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Eukaryotic Cell, November 2005, p. 1872-1881, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1872-1881.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Comparison of Cell Wall Localization among Pir Family Proteins and Functional Dissection of the Region Required for Cell Wall Binding and Bud Scar Recruitment of Pir1p

Toru Sumita, Takehiko Yoko-o, Yoh-ichi Shimma, and Yoshifumi Jigami*

Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, Tsukuba, Ibaraki 305-8566, Japan

Received 16 August 2005/ Accepted 29 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1{Delta} cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars. Stepwise deletion of the eight units of the repetitive sequence of Pir1p revealed that one unit is enough for the protein to bind bud scars and that the extent of binding of Pir1p to the cell wall depends on the number of these repetitive units. The localization of a chimeric Pir1p in which the repetitive sequence of Pir1p was replaced with that of Pir4p revealed the functional role of the different protein regions, specifically, that the repetitive sequence is required for binding to the cell wall and that the C-terminal sequence is needed for recruitment to bud scars. This is the first report that bud scars contain proteins like Pir1p as internal components.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fungal cells are surrounded by a thick cell wall, which is a tough but flexible structure that protects the cell from physical and chemical environmental factors, including pressure, various shocks, nutrients, and toxins. In the budding yeast Saccharomyces cerevisiae, the cell wall consists of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannose-containing glycoproteins (mannoproteins), together with a small amount of lipids (18). The cell wall proteins (CWPs) are mainly classified into two groups: covalently linked and noncovalently linked to the cell wall (37). The covalently linked cell wall proteins are further classified into glycosylphosphatidylinositol (GPI)-dependent CWPs (GPI-CWPs) and GPI-independent alkali-sensitive linkage CWPs (9, 17, 18, 29). GPI-CWPs have a GPI anchor at their carboxyl termini (C termini) and are immobilized on the cell wall by linking to ß-1,6-glucan, which is further coupled to ß-1,3-glucan (17, 21).

Pir proteins are the main alkali-sensitive linkage CWPs (9). Pir proteins are not modified with a GPI anchor and are directly bound to ß-1,3-glucan in the cell wall by an unknown linkage (9, 18). All Pir proteins have one (Pir4p) or several units (Pir1p, Pir2p, and Pir3p) of an internal repetitive sequence. This repetitive sequence consists of 18 to 19 amino acid residues at the amino terminus (N terminus) and is the origin of the name Pir (protein with internal repeats) (27, 42). However, the function of these repetitive sequences is currently unclear.

Genes homologous to S. cerevisiae PIR have been found in several yeasts, such as Kluyveromyces lactis, Zygosaccharomyces rouxii (42), Candida albicans (16, 26), and Yarrowia lipolytica (15), but they have not been found in the fission yeast Schizosaccharomyces pombe, suggesting that PIR genes play unique roles in budding yeasts. Although a lack of PIR genes renders cells sensitive to heat stress (42) and to tobacco osmotin (46), the function of Pir proteins in the cell wall remains unknown. Functional differences in PIR family genes have not been elucidated, although the disruption and expression profile of each PIR gene has been examined extensively (10, 22, 30, 38, 40, 42).

How the Pir proteins are bound to the cell wall is also unclear. Because Pir proteins can be liberated from the cell wall by mild alkaline extraction and because they are highly O glycosylated, it is thought that they are retained through an O-glycosidic linkage to ß-1,3-glucan (9, 19, 29). It has also been reported that Pir proteins are bound to the cell wall by disulfide bridges because they can be released by a reducing agent such as ß-mercaptoethanol or dithiothreitol (15, 28, 31). Furthermore, Castillo et al. reported that the repetitive sequence of Pir4p is necessary for its binding to the cell wall (5). These results suggest that Pir proteins may be assembled into the cell wall in several different and complex ways.

The purpose of the present study was to elucidate the functions of Pir proteins and the mechanism of their translocation to the cell wall. In this report, we show that the distribution of each Pir protein is different, and we focus on the unique localization of Pir1p at the cell wall. Further investigation revealed that Pir1p is localized inside the chitin ring of bud scars, which remain at the surface of the mother cell after completion of the budding process. Deletion of the repetitive sequence of Pir1p revealed that the downstream region is actually responsible for the recruitment of Pir1p to bud scars. Furthermore, using stepwise deletion, we found that the strength of the linkage between Pir1p and the cell wall depends on the number of repetitive sequence units.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains, growth conditions, and genetic methods. The yeast strains used in this study are listed in Table 1. We used S. cerevisiae strain MATa type W303-1A (MATa leu2-3,112 his3-11 ade2-1 ura3-1 trp1-1 can1-100; called JHY5) (41) as a wild-type strain. MAT{alpha} type haploid cells (JHY6) were used to make diploid cells. PIR1, PIR2, PIR3, and PIR4 were disrupted by LEU2, S. pombe his5+, TRP1, and URA3, respectively. The pir1{Delta} (JTS1), pir2{Delta} (JTS2), pir3{Delta} (JTS3), and pir4{Delta} (JTS4) strains were generated as follows. DNA fragments to disrupt PIR1, PIR2, PIR3, and PIR4 were amplified by PCR using primers PIR1-F1/PIR1-R1, PIR2-F1/PIR2-R1, PIR3-F1/PIR3-R1, and PIR4-F1/PIR4-R1 (Table 2) in combination with pFA6a-LEU2 (J. Horecka, unpublished data), pFA6a-His3MX6, pFA6a-TRP1 (24), and pFA6a-URA3 (J. Horecka, unpublished data) as templates, respectively. The resulting DNA fragments were introduced into W303-1A to create JTS1, JTS2, JTS3, and JTS4, respectively. The compositions of YPD and SD media were as described previously (36), although the YPD medium in the present studies was supplemented with 20 µg/ml adenine sulfate and is referred to as YPAD medium. YPAD and SD media were used to cultivate yeast cells and to select yeast recombinant transformants, respectively. When necessary, SD medium was supplemented with amino acids, adenine sulfate, and uracil. All yeast cells were grown at 30°C. Yeast transformation was carried out by the lithium acetate method (20).


