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Journal of Bacteriology, February 1999, p. 1356-1359, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received 26 October 1998/Accepted 30 November 1998

	ABSTRACT

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We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1 cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.

	TEXT

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Flocculation of Saccharomyces cerevisiae is a nonsexual aggregation of the cells which is calcium dependent and reversible. Flocculation and sedimentation of the cells to the bottom of the fermentor are desirable properties of S. cerevisiae strains used in industrial fermentation. Therefore, the mechanism by which S. cerevisiae cells flocculate has been extensively studied both biochemically and genetically. This phenomenon is thought to involve cell surface components and results from a lectin-like interaction between a cell wall sugar-binding protein and cell-surface mannan (11). Several dominant flocculation genes such as FLO1, FLO5, FLO9, and FLO10 have been defined in S. cerevisiae by classical genetics, and these genes have been cloned and sequenced by several groups (3, 21, 25). These proteins are cell wall-bound proteins with a high degree of homology (22) that are anchored in the cell wall via their C termini, while their N-terminal domains are exposed in the medium and are essential for flocculation (4, 5). Although the N-terminal domain is believed to react with neighboring cell wall mannoprotein, the precise functions of FLO gene products in cell-cell interactions are still unclear.

Heterothallic strains of the fission yeast Schizosaccharomyces pombe, h⁺, and h, are usually nonflocculent when cultured separately. In contrast, cells of the homothallic strains of S. pombe become intensely flocculent (sexual flocculation) after prolonged cultivation. Sexual flocculation by homothallic S. pombe has been extensively studied (6). However, little attention has been given to flocculent mutants from heterothallic strains and to the mechanism of nonsexual flocculation in S. pombe.

This study was performed to elucidate the mechanism of nonsexual flocculation in S. pombe. We have isolated mutants that constitutively flocculate upon growth in yeast extract-peptone-dextrose (YPD) liquid medium. One of the isolated mutants, the gsf1 mutant, causes calcium-dependent flocculation like that of S. cerevisiae FLO mutants. However, the flocculation of the gsf1 strain was not inhibited by the addition of mannose but was prevented by galactose, which is a major component of cell wall galactomannoproteins in S. pombe. Moreover, the gsf1 mutant cells coflocculated with wild-type S. pombe strains but not with the galactose-deficient gms1 mutant. These results indicated that the galactose-specific binding proteins are involved in flocculation in S. pombe. This study is the first to elucidate one of the physiological roles of galactose residues of glycoconjugates in S. pombe.

Isolation and characterization of an S. pombe flocculation mutant. To isolate flocculent mutants of S. pombe, mutagenized cells from the wild-type TP4-1D (h⁺ leu1 his2 ura4 ade6-M216) (14) strain were screened. The procedure used for mutagenesis to isolate flocculation mutants was described previously (1). The haploid S. pombe TP4-1D was grown in synthetic minimal medium (MM), and the cells were harvested and exposed to 2% ethyl methanesulfonate for 3 h at 30°C. Then the cells were washed three times with MM, inoculated into YPD medium and grown overnight in YPD medium at 28°C. The ethyl methanesulfonate-treated cells were resuspended in saline and kept at room temperature for 10 min. Small clusters of agglutinated cells were recovered from the bottom of tubes with a standard 200-µl pipette and spread onto YPD plates. After 4 days at 28°C, each colony was picked up and grown in YPD medium. The flocculent colonies were isolated and further investigated. We isolated three mutants that constitutively flocculated in YPD medium as the cultures entered stationary phase. One mutant (AAD-1), which showed extensive and stable flocculation (Fig. 1B), was then analyzed by standard yeast genetics. The flocculent AAD-1 mutant was crossed with the parent strain (TP4-5A; h leu1 ura4 ade6-M210). The heterologous diploid cells still flocculated on MM-adenine plates, and we therefore concluded that the flocculation activity of the AAD-1 strain was dominant. Tetrad analysis following sporulation of the heterologous diploid was performed to examine the flocculent phenotype. Ten or more tetrads were examined for each cross, and the flocculent trait of the mutant proved to be due to a single mutation, because tetrads uniformly gave rise to two flocculent and two nonflocculent colonies (data not shown). We isolated flocculent strains of both mating types during tetrad analysis, indicating that the flocculent phenotype is not dependent on the mating type.

View larger version (57K): [in this window] [in a new window]   FIG. 1.   Flocculation of AAD-1 (gsf1) cells. (A) Wild-type (TP4-1D) haploid cells showed no significant flocculation and remained in suspension after 48 h of cultivation at 28°C. (B) In contrast, AAD-1 (gsf1) cells flocculated and settled rapidly under the same conditions.

