The ER-Golgi v-SNARE Bet1p is required for cross-linking {alpha}-agglutinin to the cell wall in yeast

Pearl Kipnis, Naomi Thomas, Rafael Ovalle and Peter N. Lipke

Dept of Biological Sciences and the Center for Gene Structure and Function, Hunter College of the City University of New York, 695 Park Ave, New York, NY 10021, USA

Correspondence
Peter Lipke
lipke{at}genectr.hunter.cuny.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins, including {alpha}-agglutinin, are secreted to the cell surface through vesicular transport pathways. At the cell surface the GPI anchors are cleaved within the glycan, then transglycosylated to form a covalent cross-link to 1,6-{beta}-glucan. Among mutants that were temperature-sensitive for growth and for ability to cross-link the mannoprotein {alpha}-agglutinin to the cell wall, one strain was complemented by BET1, which encodes an ER-Golgi v-SNARE. Temperature-sensitive mutations in BET1 caused aberrations in cell wall structure, including excretion of {alpha}-agglutinin into the medium, sensitivity to lysis with Zymolyase and hypersensitivity to Calcofluor White. At restrictive temperatures, bet1 mutations block secretion of invertase and other proteins, but {alpha}-agglutinin was excreted into the extracellular medium. In wild-type parental or bet1 cells, secretion of {alpha}-agglutinin also continued after protein synthesis was blocked with cycloheximide. This secretion was due to continued export of a significant amount of {alpha}-agglutinin from compartments distal to the BET1-dependent secretion step. Thus, in bet1 cells the ER-Golgi block allowed secretion to continue, but prevented cell wall incorporation of the {alpha}-agglutinin. Therefore, a mutation early in the secretion pathway caused aberrant cell wall synthesis by preventing localization of key components required in wall cross-links.


Abbreviations: CFW, Calcofluor White; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell walls in Saccharomyces cerevisiae are composed of an outer layer of mannoproteins and an inner layer of 1,3-{beta}-glucans, 1,6-{beta}-glucans and a small amount of chitin. The four components of the cell wall are linked together through the highly branched 1,6-{beta}-glucans (Kapteyn et al., 1996, 1999; Kollar et al., 1995, 1997; Lipke & Ovalle, 1998). Of these components, mannoproteins are synthesized in the endoplasmic reticulum (ER) and secreted through the secretory pathway, being glycosylated in the ER and the Golgi. 1,3-{beta}-glucan and chitin are synthesized at the cell surface, apparently in membrane-bound complexes that utilize cytoplasmic UDP-sugar donors to form a polysaccharide chain that is extruded through the membrane into the exoplasmic space (Orlean, 1997). The site and mode of synthesis of the 1,6-{beta}-glucan cross-linking polysaccharide are unknown, but there is evidence favouring initial synthesis either in the ER (Shahinian & Bussey, 2000) or at the plasma membrane (Montijn et al., 1999).

{alpha}-Agglutinin is a wall mannoprotein that facilitates mating by mediating specific and kinetically irreversible adhesion of mating type {alpha} cells to cells of mating type a (Lipke et al., 1987; Zhao et al., 2001). This adhesin is a member of a large class of wall glycoproteins that are synthesized with glycosylphosphatidylinositol (GPI) anchors and are subsequently transported to the cell surface via the secretory pathway (De Groot et al., 2003; Gaynor et al., 1999; Hamada et al., 1998; Lu et al., 1994, 1995; Wojciechowicz et al., 1993). From the Golgi, such proteins are secreted to the exoplasmic face of the cell membrane. In a set of reactions that have not been characterized, the GPI glycan is cleaved, then the remnant attached to the glycoprotein is transglycosylated to 1,6-{beta}-glucan so that the mannoprotein is covalently integrated into the wall complex (Kollar et al., 1995, 1997; Lipke & Ovalle, 1998; Lu et al., 1995).

GPI anchors are essential for wall localization of this class of mannoprotein. Mutations that delete the GPI anchor signal of {alpha}-agglutinin or other GPI wall proteins result in secretion of the unanchored protein to the cell surface and excretion into the media (De Groot et al., 2003; Tsukahara et al., 2003; Vossen et al., 1997; Wojciechowicz et al., 1993). Anchorage of {alpha}-agglutinin to the cell wall is also defective in kre mutants, which have defects in synthesis of 1,6-{beta}-glucan (Lu et al., 1995).

The mechanisms responsible for the extracellular assembly and modifications of the cell wall are poorly understood. GPI-defective mutants are growth-impaired (Costello & Orlean, 1992; Kostova et al., 2003; Leidich et al., 1995; Orlean, 1997) and 1,6-{beta}-glucan-deficient strains have disorganized walls as well as defects in mannoprotein incorporation. Analysis of rho1 mutants defective in synthesis of 1,3-{beta}-glucan reveals that this glucan is essential for synthesis and anchorage of 1,6-{beta}-glucan and mannoproteins. Conversely, inhibition of GPI synthesis does not affect assembly of glucan (Roh et al., 2002). Thus it is likely that 1,3-{beta}-glucan synthesis precedes mannoprotein assembly into the wall.

