Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7,CH-8092 Zürich, Switzerland1
Department of Molecular Biology and Genetics, University of Guelph, Guelph, ON, N1G 2WO Canada2
Author for correspondence: Ursula Kües. Tel: +41 1 632 4925. Fax: +41 1 632 1148. e-mail: kues{at}micro.biol.ethz.ch
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ABSTRACT |
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Keywords: basidiomycete, Coprinus, fruiting body, galectin, secretion
Abbreviations: ECM, extracellular matrix
The GenBank accession number for the sequence reported in this paper is AF130360
a Present address: Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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INTRODUCTION |
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In the initial stage of fruiting body (basidiocarp) development, hyphae form localized, highly branched structures termed hyphal knots (Matthews & Niederpruem, 1972 ; reviewed by Kües, 2000
) (Fig. 1
). Without a light stimulus, hyphal knots develop into globose structures, called sclerotia. These long-term survival structures are hyphal aggregates consisting of an inner medulla and a melanized rind (Moore & Jirjis, 1976
; Moore, 1981
; Clémençon, 1997
; Kües et al., 1998
) (Fig. 1
). When exposed to light, hyphal knots develop into a compact hyphal mass, termed an initial (reviewed by Moore et al., 1979
; Kües, 2000
) (Fig. 1
). The hyphae of the initial eventually differentiate to form a primordium that is essentially an embryonic fruiting body. At this stage, structures composing the stipe and cap (pileus) of the immature fruiting body are clearly discernible (for reviews see Clémençon, 1997
; Moore, 1995
, 1998
) (Fig. 1
).
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Studies of the biosynthesis and architecture of the fungal cell wall, in particular of the cell wall of the ascomycete Saccharomyces cerevisiae, revealed that the outermost layer of the wall is composed of oligosaccharides covalently linked to asparagine, serine or threonine residues of cell-wall proteins (mannans) (reviewed by Kapteyn et al., 1999 ). Higher fungi can have more complex type oligosaccharides (xylomannan and galactomannan) at their surface (reviewed by Gooday, 1995
). This suggests that hyphal interactions might be mediated by lectins, a family of oligosaccharide-binding proteins. Indeed, many basidiomycetes produce low-molecular-mass lectins (see reviews by Guillot & Konska, 1997
; Wang et al., 1998
) and two lectins, Cgl1 and Cgl2, isolated from fruiting bodies of the basidiomycete C. cinereus, were partially characterized (Charlton et al., 1992
; Cooper et al., 1997
). These lectins show binding specificity towards ß-galactosides and do not share sequence homology with any other known fungal lectin. Surprisingly, the Cgl lectins were found to be homologous to the family of galectins, and were the first galectins identified outside the animal kingdom (Cooper et al., 1997
). Galectins, or S-type lectins, specifically bind ß-galactoside sugars in a calcium-independent manner and share sequence homology within the carbohydrate-recognition domain. These criteria differentiate the galectins from all other lectins (see review by Barondes et al., 1994
). In animals, galectins are involved in many different cellular processes such as muscle differentiation, olfactory development, embryo implantation, metastasis, apoptosis and mRNA splicing (Barondes et al., 1994
). Mammalian galectin-1 and galectin-3 were shown to be secreted and interact with components of the ECM (Cooper & Barondes, 1990
; Sato et al., 1993
). They lack typical secretory signal sequences and are secreted via non-classical secretory pathways (Sato et al., 1993
; Cleves et al., 1996
; reviewed by Hughes, 1999
).
The presence of galectin proteins in fruiting bodies of C. cinereus prompted us to speculate that galectins are involved in cellular aggregation, not only in the animal kingdom but also in fungi. To address a potential role of galectins in fruiting body development, we defined more closely the initial developmental phases of fruiting body formation, then analysed the expression and localization of these proteins during this process. We show that expression of the galectins is highly regulated during fruiting body development and can be correlated to external signals that control this process. Importantly, the fungal galectins are ECM proteins that are secreted by a pathway independent of the normal secretory pathway.
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METHODS |
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Cosmid isolation and plasmid construction.