The flocculent AAD-1 mutant was further characterized. Colonies of AAD-1 cells grew well at both 26 and 36°C, and therefore these cells did not exhibit a temperature-sensitive growth defect. The AAD-1 mutant had no obvious mutant phenotype with regard to ion or drug sensitivity or cell morphology. S. cerevisiae Flo1 protein is synthesized at an early stage of growth and appears to be secreted from the cells by the secretory pathway (5). We investigated whether the flocculent phenotype of the AAD-1 strain was caused by the unusual secretion of proteins by comparing the secretion of invertase, which is a typical secreted protein in S. pombe (20). Mutant and wild-type cultures were grown at 28°C to log phase and then transferred to derepression medium for 2 h, and the secreted invertase activity was measured. The invertase activity of AAD-1 cells was indistinguishable from that of wild-type cells (data not shown), indicating that the protein secretion of this mutant was normal.

Effects of cation requirement and pH on flocculation of AAD-1. When the flocculent AAD-1 cells (10⁸ cells/ml) were washed with 10 mM EDTA, flocculation was completely repressed. Deflocculated cells were washed with excess water, and CaCl₂ was added to the cell suspension (final concentration, 10 mM). The AAD-1 cells showed restoration of flocculation activity. Therefore, S. pombe flocculation is calcium dependent, similar to S. cerevisiae. In the S. cerevisiae flocculent FLO1 mutant, only Ca²⁺ was required for maximal flocculation; however, Mg²⁺ and Mn²⁺ partially substituted for Ca²⁺ (11). We examined the cation requirements of S. pombe flocculation by assaying the flocculation of EDTA-treated AAD-1 cells after the addition of various cations (250 mM). We found that flocculation activity was restored by the addition of Mn²⁺, Zn²⁺, Cu²⁺, or Li⁺ as well as Ca²⁺. However, addition of Mg²⁺, Hg²⁺, Sn²⁺, Co²⁺, or Fe²⁺ was not effective. The effects of pH on the flocculation activity were also examined. Flocculation of AAD-1 exhibited a broad pH range and was induced above pH 3.0.

S. pombe flocculin shows galactose-specific lectin-like activity. Flocculation of S. cerevisiae FLO1 mutant cells was inhibited by the addition of mannose, and therefore Flo1 is believed to possess mannose-specific lectin-like activity. To examine whether mannose inhibits the flocculation of AAD-1 cells, 100 mM mannose was added to EDTA-treated AAD-1 cells and flocculation was initiated in the presence of CaCl₂. However, flocculation of AAD-1 cells was not inhibited by the addition of mannose. Surprisingly, this flocculation was completely inhibited by the addition of 100 mM galactose (Fig. 2A). This result suggested that the flocculation of S. pombe is mediated by a galactose-specific recognition mechanism. The inhibitory effect of galactose on AAD-1 mutant cells increased with increasing amounts of galactose added to the solution, reaching a maximum at 25 mM (Fig. 2B). Therefore, we have named this mutant gsf1 for galactose-specific flocculation phenotype.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   (A) Flocculation of gsf1 cells was inhibited by the addition of galactose. Flocculation of EDTA-treated gsf1 cells was initiated by the addition of CaCl2 in the absence (a) or presence of 100 mM mannose (b) or 100 mM galactose (c). (B) Effects of the concentration of related sugars on flocculation of gsf1 cells. Flocculation assays were performed by the standard method except that different amounts of galactose (), methyl--galactose (), lactose (), or mannose () were used, and the optical density at 610 nm was determined after 5 min of settling. The percentage of cells in the supernatant was proportional to the difference in the mean optical density between the experimental tubes and the control tubes without added CaCl2.

The sugar specificity of the sugar-binding activity was examined by adding various mono- and oligosaccharides under flocculation conditions. The flocculation of gsf1 cells was inhibited by the addition of methyl-,-galactose, as well as by the addition of galactose, but not by the addition of galactosamine, N-acetylgalactosamine, or galacturonic acid (Table 1). In addition, flocculation was not inhibited by the addition of sugar alcohols such as galactitol. The flocculation activity was also inhibited by the addition of galactose-containing di- or trisaccharides such as lactose (Gal14Glc), melibiose (Gal16Glc), and raffinose (Gal16Glc12Fru), indicating that the S. pombe gsf1 cells recognize the nonreducing sugars of galactose residues (Table 1).

View this table: [in this window] [in a new window]   TABLE 1.   Effects of various sugars on flocculation of gsf1 cells

Galactose-deficient S. pombe cells were not coflocculated with gsf1 cells. The carbohydrate component of S. pombe glycoproteins consists of galactose in addition to the mannose ubiquitously found in mammalian glycoproteins. Therefore, the physiological role of galactose residues in S. pombe has recently been the focus of intense research interest. We recently isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins, and the galactose content in the gms1 mutant polysaccharides was significantly reduced (19). The gms1⁺ gene encodes a UDP-galactose transporter, and the cell surface glycoproteins of gms1 cells completely lack galactose residues (18). Although the gms1 strain does not contain any galactose residues in the cell wall glycoproteins, this gms1 strain is viable and therefore the role of galactosylation in the cell wall glycoproteins in S. pombe is still unclear (18).