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  		TABLE 1. Yeast strains used in this study

 

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  		TABLE 2. Synthetic oligonucleotides used for gene deletion, cloning, and tagging

 
Escherichia coli strain JM109 {recA1 endA1 gyrA96 thi-1 hsdR17(rK–mK–) e14–(mcrA–) supE44 relA1 {Delta}(lac-proAB) [F' traD36 proAB+ lacIq lacZ{Delta}M15]} was used for plasmid preparations and subcloning.

Visualization of Pir proteins and bud scars. To visualize Pir1p, Pir2p, Pir3p, and Pir4p, a gene encoding monomeric red fluorescent protein (mRFP) (4) was fused with PIR1, PIR2, PIR3, and PIR4, respectively. PIR1 and PIR4 open reading frames (ORFs) were fused with the mRFP gene at their 3' ends (see Fig. 1A and 6A), and PIR2 and PIR3 ORFs were fused with the mRFP gene downstream of the site recognized by Kex2p protease (see Fig. 5A and 6A). A BamHI site was created upstream of the stop codon of PIR1 and downstream of the Kex2p cleavage site of PIR2 and PIR3. The mRFP gene, which has a BamHI site both upstream of the start codon and at the end of the ORF without the stop codon, was introduced in frame into the above BamHI sites. Similarly, a SpeI site was created upstream of the stop codon of PIR4. The mRFP gene, which has a SpeI site both upstream of the start codon and at the end of the ORF without the stop codon, was introduced in frame into the above SpeI site. PIR1-mRFP, mRFP-PIR2, and PIR4-mRFP fusion genes were expressed under the control of their own promoters on pRS304 in JTS1, JTS2, and JTS4, respectively. The mRFP-PIR3 fusion gene was expressed under the control of PIR3 promoter on pRS305 in JTS3. The resulting plasmids (pRS304-PIR1-mRFP, pRS304-mRFP-PIR2, pRS305-mRFP-PIR3, and pRS304-PIR4-mRFP) were linearized and introduced into JTS1, JTS2, JTS3, and JTS4 to create JTS1-1m, JTS2-m2, JTS3-m3, and JTS4-4m, respectively.



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  		FIG. 1. (A) PIR1-mRFP fusion gene. Shown are the N-terminal region cleaved by Kex2p protease after translation (hatched box), the cleavage site recognized by Kex2p protease (triangle), eight units of the repetitive sequence (shaded boxes), and the mRFP gene (black box). (B) The position of bud scars stained with CFW is identical to that of Pir1p. The Pir1p-mRFP fusion protein was expressed under the control of the PIR1 promoter in JTS1. The images were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 


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  		FIG. 6. (A) mRFP-PIR3 and PIR4-mRFP fusion genes. Shown are the N-terminal region cleaved by Kex2p protease after translation (hatched box), the cleavage site recognized by Kex2p protease (triangle), the eight units (Pir3p) or one unit (Pir4p) of the repetitive sequence (shaded boxes), and the mRFP gene (black box). (B) Localization of mRFP-Pir3p. The mRFP-Pir3p fusion protein was expressed under the control of the PIR3 promoter in JTS3. (C) Localization of Pir4p-mRFP. The Pir4p-mRFP fusion protein was expressed under the control of PIR4 promoter in JTS4. The images shown in panels B and C were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 


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  		FIG. 5. (A) mRFP-PIR2 fusion gene. Shown are the N-terminal region cleaved by Kex2p protease after translation (hatched box), the cleavage site recognized by Kex2p protease (triangle), the 11 units of the repetitive sequence (shaded boxes), and the mRFP gene (black box). (B) Pir2p was expressed both on the cell surface (upper left panel) and at the bud scars (upper right panel). Both upper panels show the same cells expressing mRFP-Pir2p on different focal planes. The chitin rings of bud scars were stained with FITC-WGA. The mRFP-Pir2p fusion protein was expressed under the control of the PIR2 promoter in JTS2. The images were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 
Pir1p-mRFP in diploid cells was expressed as follows. The PIR1 of strain JHY6 was disrupted with S. pombe his5+ to generate JTS1{alpha}. pRS306 carrying the PIR1-mRFP gene was linearized by cleavage with EcoRV and introduced into JTS1{alpha} for chromosome integration at the URA3 locus. The resulting strain was mated with JTS1-1m to generate strain JTSD-1m.

