicating that the tunicamycin treatment had been effective.

DISCUSSION

FIG. 3. Expression of the YWP1 gene in S. cerevisiae AN1.7D (sec18 mutant). (A) Northern blot analysis of S. cerevisiae AN1.7D transformed with pEMBLyex4 (lane 1) or pYAE34CG (lane 2). 25 µg of total RNA was processed as in Fig. 1. (B) Immunodetection by Western blot of Ywp1 in S. cerevisiae AN1.7D. S, SDS-released cell wall material from cells grown at room temperature. M, Membraneous fraction and C, cytosolic fraction, from cells grown for 30 min at 37°C.

We have previously cloned a gene (YWP1) which is expressed only in mycelial cells of Y. lipolytica. This gene encodes a protein covalently bound to the cell wall glucan, and with an apparent molecular mass of 70 kDa (Ramo´n et al., 1996). In order to know Ywp1 processing and localization in the cell wall of other yeast, we transformed S. cerevisiae with different plasmids containing the YWP1 gene. Transcription of this gene, under the control of its own upstream promoter sequences (plasmids pYAE35BB and pYAE35ES), did not take place in S. cerevisiae transformants. Although this could mean that factors regulating differential gene expression in Y. lipolytica, at least for the YWP1 gene, are not present (or active) in S. cerevisiae, the precise mechanism is as yet unknown. Other genes from Y. lipolytica have been cloned and

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expressed in S. cerevisiae under control of their own promoter, for instance the URA5 gene (Sa´nchez et al., 1995). As the YWP1 gene alone was not transcribed in S. cerevisiae, other plasmids were constructed containing the YWP1 gene cDNA under control of different promoters: GAL/CYC (inducible by galactose) in plasmid pYAE34CG and ACT (constitutive actin promoter) in pYAE34ACT. When S. cerevisiae was transformed with pYAE34CG, a transcript of around 1.2 kb was detected by Northern blot analysis (as in Y. lipolytica), while when using pYAE34ACT a transcript greater than 3.4 kb was found. This could be due to the fact that YWP1 cDNA did not have the signals for transcriptional termination in pYAE34ACT. This difference in transcription did not affect the translation product because in both cases S. cerevisiae transformed with either pYAE34CG or pYAE34ACT; the proteins detected by Western blot were identical. Surprisingly, Ywp1pAbs recognized two bands, a main one with an apparent molecular mass of 70 kDa and a minor one of 81 kDa, only in the SDS-released material from the cell wall. In Y. lipolytica Ywp1 has an apparent molecular mass of 70 kDa, so we can assume that the main band of 70 kDa recognized by Ywp1pAbs in transformed S. cerevisiae is Ywp1. In Y. lipolytica, Ywp1 was covalently bound to the glucan of the cell wall, but in transformed S. cerevisiae it was not, being an SDS-released molecule. The original protein showed a mobility equivalent to a 70-kDa protein, and it was suggested that this behavior is inherent to Ywp1 as the high content in proline, serine, and threonine increase the apparent size of proteins in the Laemmli gel system (Aldovini et al., 1986; Rivero-Lezcano et al., 1995). To determine whether the signals (N-glycosylation and signal peptide) present in Ywp1 were similar in S. cerevisiae, a sec18 mutant was transformed with pYAE34CG. The ability of the S. cerevisiae sec18 mutant to accumulate secreted proteins at the endoplasmic reticulum has permitted us to demonstrate that Ywp1 was translocated into membranes and secreted to the cell wall without any modifications. Using tunicamycin we have seen that, as in the case of Y. lipolytica, Ywp1 is not N-glycosylated in S. cerevisiae. The fact that Ywp1 is released by SDS from the S. cerevisiae cell wall, whereas it is solubilized by Zymolyase from Y. lipolytica cell walls, hints that the recognition or anchoring sequences required for binding a protein to the cell wall glucans is different, at least for Ywp1, in both species. Another possibility is that the glucan receptors are missing or cryptic. In S. cerevisiae the C-terminal half of the a-agglutinin (very rich in serine and threonine) is

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Ramo´n et al.

responsible following GPI modification (see below) for its anchoring in the cell wall (Leidich et al., 1995; Schreuder et al., 1993; Vossen et al., 1997). Recently it has been proposed that the GPI anchor is added to cell wall proteins before secretion and that liberation from the anchor would produce a periplasmic intermediate that subsequently would be linked to the cell wall glucan via a transglycosylation reaction (Lu et al., 1994; De Nobel and Lipke, 1994). This type of anchor has been reported to be present in the a-agglutinin (Lu et al., 1995) and other cell wall proteins. In the case of Ywp1 the situation is apparently different, as it lacks the canonical signals for GPI anchor addition. Nevertheless it is linked covalently to the b-(1,3)-glucan, as it is released only following b-(1,3)-glucanase (Zymolyase) digestion. We know nothing about the signals for anchoring Ywp1 in the wall of Y. lipolytica, and further studies with modified versions of YWP1 are needed to learn about cell wall signaling in Y. lipolytica.

ACKNOWLEDGMENTS We thank Dr. P. Sanz for the plasmid YEplacACT and Dr. A. Nieto for S. cerevisiae AN1.7D. We are especially grateful to I. Rogla´ and E. Cebolla for their patient and expert technical assistance. A.M.R. is supported by a predoctoral fellowship from the Conselleria de Cultura, Educacio´ i Ciencia de la Generalitat Valenciana, Spain. S.M. is the recipient of a predoctoral fellowship from the Ministerio de Educacio´n y Ciencia, Spain. This work was partially supported by grants from Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (PB93-0051); Fondo de Investigacio´n Sanitaria de la Seguridad Social (95/1602), Spain; and BMH4-CT96-0310, Brussels.

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1997 FGB  

1997 Fungal Genetics and Biology