The galectin region encompassing cgl1 and cgl2 was isolated from a cosmid library of homokaryon AmutBmut (Bottoli et al., 1999 ). A 7068 bp BamHI fragment of galectin cosmid 27A9 harbouring both cgl1 and cgl2 genomic DNA was subcloned into pBluescript KS- (Stratagene) and sequenced completely on both strands (Microsynth). Fragments of a HindIII digest containing all of cgl1 and most of the cgl2 sequences (Fig. 2
) were also subcloned into pBluescript KS- to yield plasmids pBCG1 and pBCG2, respectively.
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Isolation of galectins and generation of antibodies.
Galectins were isolated from fruiting bodies of homokaryon AmutBmut. Lyophilized fungal tissue was ground with a mortar and pestle and the resulting powder was resuspended in 50 mM Tris/HCl pH 7·0, 150 mM NaCl. This suspension was centrifuged at 10000 g to remove particulate material. The protein concentration of the supernatant was determined with a Bradford assay (Bio-Rad) and adjusted to 1 mg ml-1. The sample was applied to an affinity column composed of lactosyl-Sepharose (Pharmacia) with a bed volume of 5 ml at a flow rate of 1 ml min-1. Following complete elution of the flow-through peak, a 0·1 M lactose solution was applied to the column and the lectins eluted from the column were collected, dialysed and concentrated by lyophilization. The pure galectins were sent to ReadySysteme AG where
-galectin (
-Cgl) antibodies were raised in rabbits.
Detection of galectins by Western blotting.
Protein was extracted from lyophilized mycelium or tissue as outlined above. Five microgrammes of total saline-soluble protein for each sample were separated on a 15% SDS-PAGE gel (Laemmli, 1970 ) using the Protean II xi cell (Bio-Rad). Proteins were transferred to Immobilon P membranes (Millipore) in 48 mM Tris/HCl (pH 9·2), 39 mM glycine, 0·0375% SDS, 20% methanol) using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). The membrane was incubated for 20 min in TBS-T [20 mM Tris/HCl, pH 7·5, 0·5 M NaCl, 0·1% (w/w) Tween-80] containing 10% (w/v) dry milk powder at room temperature. Binding of the
-galectin antibodies was performed for 16 h in TBS-T plus 1% (w/v) dry milk powder using a 1:3000 dilution of the serum. The membrane was washed three times in TBS-T and then incubated in TBS-T plus 1% (w/v) dry milk powder and Protein A coupled to horseradish peroxidase (125 ng ml-1; Sigma). Horseradish peroxidase was detected by ECL chemiluminescence (Amersham Life Science).
Determination of mRNA levels by RT-PCR.
Total RNA was isolated from powdered, lyophilized Coprinus tissues by a guanidinium isothiocyanate procedure (Chomczynski & Sacchi, 1987 ) and genomic DNA of homokaryon AmutBmut was obtained by the method of Zolan & Pukkila (1986)
. The cDNA templates were generated using a DNase I and cDNA synthesis kit (Gibco-BRL) starting with 2 µg total RNA and an oligo-poly(dT)15 primer. An equal portion of each cDNA as well as 10 ng samples of pBCG1, pBCG2 and total genomic DNA of C. cinereus homokaryon AmutBmut were added to 50 µl PCR reaction mixtures [20 mM Tris/HCl pH 8·3, 1·5 mM MgCl2, 15 mM each dNTP and 1 U Taq polymerase (MBI Fermentas)]. The cgl1- (CGL1F, 5'-ACAGCAGGACCAAGGGT-3'; CGL1R, 5'-GTACCGACAGCTAGCAAGCA-3'), cgl2- (CGL2F, 5'-AACAGCAGGCTCAAGAAC-3'; CGL2R, 5'-AATATGTTGGTGTGGCT-3') and ß-tubulin- [ß-tub; (Matsuo et al., 1999
)] (ß-tubF, 5'-CTCGTCTCCACTTCTTCATG-3'; ß-tubR, 5'-CGTCCTGGTATTGCTGGTACTCAGC-3') specific primers all flanked introns and were used at a final concentration of 0·5 µM. The reaction mixtures were subjected to 1 cycle of 5 min at 95 °C, 2 min at 50 °C and 2 min at 72 °C, followed by 29 cycles of 30 s at 95 °C, 30 s at 50 °C and 2 min at 72 °C. Aliquots of the samples were then separated on 2% agarose gels.
Immunolocalization of galectin protein.