Bud scars were stained with calcofluor white (CFW; Sigma-Aldrich, St. Louis, MO) at a final concentration of 1 µg/ml for 10 min or with fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA, lectin from Triticum vulgaris; Sigma-Aldrich, St. Louis, MO) (FITC-WGA) (32) at a final concentration of 40 µg/ml for 10 min.

Gene disruption. BNI1, SPA2, PEA2, AXL2, BUD3, BUD8, RGA1, BEM1, BEM2, VMA6, APN1, CHS2, and CHS3 in strain JTS1-1m were disrupted with S. pombe his5+. The disruption cassette was PCR amplified using synthetic oligonucleotides as primers (Table 2) and pFA6a-His3MX6 as a template. For example, the BNI1 disruption cassette was PCR amplified using the primers BNI1-F1 and BNI1-R1. Each resulting DNA fragment was introduced into JTS1-1m.

Microscopic observation. Yeast cells were cultivated in 2 ml of YPAD medium until the late exponential phase at 30°C. They were then centrifuged (2,500 x g, room temperature, 5 min), washed twice, and suspended in 500 µl of phosphate-buffered saline buffer (10 mM sodium phosphate and 150 mM sodium chloride, pH 7.4). For three-dimensional (3-D) observation, the cells were suspended in 500 µl of phosphate-buffered saline containing 0.1% agarose.

We used a BX50 fluorescence microscope (Olympus, Tokyo, Japan) and MicroMAX cooled charge-coupled device (CCD) camera (Roper Scientific, Duluth, GA) for observation of staining. For 3-D observation, we used an IX71 fluorescence microscope (Olympus) with a confocal scanner, a piezo-actuated prototype 3-D system (Yokogawa Electric, Tokyo, Japan), and a high-gain avalanche rushing amorphous photoconductor camera system (Hitachi Kokusai Electric and NHK, Tokyo, Japan). We also used an EVM285SPD cooled CCD camera (Texas Instruments, Dallas, TX) instead of a high-gain avalanche rushing amorphous photoconductor camera for enlarged images of the bud scars. The photographic images were analyzed using Adobe Photoshop, version 5.0.

Deletion of the repetitive sequence of PIR1. The repetitive sequence of PIR1 was deleted stepwise by PCR as follows. A BamHI site was created downstream of the Kex2p cleavage site of PIR1 on pUC118 to generate pUC-PIR1B. Several regions of the PIR1 ORF were PCR amplified using primers PIR1-A, PIR1-B, PIR1-C, PIR1-D, PIR1-E, and PIR1-F in combination with primer PIR1-2550 (Table 2). Each amplified DNA fragment was digested with BamHI and ClaI and then ligated into the BamHI-ClaI region of pUC-PIR1B. Each resulting plasmid was digested with BglII and ClaI and ligated into the BglII-ClaI region of pRS304-PIR1-mRFP. These plasmids were linearized by EcoRV digestion and introduced into JTS1 for chromosome integration at the TRP1 locus. These transformants contain 5, 4, 3, 2, 1, and 0 repetitive sequence units and were named JTS1-A, JTS1-B, JTS1-C, JTS1-D, JTS1-E, and JTS1-F, respectively (see Fig. 7A).



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  		FIG. 7. (A) Stepwise deletion of the repetitive sequence of Pir1p. Shown are the N-terminal region cleaved by Kex2p protease (hatched box), the cleavage site recognized by Kex2p protease (triangle), the repetitive units (shaded boxes), and the mRFP gene (black box). The circles indicate N glycosylation sites. (B) Localization of deleted versions of Pir1p-mRFP (left panels) and CFW staining (right panels). The images were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 
Expression of the Pir4/Pir1 chimeric protein. The repetitive sequence of PIR4 and the downstream region of the repetitive sequence of PIR1 were fused together. PIR4-RepA and PIR4-RepB (Table 2) were chemically synthesized, annealed, phosphorylated with T4 polynucleotide kinase, and inserted into the BamHI-SmaI site of pBluescript II SK+ (pBS) to generate pBS-PIR4Rep. The insert of this plasmid contains the repetitive sequence of PIR4, which has an HpaI site at its 3' end. The downstream region of the repetitive sequence of PIR1 was PCR amplified using primers PIR1-HpaI and M13 with pUC-PIR1B as a template. The amplified fragment was digested with HpaI and HindIII and inserted into the HpaI-HindIII site on pBS-PIR4Rep. The resulting plasmid was digested with BamHI and ClaI, and the resulting fragment containing the repetitive sequence of PIR4 was ligated into the BamHI-ClaI region on pUC-PIR1B. The plasmid thus generated was digested with BglII and ClaI and then ligated into the BglII-ClaI region of PIR1-mRFP on pRS304. This chimeric gene was expressed under the control of PIR1 promoter in JTS1 and was referred to as JTS1-41 (see Fig. 9A).



