Angewandte Molekularbiologie, Institut für Mikrobiologie (FR 8.3), Universität des Saarlandes, Gebäude 2, Postfach 151150, D-66041 Saarbrücken, Germany
Correspondence
Manfred J. Schmitt
mjs{at}microbiol.uni-sb.de
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ABSTRACT |
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This paper is dedicated to Professor Dr Ferdinand Radler on the occasion of his 75th birthday.
Present address: Lehrstuhl für Bioinformatik, Biozentrum der Universität Würzburg, D-97074 Würzburg, Germany.
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INTRODUCTION |
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Yeast KRE1 encodes a serine/threonine-rich, highly O-glycosylated protein of the cell surface that is involved in a late stage of the synthesis and/or assembly of yeast cell wall 1,6--D-glucan (Boone et al., 1990
). In a
kre1 null mutant, the total amount of cell wall 1,6-
-glucan is 40 % lower than in the wild-type and the resulting glucan polymer shows a lower degree of polymerization and possesses significantly fewer
-1,3-glycosidic branches. It has been speculated that during its secretion Kre1p is glycosylphospatidylinositol (GPI)-anchored via its C-terminal hydrophobic part, but so far this hypothesis has not clearly been proven. Recently, the membrane-bound form of Kre1p has been shown to function as a plasma membrane receptor for the yeast K1 killer toxin (Breinig et al., 2002
). Here, we extend our previous finding by investigating Kre1p with respect to its function in cell wall assembly and composition. The presented data imply a structural, rather than enzymic, function of yeast Kre1p within cell wall
-1-6-glucan assembly and architecture.
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METHODS |
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The K1 and K28 toxins were prepared as described by Pfeiffer & Radler (1982) and Schmitt & Tipper (1990)
using S. cerevisiae strains T158C and MS300b, respectively, as toxin producer. Zygocin was prepared from the Zygosaccharomyces bailii killer strain 412 essentially as previously described (Radler et al., 1993
; Weiler & Schmitt, 2003
). In brief, the corresponding killer yeast was grown at 20 °C in synthetic B-medium (pH 4·7 for K1 and K28, pH 4·0 for zygocin). After entering the stationary growth phase, cells were pelleted by centrifugation and the cell-free culture supernatant was concentrated 1000-fold by ultrafiltration using a Sartorius ultrafiltration system (SM 16525) and trifluoracetate membranes with a molecular mass cut-off of 10 kDa. The biologically active toxins were filter-sterilized and kept frozen at 20 °C. Toxin activity was determined in a standard killing-zone assay as decribed previously (Breinig et al., 2002
) and toxin activity is expressed in arbitrary units (Schmitt & Tipper, 1990
).
For spheroplast preparation, the appropriate yeast was cultivated at 30 °C in YEPD medium until late exponential growth phase (5x107 cells ml1), harvested and washed twice with sterile water. Cells were resuspended in Tris/HCl buffer (0·1 M Tris/HCl, pH 8·0, 5 mM DTT, 5 mM EDTA), incubated at 30 °C for 30 min and washed twice in 1·2 M sorbitol. Washed cells were finally resuspended in 1·2 M sorbitol containing 0·5 M Na2HPO4 and yeast cell spheroplasts were generated by zymolyase 20T treatment (Schmitt & Tipper, 1990).
Escherichia coli strains, plasmids and DNA manipulations.
Standard molecular manipulations were performed as described by Sambrook et al. (1989). For cloning, E. coli strain DH5
[F recA1 endA1 gyrA96 thi hsdR17 supE44 relA1
(argFlacZYA)U169 (
80dlacZ
M15)
] was used. All Kre1p expression plasmids used in this study are derivatives of the episomal 2µ vector pPGK-M28-I (Schmitt & Tipper, 1995
) in which the entire K28 pptox open reading frame (M28) was replaced by the indicated KRE1 gene fusion and placed under transcriptional control of the constitutive PGK1 promoter (Breinig et al., 2002
).
Extraction of cell wall mannoproteins.