For light microscopy, small 1 to 2 µm thick hand-cut sections of fungal tissue were fixed for 2 h at 4 °C in 20 mM Tris/HCl (pH 7·2), 0·5 M NaCl, 4% (v/v) paraformaldehyde. Following fixation, the samples were dehydrated in a series of ethanol washes and then washed with xylene. The samples were embedded in Paraplast Plus (Sigma) overnight in a 1:1 solution of wax/xylene. The following day the wax/xylene mixture was melted at 60 °C and replaced with fresh molten wax. The samples were kept at 60 °C for 1 h and then the wax was replaced with fresh molten wax. After repeating this last step two more times, the samples were placed into blocks containing fresh molten wax and allowed to harden. The embedded samples were trimmed and 58 µm sections were cut from the samples (Leica sectioner), and the ribbon of sections was placed onto a water droplet on slides coated with polylysine (Sigma) and incubated at 42 °C until the sections had stretched. The water was removed from the slides and the samples were incubated at 42 °C overnight to firmly affix the samples to the polylysine-coated slides. The paraffin was removed from the sections through a series of xylene washes and the samples were rehydrated through a decreasing series of ethanol washes. Once rehydrated, the samples were blocked for 20 min in 10% milk powder in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl, 0·5% (w/w) Tween-80 (blocking buffer). The -Cgl antibodies and pre-immune serum were incubated on the sections in blocking buffer for 2 h at room temperature. The sections were rinsed in blocking buffer and then incubated with the secondary antibody (rhodamine-conjugated goat anti-rabbit antibody) in blocking buffer for 1 h in the dark. The sections were rinsed in blocking buffer and then in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl, 0·5% Tween-80 and a drop of antifade reagent (Bio-Rad) was placed on each of the slides. The immunofluorescence was observed using a Zeiss Axiophot Photomicroscope with a Zeiss filter set 15.
For electron microscopy, tissue samples were fixed in 2·5% formaldehyde/0·5% glutaraldehyde in 0·1 M cacodylate buffer. The samples were dehydrated in an increasing series of ethanol washes and then embedded in LR white resin (Sigma). The embedded samples were trimmed and sectioning was performed on a Reichert-Jung Ultracut E microtome with a diamond knife (Diatome). Ultrathin sections were transferred to 200 mesh carbon coated copper grids (Marivac). To immunolabel the sections, they were equilibrated in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl, 0·5% Tween-80, then blocked in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl, 0·5% Tween-80, 0·2% (v/v) Tween-20, 0·2% (w/v) glycine, 2% BSA (TBS/TG/BSA) and then washed in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl, 0·5%, 0·2% (v/v) Tween-20, 0·2% (w/v) glycine (TBS/TG). The sections were incubated in -Cgl antibodies (1/100 dilution) or pre-immune serum (1/100 dilution) in TBS/TG/BSA for 16 h. The sections were washed in TBS/TG and then incubated in gold-conjugated protein A (in TBS/TG/BSA). After washing the grids in TBS/TG, then TBS, bound antibodies were fixed to the sections for 5 min in 3% (v/v) glutaraldehyde, 2% formaldehyde (v/v) in 25 mM Tris/HCl (pH 8·0). The sections were washed in water, air-dried, stained in saturated uranyl acetate in 70% ethanol for 15 min and washed in 70% ethanol and water. The grids were allowed to air dry and were viewed with a Hitachi electron microscope.
Immunoprecipitations and low-pH extraction of yeast cell-wall associated proteins.
Yeast cells were grown to stationary phase in plasmid-selective minimal medium. Cells (2·5 OD546 units) were harvested by centrifugation, resuspended in 0·5 ml minimal medium and incubated at either 25 °C or 37 °C for 30 min (to set the sec18ts block). Trans35S label [250 µCi (9250 kBq); ICN Radiochemicals] and protease inhibitors (1 µg chymotrypsin ml-1, 1 µg pepstatin ml-1 and 1 mM PMSF final concentration) were added to the cells. Following 30 min incubation at the appropriate temperature with shaking, the labelling reactions were stopped by the addition of an excess of non-radioactive methionine and cysteine. After a 1·5 h chase, the cells were pelleted, washed in 10 mM NaN3, resuspended in low-pH extraction buffer (50 mM sodium citrate, pH 3·7, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF) and incubated at 37 °C for 45 min. The cells were vortexed for 5 s and the extracted proteins were separated from the intact cells by centrifugation at 10000 g for 10 s. The supernatant was centrifuged at 10000 g for 1 min to ensure that it was free of cells and proteins were precipitated by adding a one-tenth volume of 100% TCA. After centrifugation, both the cells and TCA precipitated samples were resuspended in 200 µl 50 mM Tris/HCl pH 7·5, 1% SDS. The cells were lysed by vortexing for 1 min with glass beads. Both the total cell extracts and the low-pH extracted cell-wall proteins were boiled for 3 min. Half of the total cell extract was used for immunoprecipitation with a mixture of -Cgl and
-hexokinase antibodies, the other half with
-CPY antibodies. The cell-wall extracts were incubated with a mixture of
-Cgl and
-hexokinase antibodies. Immunoprecipitations, SDS-PAGE and fluorography were carried out as previously described (Franzusoff et al., 1991
; te Heesen et al., 1992
).