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  		FIG. 9. (A) A chimeric Pir1p containing the repetitive sequence of Pir4p instead of the Pir1p repetitive sequence (Pir4/Pir1 chimeric protein) was produced under the expression of the PIR1 promoter. Shown are the N-terminal region of Pir1p cleaved by Kex2p protease (hatched box), the cleavage sites recognized by Kex2p protease (triangle), the Pir4p repetitive sequence (shaded box), and the mRFP gene (black box). (B) The Pir4/Pir1 chimeric protein fused with mRFP was localized to bud scars (middle panel). The chitin ring was stained with CFW (left panel), and a phase-contrast view of cells is shown (right panel). The images were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 
Preparation of Pir1p-mRFP. Pir1p-mRFP fusion proteins were directly prepared from whole cells by a mild alkaline treatment. The cells were cultivated overnight in 2 ml of YPAD medium, after which 300 µl of the suspension was seeded in 15 ml of fresh YPAD medium and further cultivated for 6 h until the cell wet weight was 10 mg/ml. The cells were centrifuged and washed three times. The fusion proteins were extracted by treating the cells overnight at 4°C with 1 ml of 30 mM NaOH. The cells were centrifuged, and proteins in the supernatants were recovered by trichloroacetic acid precipitation. The proteins released into the medium during cultivation were collected overnight at 4°C by adding 111 µl of 100% (wt/vol) trichloroacetic acid to 1 ml of the medium, followed by centrifugation (13,000 x g, 4°C, 20 min) to precipitate the protein.

Western blotting. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 5 to 20% gradient gel (Atto, Tokyo, Japan) and then transferred to a Hybond-P polyvinylidene difluoride membrane (Amersham, Piscataway, NJ). Pir1p-mRFP and the truncated derivatives were detected with an immunopurified polyclonal antibody against mRFP followed by horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham). The polyclonal antibody against mRFP was obtained from TaKaRa Co., Ltd. (Tokyo, Japan). The C-terminal region of the mRFP protein (Glu212-Ala225) was chemically synthesized and coupled to keyhole limpet hemocyanin through the cysteine residue of the peptide. This conjugate was injected into a rabbit to obtain an antipeptide antibody, which was purified by sequential chromatographic steps on protein A and peptide columns. Immunoreactive bands were visualized using the ECL plus Western blotting detection system (Amersham) and with a LAS-1000 luminescence image analyzer (Fuji Photo Film, Tokyo, Japan).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pir1p localizes inside the chitin rings of bud scars. Although Pir1p had been isolated as a cell wall protein, the exact localization of Pir1p at the cell wall had not been reported. We constructed a Pir1p fusion protein tagged with mRFP at the C terminus to monitor the cellular localization (Fig. 1A). We confirmed that Pir1p was expressed on the cell surface. Interestingly, Pir1p was visualized as one or several spots on the cell surface. A similar result was obtained when Pir1p was tagged with mRFP at its N terminus (data not shown), indicating that Pir1p localizes in clusters. A more precise observation revealed that some spots of Pir1p take the form of a ring-like structure (Fig. 1B).

Based on this result, we expected that Pir1p is localized at bud scars. Because the bud scar is mainly composed of chitin, we confirmed this by staining the cells with CFW, which specifically binds chitin (Fig. 1B). As expected, the positions of the Pir1p spot and of the bud scar were very similar, clearly indicating that Pir1p is localized at bud scars.

To investigate the precise localization of Pir1p around the chitin ring of bud scars, we observed the cells with a confocal microscope connected with cooled CCD camera system and then enlarged the image of the bud scars using digital imaging software. Merging of the chitin ring and Pir1p localization revealed that they did not overlap; the ring structure of Pir1p is obviously located inside the chitin ring (Fig. 2A). We obtained similar results using FITC-WGA (lectin from Triticum vulgaris) instead of CFW to stain the chitin ring (Fig. 2B). These results indicate that Pir1p localizes inside the chitin ring of bud scars.



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  		FIG. 2. Pir1p localizes inside the chitin ring. The chitin rings of bud scars were stained with CFW (A) or FITC-WGA (B). The Pir1p-mRFP fusion protein was expressed under the control of the PIR1 promoter in JTS1. The images were taken using an IX71 fluorescence microscope with a confocal scanner and an EVM285SPD cooled CCD camera.

 
We further observed the chitin ring of bud scars and Pir1p-mRFP stereoscopically by confocal fluorescent microscopy. The 3-D observation indicates not only that the ring structure of Pir1p is located inside the chitin ring but also that not all bud scars contain Pir1p (see the supplemental material at http://unit.aist.go.jp/rcg/rcg-gb/pir1.html). Because the ring structure of Pir1p-mRFP cannot be observed at the budding site before cell separation is completed, this result suggests that Pir1p localizes inside the chitin ring of bud scars after cell separation has completed.