Crude cell wall mannoproteins were extracted using a method according to Nakajima & Ballou (1974a, b)
modified as previously described (Schmitt & Radler, 1987
). Briefly, cells were cultivated at 30 °C in YEPD containing 5 % glucose until late exponential growth phase, harvested and washed three times with sterile water. Cells were resuspended in 20 mM citrate/phosphate buffer (pH 7·0; 1 ml cell suspension per g wet weight of cells) and autoclaved for 90 min at 121 °C. After centrifugation the pellet was resuspended in 20 mM citrate/phosphate buffer and autoclaved again. This procedure was repeated and the supernatants were pooled. After the addition of 3 vols ice-cold ethanol, the obtained crude mannoprotein precipitate was pelleted at 4 °C, resuspended in 70 ml extraction buffer and dialysed overnight against deionized water. The amount of mannoprotein was determined after lyophilization. For partial purification of the crude mannoproteins, a cetavlon fractionation was carried out by the method of Lloyd (1970)
. The crude mannoprotein was dissolved in double-distilled water (25 ml g1) and combined with a solution of 1 g cetavlon (cetyl-N,N,N-trimethyl-ammonium bromide) in 10 ml double-distilled water. After stirring for 1 h at 20 °C, the precipitate was pelleted and washed with 50 ml distilled water. In order to precipitate cell wall mannoproteins as a boric acid complex, the pooled supernatants were acidified by the addition of 100 ml boric acid (1 %) and the pH was adjusted to pH 8·8 by the addition of 2 M NaOH. The resulting complex was pelleted, washed twice with sodium acetate (0·5 %, pH 8·8) and resuspended in acetate (0·5 %). Another precipitation was performed by the addition of 1 g solid sodium acetate and 3 vols ethanol. The precipitate was pelleted, washed twice with acetate (2 % in ethanol), and resuspended in double-distilled water. After neutralization with 2 M NaOH and subsequent dialysis against 200 vols of distilled water, the cetavlon mannoprotein was maintained and lyophilized for performing further studies.
Adsorption of killer toxins K1 and K28 to cetavlon mannoprotein in vivo competition assay.
Cetavlon mannoproteins were resuspended at concentrations from 1 to 10 mg ml1 in 880 µl B-medium, pH 4·7 (Pfeiffer & Radler, 1982). Each sample also contained 20 µl of the appropriate killer toxin (about 104 U ml1) and 100 µl of S. cerevisiae SEY6210 with a cell concentration of about 107 cells ml1. After incubation of the samples for 24 h with gentle shaking at 20 °C, colony-forming units of the sensitive strain were determined by plating a range of dilutions onto YEPD agar, pH 7·0. The remaining toxin in the samples was rapidly inactivated due to the high pH of the agar. Competition assays were performed according to the methods of Hutchins & Bussey (1983)
and Schmitt & Radler (1987)
, with S. cerevisiae 518 as sensitive test strain.
Immunoblot analysis.
After transformation with the indicated Kre1p expression construct, approximately 2x107 yeast cells were grown either at the permissive (20 °C) or at the semi-restrictive (25 °C) temperature in minimal medium under conditions of plasmid selection. Cells were harvested and the extracellular proteins secreted into the cell-free culture medium were concentrated by ethanol precipitation and incubated overnight at 20 °C. Protein samples were resuspended in water and separated by Tricine-SDS-PAGE according to the method of Schagger & von Jagow (1987). After electrotransfer of the proteins onto a nitrocellulose membrane, blots were incubated with a monoclonal anti-GFP (green-fluorescent-protein) antibody (Roche; diluted 1/10 000) followed by treatment with an alkaline phosphatase-coupled anti-rabbit antibody (Sigma; diluted 1/3000), and developed with NBT/BCIP (Roche).
Preparation of cell wall 1,6--D-glucan.
1,6--D-Glucan was isolated and quantified as described by Boone et al. (1990)
. In brief, yeast cells were cultivated in SC medium (or the appropriate D/O-medium if plasmid selection was required) until stationary growth phase. Cells were harvested, washed twice with distilled water and then extracted three times with 0·5 ml 3 % NaOH at 75 °C (1 h per extraction) to remove alkali-soluble glucan and mannoproteins. After alkali extraction, cells were neutralized and washed by the addition of 1 ml 0·1 mM Tris/HCl (pH 7·5) and 1 ml 10 mM Tris/HCl (pH 7·5). Washed cells were resuspended in 1 ml 10 mM Tris/HCl (pH 7·5) containing 5 mg zymolyase 20T (ICN Biomedicals) and incubated overnight at 37 °C. After digestion of the cell wall, the insoluble cell pellet was removed by centrifugation (13 000 r.p.m., 15 min) and the supernatant was dialysed against distilled water. Analysis of the carbohydrate retained after dialysis yielded the proportion of alkali-insoluble 1,6-
-glucan. Total carbohydrate in each fraction was measured as hexose by the phenol/sulfuric acid method of Dubois et al. (1956)
. Each experiment was repeated at least three times.