-Cgl and
-hexokinase antibodies were used at a 1:500 dilution while the
-CPY antibodies were used at a 1:100 dilution.
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RESULTS |
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The protein-coding sequences of the two galectin genes were not interrupted by introns, but two introns containing the typical Coprinus 5'- and 3'- splice site and branch-acceptor sequences (Seitz et al., 1996 ) were identified in the 5' and the 3' untranslated regions (Fig. 2b
). The removal of each intron from the primary RNA transcripts was verified by RT-PCR using specific primers flanking each of the introns (data not shown for the 5' introns, see below for evidence of splicing of the 3' introns).
Differential regulation of cgl1 and cgl2 expression during fruiting body development
To assess the level of galectin proteins during fungal development, we generated -Cgl antibodies against galectin protein purified from fruiting bodies of strain AmutBmut. This serum recognized the two galectin proteins in Western blot experiments, and it was possible to separate the faster moving Cgl1 from Cgl2 by SDS-PAGE (Cooper et al., 1997
) (Fig. 3c
, lane 1). These antibodies made it possible to examine Cgl1 and Cgl2 expression during fruiting body development of the homokaryotic strain AmutBmut.
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To determine the expression of galectins in the mature fruiting body, different tissues of this structure were examined (Fig. 3c). The basidiocarp is composed of two major parts, the stipe (stalk) and the pileus (cap). Present on the surface of the pileus is a fluffy protective tissue called the veil. Under the veil tissue of the pileus is a rigid layer of cells called the pileus-cortex that supports the gill tissue containing the trama, subhymenium and hymenium (Clémençon, 1997
). Both Cgl1 and Cgl2 were observed in the different tissues and both were present in relatively equal quantities, with the highest levels in the veil cells and the lowest levels in the gill tissue (Fig. 3c
).
These data suggest that the two galectin genes are differentially expressed during fruiting body development: Cgl2 expression was correlated with the formation of hyphal knots and was also found in developing fruiting bodies, whereas Cgl1 was found exclusively in fruiting mycelium and was present at high levels in the tissue of fruiting bodies.
Differential regulation of cgl1 and cgl2 expression by environmental signals
Our data suggested that expression of the galectin proteins correlated with the induction of differentiation processes in the mycelium. Since differentiation of the mycelium is a response to external stimuli, we modulated the developmental processes by specific growth conditions. Thereby, it was possible to define more precisely the signals required for fruiting body development and to monitor the expression of the two galectin genes.
Recent results showed that hyphal-knot formation is repressed by blue light (Kües et al., 1998 ) (Fig. 1
). When kept in constant dark, fully grown cultures develop hyphal knots as a response to nutritional depletion. If exposed to light at 25 °C, hyphal knots can develop into fruiting body initials (Fig. 1
). If maintained in darkness at 37 °C, these hyphal knots will differentiate into sclerotia (Fig. 1
). When cultures were grown in continuous light at 37 °C, hyphal-knot development was repressed. Hence, neither sclerotia nor fruiting bodies are formed on such cultures (Kües et al., 1998
). We followed the expression of galectins in cultures grown at 37 °C either in continuous dark or continuous light (Fig. 4
). Galectin expression was observed only in cultures grown in continuous darkness, whereas continuous light repressed induction of both hyphal-knot formation and galectin expression. Galectin expression in dark-grown cultures coincided with the appearance of hyphal knots on the cultures (Fig. 4
). With increasing age, the number of hyphal knots per culture increased, levelling off after 8 d incubation. By day 8, sclerotia were starting to develop from the hyphal knots, and by day 10 most hyphal knots had developed into the light-brown immature and dark-brown mature sclerotia. In the dark-grown cultures, Cgl2 was the predominant galectin detected, while only very low levels of Cgl1 were observed in the oldest cultures containing mature sclerotia. We analysed transcript levels of cgl1 and cgl2 by RT-PCR using cgl1- and cgl2-specific primers flanking the 3' introns (Fig. 4b
). This analysis indicated that cgl2 was expressed in all cultures that formed knots and sclerotia, while cgl1 was only weakly expressed in the older cultures with mature sclerotia, but isolated sclerotia did not contain detectable levels of Cgl1 and Cgl2 protein (data not shown). In cultures grown in continuous light neither cgl1 nor cgl2 transcripts were detected (Fig. 4b
).