The budding pattern of diploid cells is different from that of haploid cells (8). To further investigate the localization of Pir1p, Pir1p-mRFP was expressed in diploid cells. PIR1 was disrupted in JHY6 (MAT{alpha} type) cells, and the fusion gene encoding Pir1p-mRFP was introduced. After mating of this strain and JTS1-1m, we observed the distribution of Pir1p in the resulting diploid. As shown in Fig. 3, Pir1p localized inside the chitin ring of bud scars in diploid cells as expected. A similar result was obtained when the chitin ring was stained with FITC-WGA instead of CFW (data not shown). These findings confirm that Pir1p is expressed at bud scars both in haploid and diploid cells.



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  		FIG. 3. Pir1p in diploid cells also localizes inside the bud scars. The chitin rings of bud scars were stained with CFW. The Pir1p-mRFP fusion protein was expressed under the control of the PIR1 promoter in the PIR1 disruptant (JTSD-1m). The images were taken using an IX71 fluorescence microscope with a confocal scanner and an EVM285SPD cooled CCD camera.

 
Translocation of Pir1p to the budding site is not affected in chs2{Delta} and chs3{Delta} cells. Next, we investigated the relationship between Pir1p and chitin. Before mother and daughter cells separate, chitin synthase III (CSIII) catalyzes the synthesis of the chitin ring at the base of an emerging bud. The primary septum is then formed by CSII, which is the first step in the division of these cells. After the primary septum is completed, secondary septa are laid down from the mother and daughter cells. Cell separation is facilitated by a partial digestion of the primary septum by a chitinase, and bud scars are the remains of the septum formed during these processes (see reference 3 for a review). CHS3 encodes the putative catalytic subunit of CSIII, and CHS3 mutants do not exhibit a ring structure at bud emergence (35). CHS2 encodes CSII, which is specific for primary septum formation (35). The chs2 mutants also exhibit an abnormal pattern of separation due to the lack of the primary septum, but the chitin ring at bud emergence is formed normally (34, 35). If Pir1p recognizes the primary septum or the chitin ring, Pir1p-mRFP is expected to be invisible in chs2{Delta} and chs3{Delta} cells, respectively. Therefore, we investigated the localization of Pir1p both in chs2{Delta} and chs3{Delta} cells. The ring structure of Pir1p-mRFP was observed at the chitin rings in chs2{Delta} cells (Fig. 4A). In contrast, although Pir1p-mRFP was not detected as a ring structure, it was still observed as a cluster at the budding site in chs3{Delta} cells (Fig. 4B), suggesting that Pir1p is transported to bud scars but cannot form a ring structure because of the lack of a chitin ring. This result indicates that Pir1p is transported to the budding site and is located there even when the primary septum formation or the chitin ring is not normal.



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  		FIG. 4. Pir1p is localized at the budding site both in chs2{Delta} cells (A) and in chs3{Delta} cells (B). The Pir1p-mRFP fusion protein was expressed under the control of the PIR1 promoter in pir1{Delta}chs2{Delta} and pir1{Delta}chs3{Delta} cells, respectively. The ring structure of Pir1p-mRFP was not observed in chs3{Delta} cells due to the lack of a chitin ring. The chitin rings of bud scars were stained with CFW. The images were taken with a BX50 fluorescence microscope and a MicroMAX cooled CCD camera.

 
Pir2p also localizes at bud scars. Next, we investigated the localization of Pir2p. Pir2p was fused with mRFP at its N terminus (mRFP-Pir2p) (Fig. 5A). The fusion site was placed downstream of the site cleaved by Kex2p because Pir2p fused with mRFP at its C terminus (Pir2p-mRFP) was not detected at the cell surface, probably due to degradation of the fusion protein (data not shown). The fusion protein mRFP-Pir2p was expressed on the cell surface, but some of it was concentrated into a unique circle in the cell wall, suggesting that Pir2p is also present in bud scars. As shown in Fig. 5B, the ring structures were observed in cells expressing mRFP-Pir2p, confirming the localization of Pir2p at bud scars.

We also investigated the distribution of Pir3p and Pir4p in detail. Pir3p and Pir4p were fused with mRFP at the N and C terminus, respectively (Fig. 6A). As a result, both proteins were uniformly expressed on the cell surface irrespective of bud scars (Fig. 6B and 6C). The 3-D imaging of mRFP-Pir3p and Pir4p-mRFP revealed that these proteins are distributed on the cell surface almost uniformly and are not likely to be related to bud scars (data not shown).