To determine if galactose-containing cell surface glycoproteins are required for flocculation in S. pombe, we tested the coflocculation between the gsf1 and gms1 cells. Mixtures of flocculent gsf1 and nonflocculent cells (wild type or gms1), in a 20:1 ratio, were prepared in 2 ml of succinate buffer (pH 4.0). Flocculation was initiated by the addition of CaCl₂ (final concentration, 50 mM) and agitation at 28°C for 5 h, and the cell clumps were allowed to settle for 5 min. When wild-type cells were mixed with gsf1 cells and allowed to co-flocculate, the supernatant was clear indicating that the wild-type and gsf1 cells were combined into flocs (Fig. 3A). In contrast, very little coflocculation with gms1 cells was observed and the supernatant remained turbid (Fig. 3A). These nonflocculent cells were confirmed to be derived from gms1 cells by microscopic analysis, because the morphology of gms1 cells was different from that of gsf1 cells (data not shown). Therefore, the binding sites of gsf1 cells may be present on the surface of nonflocculent wild-type cells. In contrast, the cell surface of the gms1 strain did not include recognition and binding sites involved in flocculation interactions.

View larger version (79K): [in this window] [in a new window]   FIG. 3.   (A) Coflocculation between gsf1 cells and wild-type (a) or gms1 (b) cells. (B) Flocculation of gsf1 cells was inhibited by the addition of galactomannan from wild-type cells (a) but not by that from gms1 cells (b).

To identify the lectin-like interaction between the gsf1 cells and cell surface galactomannan, we prepared cell surface galactomannans from wild-type and gms1 cells and tested the inhibitory effect on gsf1 flocculation. N-linked polysaccharides of cell surface glycoproteins from wild-type and gms1 mutant cells were prepared as described previously (19). The EDTA-treated gsf1 cells were added to both wild-type and gms1 galactomannans (final concentration, 20 mg/ml), and flocculation was initiated by adding 250 mM CaCl₂. The flocculation of gsf1 cells was inhibited by the addition of wild-type galactomannan, as expected (Fig. 3B). In contrast, the flocculation of gsf1 cells was not inhibited in the presence of gms1 (galacto)mannan (Fig. 3B). These results indicated that gsf1 cells specifically recognize cell surface galactomannan in S. pombe cells, and therefore the flocculation receptors for gsf1 cells were side branches of -linked galactose residues. The detailed roles of mannan structures as flocculation receptors of S. cerevisiae were studied by using glycosylation-defective mutant strains. Very little coflocculation was found with several glycosylation-defective mutants such as mnn2 and mnn5 strains (2), indicating that flocculation ligands of S. cerevisiae are side branches of outer-chain mannan in the yeast cell wall, two or three mannose residues in length (16). The structure of (galacto)mannan from S. pombe gms1 galactose-deficient cells was quite different from that of wild-type cells and consisted exclusively of an unbranched 16 polymannose outer chain which is very similar to that from S. cerevisiae mnn2 mutant cells (18). These results suggested that both S. pombe and S. cerevisiae flocculins specifically recognize the side chains of cell wall glycoconjugates.

Johnson et al. reported that the addition of 50% galactose dispersed nonsexual flocs of wild-type h⁺ or h haploid S. pombe cells (8). This suggested that wild-type S. pombe cells may produce small amounts of galactose-specific flocculin induced by nutrient limitation in the medium. Interestingly, the addition of galactose to the medium suppressed nonsexual flocculation and promoted sexual flocculation (12). Therefore, the mechanisms of sexual and nonsexual flocculation may be regulated by different systems. Recently, the G-protein-coupled receptor kinase homologue gene (prk1⁺) was isolated in S. pombe (26). Disruption of the prk1⁺ gene increased flocculation (26), and the Prk1 protein has significant sequence similarity to the S. cerevisiae Ume5p kinase (17). The UME5 gene is also known as SSN3 (9) and suppresses the snf1 mutant defect in SUC2 derepression (24). The SSN3 null mutation partially relieved glucose repression of SUC2 and also increased flocculation (9). Moreover, several genes involved in glucose repression, such as TUP1 (10), SSN6 (7), and MIG1 (15), were found to play important roles in nonsexual flocculation in S. cerevisiae. These findings strongly suggest that S. pombe flocculation may be controlled by the prk1⁺ gene through catabolite repression, similar to the FLO1 gene from S. cerevisiae (23). We recently found that flocculation of the prk1 mutant cells was also inhibited by the addition of galactose (unpublished results). Isolation and characterization of genes involved in the expression of gsf1⁺ should lead to a better understanding of the molecular mechanisms of flocculation in S. pombe.

	ACKNOWLEDGMENTS

We gratefully acknowledge gifts of the S. pombe strains from Takashi Toda. We thank Chikashi Shimoda, Yuko Giga-Hama, Hideki Tohda, and Shojiro Iwahara for many helpful discussions during the course of this work. We thank Osamu Iwaihara and Atsuko Nakamura for their excellent technical assistance.

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (to K.T.) and by a Sasakawa Scientific Research Grant from The Japan Science Society (to N.T.).

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. Phone: 81 87-891-3116. Fax: 81 87-891-3021. E-mail: takegawa{at}ag.kagawa-u.ac.jp.

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