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Localization of galectins within fruiting body tissues
Galectin expression was initiated by the induction of hyphal-knot formation, but galectins are most abundant in fruiting body tissues. To determine the localization of the galectins more precisely, an immunohistochemical approach was taken. A horizontal section through a developing cap (in a stage shortly after karyogamy) is shown in Fig. 6. Galectin labelling was observed throughout the fruiting body, within most tissues of the stipe and pileus. However, there were large differences in the level of labelling between some of the different tissues or cell types. The highest level of expression observed was within a group of cells that form the outer portion of the stipe, a region called the lipsanoblem (Clémençon, 1997
), and strong labelling extended into the primary gills. Another region of intense labelling was along the veil cells of the pileus. These highly vacuolated cells showed high levels of labelling along the surface of the cells. Moderate labelling was observed in the central hyphae of the stipe, and also on the hyphae of the pileus-cortex. The lowest level of labelling was observed on the basidia present along the hymenium. All of the staining observed appeared to be present at the surface and in between the hyphae.
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Cell-wall proteins from stationary-phase yeast cells harbouring a cgl2 expression vector were extracted by incubating the cells in low-pH (pH 3·7) buffer at 37 °C. With this very mild extraction procedure, it was possible to solubilize Cgl2 without releasing the highly expressed cytoplasmic protein hexokinase (Fig. 8a). We concluded that Cgl2 was secreted when expressed in S. cerevisiae and remained attached to, or trapped within, the cell wall. Based on the time course of extraction shown in Fig. 8(a)
, we chose 45 min as an optimal extraction time for further experiments.
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DISCUSSION |
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Although the expression of cgl2 was regulated by the same factors that regulate hyphal-knot development, and the protein was localized to the hyphal knots (data not shown), we do not know whether Cgl2 protein is required for hyphal-knot formation. We have isolated mutant strains that form hyphal knots but are unable to enter the fruiting body-specific differentiation pathway (Y. Liu, M. Aebi & U. Kües, unpublished) and some of the mutant strains do not express galectins. These mutant strains indicate that Cgl2 expression might not be necessary for hyphal-knot formation. However, all mutant strains unable to form fruiting body primordia were also deficient in the expression of Cgl1 protein, suggesting that this protein is involved in fruiting body formation. Specific knock-out experiments are in progress to determine whether the two proteins are essential for the developmental processes.
Cellular and subcellular localization of the galectins
Within the fruiting bodies of Coprinus the galectins were localized throughout most tissues, but primarily within the veil and the lipsanoblem (Fig. 6). These two tissues are distinct, yet both are subjected to enormous levels of stress during fruiting body development. During early stages of primordial development the veil tissue covers the entire embryonic fruiting body. As the fruiting body grows, the veil tissue is stretched and eventually torn. The only remnants of the veil in the mature fruiting body is a sparse web-like tissue present on the surface of the pileus (Clémençon, 1997
). Another tension is generated on the lipsanoblem of the stipe due to cap expansion. As shown in Fig. 6
, this tissue is directly connected to the primary gills and thereby provides an anchor required during expansion of the cap mediated by tangential growth of the meristemoid tissue of the cap (Reijnders, 1979
; for review see Moore, 1998
). The same tissue of the stipe is also involved in the process of rapid stipe elongation (see review by Kamada, 1994
) where a strong hyphal adherence is essential for stability of the stipe. It is tempting to speculate about the function of the two galectin proteins. The localization of the proteins in the ECM suggests that they are involved in hyphalhyphal interactions, mediated by their ability to bind specific carbohydrates of the cell wall. Homodimer formation, a prerequisite of this hypothesis, has been demonstrated for the Cgl2 protein (Cooper et al., 1997
). The tension applied on the outer stipe and cap tissues requires an increased connectivity of the hyphae forming them. The high level of galectins in the extracellular matrix of the lipsanoblem suggests that they contribute to a strong hyphalhyphal interaction.