Disruption of genes involved in bud site selection or cell polarity does not affect the localization of Pir1p. The budding site in haploid cells has been reported to be different from that in diploid cells. The a and {alpha} haploid cells bud with an axial pattern, choosing new bud sites adjacent to the previous site of bud emergence, while a/{alpha} diploid cells bud in a bipolar pattern, choosing new bud sites at either end of the cell (8). The position where a new bud emerges on the cell surface is determined by several factors. Mutations in BUD3, BUD4, AXL1, or AXL2/BUD10 cause a defect in the axial budding pattern in haploid cells, but they have no effect on the bipolar budding pattern of diploid cells (6, 7, 11, 12, 33). In contrast, mutations in BNI1, SPA2, PEA2, BUD6, BUD7, BUD8, or BUD9 in diploid cells cause a defect in the bipolar budding pattern but have no effect on the axial budding pattern (2, 13, 43, 47). Therefore, it is possible that the disruption of these genes may affect the localization of Pir1p at bud scars both in haploid and diploid cells. Consequently, we individually disrupted BNI1, SPA2, PEA2, AXL2, BUD3, and BUD8 in strain JTS1-1m, which produces mRFP-tagged Pir1p. In addition, we disrupted several genes related to cell polarity: RGA1, a gene encoding a GTPase-activating protein for the polarity-establishment protein Cdc42p (39); BEM1, a gene involved in normal bud emergence and morphogenesis (23); and BEM2, a gene encoding a Rho GTPase-activating protein involved in the control of the cytoskeletal organization and cellular morphogenesis (25, 45). However, disruption of these genes had no effect on the localization of Pir1p (data not shown). The bud10{Delta} cells, for example, exhibited a bipolar pattern of bud site selection even in haploid cells, but Pir1p was observed at the incorrectly positioned bud scars. These results suggested that Pir1p localizes to bud scars independent of cell polarity and bud site selection.

Next, we disrupted two genes that reportedly associate with Pir1p: VMA6, which encodes vacuolar ATPase V0 domain subunit d (14); and APN1, which encodes a key enzyme in the base excision repair pathway in the nucleus and mitochondria (44). However, neither gene disruption had any effect on the localization of Pir1p (data not shown).

Repetitive sequence of Pir1p is essential for its binding to cell wall. The repetitive sequence of Pir1p consists of eight tandem units of 18 to 19 amino acids. To investigate the relationship between this repetitive sequence and the localization to bud scars, we removed these units stepwise from the N terminus. We created six types of Pir1p derivatives: Pir1p-A, Pir1p-B, Pir1p-C, Pir1p-D, Pir1p-E, and Pir1p-F. These proteins contain five, four, three, two, one, and no repetitive sequence units, respectively (Fig. 7A), and each was fused with mRFP at its C terminus. As shown in Fig. 7B, Pir1p-A, Pir1p-B, Pir1p-C, Pir1p-D, and Pir1p-E were localized at bud scars, but Pir1p-F was not detected anywhere on the cell surface.

Pir1p-F, which does not contain any repetitive sequence units, may not bind to the cell wall but may instead be released into the culture medium. Thus, we measured the amount of Pir1p-mRFP or its truncated derivatives that were secreted in the medium. Western blotting revealed that the amount of secreted Pir1p gradually increased as the number of repetitive units decreased (Fig. 8A). These fusion proteins appeared as smears with molecular weights higher than their predicted protein sizes, implying that they are highly glycosylated. Pir1p-E and Pir1p-F were smaller than the other Pir1p-mRFP fusion proteins due to the lack of one N-linked sugar chain (Fig. 7A). These results indicated that the repetitive sequence plays some role in the binding of Pir1p to the cell wall. It is also likely that the Pir1p derivatives containing many repetitive sequence units can bind strongly to the cell wall, whereas the Pir1p derivative containing one repetitive unit has little ability to bind to the cell wall.



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  		FIG. 8. (A) Western blotting to detect the secreted Pir1p-mRFP. Lanes: W, wild type without PIR1-mRFP; Pir1p-mRFP, JTS1-1m; A, JTS1-A; B, JTS1-B; C, JTS1-C; D, JTS1-D; E, JTS1-E; F, JTS1-F. (B) Western blotting to detect the cell wall-localized Pir1p-mRFP released from the strains shown in Fig. 7 by a mild alkali treatment. Lanes: W, wild type without PIR1-mRFP; Pir1p-mRFP, JTS1-1m; A, JTS1-A; B, JTS1-B; C, JTS1-C; D, JTS1-D; E, JTS1-E; F, JTS1-F.