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RESULTS |
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We and others have previously shown that K1 toxin resistance of a yeast kre1 mutant is due to a block in the initial interaction of the toxin with the yeast cell surface, and that susceptibility can be restored by retransformation with the wild-type KRE1 gene. This observation implies that expression of wild-type Kre1p not only increases the 1,6-
-glucan content of the disruption mutant up to wild-type level, but also restores toxin binding to the cytoplasmic membrane. In order to investigate the effect of Kre1p on cell wall glucans in more detail, we determined the amount of alkali-insoluble 1,6-
-D-glucan in a
kre1 null mutant before and after expression of either full-length Kre1p or its N-terminally truncated derivative
1225Kre1p. As summarized in Table 1
, the 1,6-
-glucan level in the null mutant compared to the wild-type was diminished by 33 %, thus confirming earlier reports of Boone et al. (1990)
. As expected, expression of
1225Kre1p did not restore 1,6-
-glucan levels, while expression of full-length Kre1p did, indicating that the N-terminal part of Kre1p is responsible for its function in 1,6-
-glucan assembly. The slightly increased 1,6-
-glucan content seen in the
kre1 mutant after expression of the full-length protein Kre1p is likely to be caused by its multi-copy expression from the strong PGK1 promoter.
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The capacity of the isolated mannoproteins to adsorb K1 as well as K28 was also determined. Mannoprotein fractions of the kre1 disruption mutant showed a decreased adsorption of K28 compared to wild-type, confirming the above-mentioned assumption that cell surface mannoproteins containing 1,3-
-mannotriose outer side-chains, which are responsible for K28 toxin binding in vivo and in vitro, are either missing or somehow under-represented in the genetic background of a
kre1 null mutation (Fig. 1
). Interestingly, isolated and partially purified cell wall mannoproteins could also adsorb significant amounts of K1 toxin, indicating that the mannoprotein fraction still contains 1,6-
-D-glucans. Thus, our data are consistent with observations of Kapteyn et al. (1996)
and Kollar et al. (1997)
that cell wall mannoproteins are embedded in a heteropolymeric matrix consisting of 1,3-
- and 1,6-
-glucans. Whereas isolated mannoproteins from an mnn2 mutant did not bind significant amounts of K28, they were still capable of binding to K1 toxin in a similar manner as Mnn2+ cells; this again implies that 1,6-
-glucan synthesis is not negatively affected in the mnn2 mutant.
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To further confirm that GPI-anchoring is necessary for the in vivo targeting of Kre1p and its subsequent incorporation into the wall, the gpi1 mutant was transformed with a plasmid expressing a GFP-tagged derivative of Kre1p and subsequently examined for secretion of the K28/GFP/Kre1p fusion into the culture medium before and after shifting the cells to the semi-restrictive temperature. As illustrated in Fig. 3, a signal for the secreted K28/GFP/Kre1p fusion was detectable in the culture supernatant of cells grown at 25 °C, but not at the permissive temperature, confirming that GPI1 is involved in the transient GPI-anchoring of Kre1p.
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DISCUSSION |
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Moreover, the observed K1/K28 cross-resistance at the cell wall level in a yeast kre1 mutant suggests that resistance to K28 toxin can be attributed to the decreased amount of cell wall mannoproteins which represent the primary toxin receptors for K28 (Schmitt & Radler, 1988
). This finding is consistent with data from Kapteyn et al. (1997)
and Kollar et al. (1997)
showing that a major group of mannoproteins is covalently anchored in the cell wall by 1,6-
-glucans. Furthermore, it has been reported that severe 1,6-
-glucan defects in several kre mutants lead to the secretion of certain cell wall mannoproteins, e.g.
-agglutinin (Lu et al., 1995
). This result is somewhat contradictory to that of Hutchins (1982)
, who found that kre1 mutants do not show a significant reduction in total cell wall mannoproteins. Our data confirm the latter observation since deletion of KRE1 did not significantly decrease the overall amount of mannoproteins. The regained K28 toxin sensitivity of the
kre1 mutant after removal of the cell wall demonstrates the existence of two different receptor populations for the toxins K1 and K28 at the level of the cytoplasmic membrane, as formerly speculated by Schmitt & Compain (1995)
. This is also confirmed by more recent studies in which it was shown that soon after K28 has bound to the cell wall, the toxin is taken up by receptor-mediated endocytosis and traverses the secretion pathway in reverse, and finally dislocates into the cytosol, where K28-
transmits the cytotoxic signal into the nucleus (Schmitt & Eisfeld, 1999
; Eisfeld et al., 2000
).