Secretion of the galectins
Immunolocalization as well as the expression of Cgl2 protein in yeast demonstrate that the Coprinus galectins are secreted, yet both Cgl1 and Cgl2 lack the hydrophobic N termini typical of proteins secreted by the classical secretory pathway. In addition, the fungal galectins are not post-translationally modified (Cooper et al., 1997 ). In animals, galectin-1 and galectin-3 are secreted independently of the classical secretory pathway (Cooper & Barondes, 1990
; Sato et al., 1993
; Mehul & Hughes, 1997
; reviewed by Hughes, 1999
). Our analysis of Cgl2 secretion in yeast suggests that as found for human galectin-1 (Cleves et al., 1996
), the export does not occur via the classical Sec18p-dependent pathway. It has been suggested that alternate routes of secretion maintain the separation of ligands from their receptors until a proper contextual environment is reached outside the cell (Bejcek et al., 1989
; Dunbar et al., 1989
). Since all glycosylated derivatives emerge from the endoplasmic reticulum/Golgi, the alternate mechanism of secretion of mammalian galectin-1 has been postulated to prevent intracellular binding of galectin-1 to its galactose receptor (Cooper et al., 1991
) and to provide a means for developmental modulation of secretion by external factors present in the extracellular matrix (Hughes, 1999
). In mammalian systems, other proteins known to be secreted by non-classical mechanisms include basic fibroblast growth factor (bFGF) (Yu et al., 1993
), interleukin-1ß [IL-1ß (Rubartelli et al., 1990
)] and thioredoxin (Rubartelli et al., 1992
). Both bFGF and IL-1ß are present within cytoplasmic vesicles, and in the case of bFGF, these vesicles have been shown to fuse with the plasma membrane, discharging bFGF from the cell (Qu et al., 1998
). Our subcellular immunolocalization experiments indicated that the Coprinus galectins were also localized within cytoplasmic vesicles and in darkly staining aggregates associated with the cell membrane (Fig. 7
). As is the case with bFGF and IL-1ß, it is possible that the fungal galectins are secreted via cytoplasmic vesicles.
In filamentous fungi, classical protein secretion occurs predominantly at the growing hyphal tip (Wösten et al., 1991 ; Moukha et al., 1993
). The finding that the Coprinus galectins are capable of being secreted via a non-classical route indicates that galectin secretion may not be limited to the hyphal tip. Such a system would enable the fungus to modulate its entire hyphal surface at specific periods during development. If galectins play a role in hyphalhyphal interactions in the fruiting body, this may be an important mechanism to consider, because fungal morphogenesis is often achieved by rapid growth of cells already present within the tissue and not by cellular division (see the reviews by Gooday, 1975
; Kamada, 1994
; Moore, 1998
). Thus, hyphal growth occurs along the lateral walls of the hyphae and not at the tip. During such growth, cell-wall biogenesis occurs equally over the cell-wall surface (reviewed by Gooday, 1975
, 1982
). Non-classical secretion might provide means of delivering large amounts of cell-wall components (including galectins) to the ECM, independent of hyphal-tip growth.
Celerin et al. (1997) identified fungal collagens (fungal fimbriae), indicating that components of the ECM evolved prior to the divergence of animals and fungi. Our localization of the fungal galectins to the ECM strengthens this point and shows that two characteristic proteins of the animal ECM also exist in fungi. Such conservation of the components of the ECM suggests that the interactions present between the ECM are also conserved. In animal systems, galectins bind certain collagens (Warfield et al., 1997
; Ochieng et al., 1998
; Sasaki et al., 1998
). The fungal collagens have glyco-proteinaceous subunits (Castle et al., 1992
) and homo-logues have been identified in C. cinereus (Castle & Boulianne, 1991
). It will be interesting to see if their animal counterparts, the fungal galectins, interact with fungal collagen in the ECM of the fruiting body.
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ACKNOWLEDGEMENTS |
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Received 23 February 2000;
accepted 9 May 2000.