 
Next, we measured the amount of Pir1p on the cell wall. First, we tried to prepare Pir1p-mRFP from the isolated cell wall fractions by alkaline treatment as reported previously (1). However, almost all Pir1p-mRFP fusion proteins were degraded, probably due to the vigorous breaking of yeast cells with glass beads during the preparation of the cell wall fraction. To attempt to avoid the degradation of Pir1p-mRFP, we prepared it from intact whole cells. Yeast cells expressing Pir1p-mRFP or its truncated derivatives Pir1p-A to Pir1p-F were cultivated, collected, washed, and then resuspended in a mild alkali solution. Western blotting confirmed the isolation of Pir1p-mRFP (Fig. 8B). In addition, Pir1p-A, Pir1p-B, Pir1p-C, Pir1p-D, and Pir1p-E were detected, but Pir1p-F was not. Although we were unable to quantify the results shown in Fig. 8A, the amounts of Pir1p-A to Pir1p-E released from the cell wall were obviously higher than the amount of Pir1p-mRFP (Fig. 8B). Because each of these proteins was expressed under its own promoter and the total amount of expressed protein appeared to be identical, this suggested that the derivatives were easily released from the cell wall due to their shorter repetitive sequence. Furthermore, the inability to detect Pir1p-F indicates that little if any of it localizes to the cell wall, which is consistent with our microscopic observations (Fig. 7B).

The C-terminal region of Pir1p is required for its recruitment to bud scars. To investigate whether the repetitive sequence of Pir1p is involved in its recruitment to bud scars, we replaced the repetitive sequence of Pir4p, which distributes uniformly on the cell wall (Fig. 6C), with that of Pir1p, creating the chimeric protein Pir4/Pir1 (Fig. 9A). Pir4p has only one repetitive unit, and it was reported that this sequence is necessary for its binding to the cell wall (5). The chimeric protein with the repetitive sequence of Pir4p followed by the C-terminal region of Pir1p was fused with mRFP at its C terminus (Fig. 9A). As shown in Fig. 9B, this chimeric protein localized to bud scars, indicating that the region downstream from the repetitive sequence of Pir1p, not the repetitive sequence itself, is important for its recruitment to bud scars.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Advantage of 3-D microscopic observation of yeast cells. In the present studies, 3-D superfine fluorescence microscopy allowed the detection of Pir1p in more detail and the collection of more information than two-dimensional fluorescence microscopy. Because all of the Pir-mRFP fusion proteins were expressed under the control of their own promoters and all PIR-mRFP fusion genes were integrated into the chromosome, the fluorescence derived from Pir-mRFP fusion proteins could be used as an indication of the amount of native protein in the cells. Because of this ability of superfine 3-D microscopic systems, they will become increasingly useful for detecting the precise cellular localization of proteins. For example, 3-D fluorescence microscopy allowed us to determine that Pir1p is not always located inside the chitin ring of bud scars (see the supplemental material at http://unit.aist.go.jp/rcg/rcg-gb/pir1.html). This result suggests that Pir1p is localized and functions in the bud scars after cell separation. This is important information for elucidating the function of Pir1p. Furthermore, without 3-D fluorescence microscopic systems, it is quite difficult to detect all bud scars in a whole cell, and it is unlikely that the same results would have been obtained using conventional two-dimensional confocal microscopy. Therefore, it may be interesting to investigate the cellular localization of other proteins with 3-D fluorescence microscopic systems even if their localization has already been reported. However, our current 3-D fluorescence microscopic system still has some limitations. For example, it took more than 1 min to collect the 100 FITC and mRFP confocal images for the view shown in the supplemental material. Therefore, it will be difficult to identify the cellular localization of quickly moving proteins using current 3-D systems. Further improvement of our prototype 3-D fluorescence microscopic system should provide more precise information not only for Pir proteins but also for other proteins of interest.

Function of Pir1p. Although the PIR genes were identified by Toh-e and coworkers in 1993 (42), the function of the Pir proteins and the role of their repetitive sequences have not yet been determined. Here, we reported that Pir1p is localized inside the chitin ring of bud scars. For more than 30 years, the bud scar has been known as a ring-like structure made almost entirely of chitin, but other components of the chitin ring have not been investigated. Pir1p is the first protein shown to be specifically expressed inside bud scars. Pir2p is also located at bud scars, but it exists in other regions of the cell wall as well. Pir3p and Pir4p are uniformly expressed in the entire cell wall and are not localized at bud scars. Moukadiri et al. reported that Pir4p is distributed predominantly on the surface of growing daughter cells (27). They observed Pir4p by using an indirect immunofluorescence method with a polyclonal antibody against Pir4p. The discrepancy in the localization of Pir4p may be due to the differences in the method of visualization.

The difference in cellular localization among Pir family proteins may reflect a difference in their functional positions. Because Pir1p is not localized at the bud neck during the budding process but is present at bud scars after the completion of cell separation, it is conceivable that Pir1p is required for reinforcement of the weakened cell wall following cell budding. If this is the case, Pir3p and Pir4p may have some function that compensates or protects the entire cell wall from the various forms of environmental stress. Pir2p may reinforce the cell wall both at bud scars and in other regions. In fact, the yeast cell compensates for the decreased level of ß-1,6-glucan in its cell wall by upregulating the transcription of Pir (19, 37). In addition, it was reported that a lack of some Pir proteins renders cells sensitive to heat (42) and tobacco osmotin (46). It was also reported that cells with disrupted PIR genes showed growth defects in the presence of CFW or Congo red (30). These results support our hypothesis that Pir proteins maintain the integrity of the cell wall. Because PIR1 and PIR2 are reported to be highly expressed during G1 phase (38), Pir1p and Pir2p may be incorporated in bud scars to reinforce the budding site after the completion of the budding process.