However, the cell wall receptor of the Z. bailii virus toxin zygocin, which is also a cell wall mannoprotein (see Table 3), is still present in the
kre1 mutant, indicating that the normal composition of cell wall mannoproteins is somehow altered in a Kre1 genetic background. Since isolated mannoproteins from a
kre1 mutant showed a significantly decreased K28 toxin binding when tested in an in vivo competition experiment, it can be speculated that the serine/threonine-rich region of Kre1p is responsible for ensuring wild-type levels of those cell wall mannoproteins which contain 1,3-
-mannotriose outer side-chains that function as primary K28 toxin receptors (Schmitt & Radler, 1988
). This would suggest a more structural function of Kre1p in linking certain mannoproteins of the cell surface to cell wall glucans. The observation that a haemagglutinin-tagged derivative of Kre1p mainly localizes to the cell wall likewise supports a more structural role of Kre1p, but an enzymic function of Kre1p after its transfer to and incorporation into the wall is unlikely (Roemer & Bussey, 1995
). This speculation is further supported by recent work of Terashima et al. (2003)
, who showed that changing the normal localization of a GPI-associated yeast protein from the plasma membrane to the cell wall greatly affects its cellular function.
Taken together, the data presented in this study argue against an enzymic function of Kre1p and rather suggest a more structural role within the formation of the cell wall network, possibly by being somehow involved in covalently cross-linking 1,6--glucans to other cell wall components such as 1,3-
-glucan, chitin and certain mannoproteins (Fig. 5
). Roemer & Bussey (1995)
could not detect any covalently attached 1,6-
-glucan by using a 1,6-
-glucan antiserum and furthermore observed that Kre1p expressed in a kre5 mutant (which lacks detectable 1,6-
-glucan) shows the same electrophoretic mobility as in a Kre+ wild-type background. However, the inability to detect 1,6-
-glucan immunologically is not a definitive proof. In a similiar manner, this phenomenon was previously ascribed to a peculiar cell wall structure of certain proteins as reported for Cwp2p (van der Vaart et al., 1996
). The lack of a mobility shift of Kre1p expressed in a kre5 mutant could be due to changes to the composition within the cell wall, which is a known compensatory yeast response to a variety of internal and external stress factors (Ram et al., 1998
), but again this would not exclude a structural function of Kre1p. Montijn et al. (1999)
demonstrated that 1,6-
-glucan synthesis takes place at the cell surface, suggesting that Kre1p is not associated with 1,6-
-glucan during its passage through the secretory pathway but rather receives some so far unknown modification which could be required for its proper structural function later on. In another explanation, Kre1p expressed in a kre5 mutant could be incorporated into the wall by cross-linking other cell wall components such as 1,3-
-glucan or chitin. The fact that digests of cell wall preparations did not release significant amounts of Kre1p could be due to unusual post-translational processing of Kre1p within the secretory pathway as speculated by Roemer & Bussey (1995)
. An unusual modification such as this could be the main reason for embedding the protein as a 1,3-
-glucanase-resistant structural component into the wall, as was recently described for Gas1p (De Sampaio et al., 1999
). However, our data suggest that Kre1p might be directly involved in cross-linking certain mannoproteins of the cell surface as indicated by the unique sensitivity profile observed for the killer toxins K28 and zygocin (see Table 2
).
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The precise function of Kre1p in the in vivo synthesis and assembly of cell wall 1,6--glucan has still to be elucidated. However, the observation here of a slight decrease in cell wall mannoprotein content and an altered or reduced amount of cell surface mannoproteins containing 1,3-
-mannotriose outer side-chains in a yeast
kre1 mutant may be a useful basis upon which to further investigate the protein's function, especially if Kre1p were to be involved somehow in cross-linking of certain mannoproteins and cell wall 1,6-
-glucan. Additional experiments will be required to show if our current model of Kre1p in vivo function (as presented in Fig. 5
) holds true; we intend to address this aspect in the near future.
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ACKNOWLEDGEMENTS |
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Received 18 March 2004;
revised 25 June 2004;
accepted 1 July 2004.
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