Because Pir1p is the first protein shown to be localized to bud scars, it may have functions other than cell wall reinforcement. For example, it is possible that Pir1p acts as a marker to prevent new budding from a site where budding was just completed. It is also possible that Pir1p expands and/or breaks the chitin ring in aged cells, a process that occurs as the cell grows (32). Further analysis is required to understand the precise role of Pir proteins.

Role of the repetitive sequence of Pir proteins. The repetitive sequence of Pir proteins was first investigated by Castillo et al. using Pir4p, which has only one repetitive unit (5). Evidence was obtained that the repetitive sequence of Pir4p is necessary for its anchoring to the cell wall. Because Pir1p was found to localize to bud scars, we investigated the functional relationship between the repetitive sequence and the localization of Pir1p. The finding that a chimeric protein consisting of the repetitive sequence of Pir4p and the C-terminal region of Pir1p is present at bud scars provided us with an important clue. This result clearly demonstrates that the repetitive sequence of Pir1p is required for the binding to the cell wall and that the C-terminal region is necessary for recruitment to specific regions of the cell wall. Considering the previous results (5), we expect that the repetitive sequence of Pir proteins anchors the proteins to the cell wall. However, it is still unclear how Pir proteins are cross-linked to the cell wall through their repetitive sequences. Their release from cell walls by a mild alkaline treatment suggests that they may be cross-linked through their O-glycans. Thus, it is likely that Pir1p is cross-linked to the cell wall through O-glycans attached to the repetitive sequences.

Mechanism of Pir protein localization to bud scars. How and when Pir1p is translocated to bud scars is unknown. Because Pir1p is not detected around the bud neck and because the bud scars do not always contain Pir1p, we expect that Pir1p translocates to bud scars after the completion of cell separation. The precise localization of Pir1p in bud scars is also unclear. To distinguish whether Pir1p recognizes the primary septum or the chitin ring, we investigated the localization of Pir1p in chs2{Delta} and chs3{Delta} cells because the primary septum and the chitin ring are not formed normally in chs2{Delta} and chs3{Delta} cells, respectively. We first expected that if Pir1p recognizes the primary septum or the chitin ring, Pir1p is not detected at bud scars in chs2{Delta} and chs3{Delta} cells, respectively. However, Pir1p is translocated to bud scars both in chs2{Delta} and chs3{Delta} cells (Fig. 4). The different appearance of Pir1p in chs2{Delta} and chs3{Delta} cells may be due to the different morphology of the bud scars in these mutants. This finding indicates that Pir1p is translocated to the budding site even when septum formation or the chitin ring is not normal. Further investigations, for example by immunoelectron microscopy, are necessary to determine the precise location of Pir1p. This information and determination of the period when Pir1p is localized to bud scars will lead to a better understanding of Pir1p function.

We disrupted several genes involved in bud site selection and cell polarity. As expected, due to the disruption of these genes, budding patterns and cell polarity became abnormal. However, Pir1p still localized at incorrectly positioned bud scars. These results indicate that Pir1p is transported and localized to bud scars independent of the mechanisms of cell polarity and bud site selection. We expect that there is an as-yet-unidentified mechanism for transporting proteins such as Pir1p to bud scars. The truncation of Pir1p indicates that the region responsible for its recruitment to bud scars lies in the C terminus. Combined with the finding that Pir2p is also transported to bud scars, this result indicates that Pir1p and Pir2p may have the same or similar signal sequences in their C termini for transport to bud scars. An alignment of the amino acid sequences downstream from the repetitive sequences of Pir1p to Pir4p is shown in Fig. 10. Although the C-terminal regions of the four Pir proteins are similar, Pir1p and Pir2p have highly conserved sequences that are not conserved in Pir3p and Pir4p. It is likely that the amino acids around this sequence act as a bud scar localization signal, although a more detailed identification of the targeting sequence will be necessary.



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  		FIG. 10. An alignment of the C-terminal regions of Pir1p to Pir4p. The sequences highly conserved in Pir1p and Pir2p but not in Pir3p and Pir4p are indicated by asterisks. The numbers correspond to the amino acid residues of the Kex2p-processed form of each Pir protein.

 


   ACKNOWLEDGMENTS
 
We are grateful to Roger Tsien (Howard Hughes Medical Institute Laboratories, University of California at San Diego) for providing the mRFP gene and to K. Nakayama, Y. Chiba, X. Gao, and H. Abe for helpful discussions.

This work was partly supported by the Japan Society for the Promotion of Science (JSPS) and a grant-in-aid for the DB project by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, Tsukuba, Ibaraki 305-8566, Japan. Phone: 81-29-861-6160. Fax: 81-29-861-6161. E-mail: jigami.yoshi{at}aist.go.jp. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Eukaryotic Cell, November 2005, p. 1872-1881, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1872-1881.2005
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