To gain insight into the wall assembly process, we have further analysed a set of mutants defective in cell wall incorporation of the GPI-anchored protein {alpha}-agglutinin. These mutants were isolated in a screen for temperature-sensitive growth and failure to cross-link {alpha}-agglutinin to the wall (Benghezal et al., 1995). A subset of these mutants was screened for normal synthesis of GPI anchors and we have further characterized some of these. We report here on phenotypes and identification of the mutated gene of AC59, a strain with multiple cell wall defects. The identification of AC59 as a bet1 mutant leads to a novel role for the ER and Golgi in cell wall assembly.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
Reagents were purchased from Sigma unless otherwise stated. Growth medium conditioned with S. cerevisiae X2180-1A (MATa) served as the source of the sex pheromone a-factor. Medium conditioned with X2180-1B (MAT{alpha}) served as control.

Strains, plasmids and culture conditions.
Strains and plasmids are listed in Table 1. The YCp50-based yeast genomic library 3JDAF2 was kindly provided by Dr Jeanne Hirsch, Mt Sinai School of Medicine, New York, USA (Hirsch & Cross, 1993). Transformation of yeast cells and recovery of plasmids from yeast were performed by published methods (Hoffman & Winston, 1987; Ito et al., 1983). Plasmid purification kits (Qiagen) were used to purify plasmid DNA for yeast transformation and sequencing. Escherichia coli was grown in LB-Amp under standard conditions. Yeast was grown in rich YEPD or defined YNB-based media as appropriate. For temperature-sensitive strains the permissive was 23 °C, the semi-permissive temperature was 30 °C and the restrictive temperature was 37 °C. Yeast cell densities were determined by light scattering at 660 nm in a Spectronic 21 DV.


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Table 1. Strains and plasmids

 
Quantitative agglutination assay for whole cells and soluble {alpha}-agglutinin.
Agglutination assays were performed as described previously (Chen et al., 1995; Terrance & Lipke, 1981). {alpha}-Agglutinin activity is reported in Units, determined as the amount that inactivates the a-agglutinin on 2x106 cells of mating type a, 2x10–8 g purified protein (Chen et al., 1995). Unless otherwise stated, error bars are the standard error from triplicate tubes.

Qualitative agglutination assay.
Strains to be tested were grown at 23 °C and 106 cells in 0·1 ml were transferred to 3 ml YEPD with a similar number of W303-1A tester cells (Lipke et al., 1989). The tubes were vortexed and incubated overnight at room temperature, shaking at 120 r.p.m. If {alpha} and a cells agglutinated, a broad lacy pellet appeared at the bottom of the tube; with no agglutination, a compact round pellet resulted.

Cell lysis by Zymolyase.
Enzyme preparation and cell wall lysis assays were based on the method described previously (Ovalle et al., 1999). Cells were grown in 5 ml YEPD to an OD660 of 0·4, harvested and washed three times with deionized water. The pellets were resuspended to an OD660 of 0·6 in TE buffer, pH 7·5 (50 mM Tris/HCl, 5 mM EDTA), plus 5 % PEG 8000 which was added just before use. The cells were then incubated at 23 °C for 30 min. A volume (200 µl) of each sample was added to microtitre plate wells in sets of three wells per sample. Zymolyase 100 T (ICN) was added to each set of three wells to a final concentration of 40 µg ml–1. As a control, TE buffer only was added to one set of wells. The Zymolyase and substrate were mixed in the wells and the microtitre plate was immediately inserted into the spectrophotometer. Optical density was recorded at 1 min intervals for 1 h.

Invertase assays.
Cell surface and soluble invertase activity was assayed as reducing sugar production from sucrose (Jue & Lipke, 1985; Kwon-Chung et al., 1990). AC59 was grown to an OD660 of 0·235, pelleted, resuspended in 1 % yeast extract and 1 % peptone (YP) containing 0·1 % glucose, and incubated at 120 r.p.m. at room temperature for 35 min to derepress the expression of invertase. The culture was divided into two flasks and incubated at 37 and 23 °C, respectively, for 2 h. The cells were harvested and the pellets and supernatants saved. Culture supernatants were dialysed in 0·01 M sodium acetate, pH 5·5, lyophilized and resuspended in H2O to one-quarter of the original volume. The pellets were resuspended in 1 ml 0·1 M sodium acetate, pH 5·5, and 10 mM sucrose. Tubes were incubated at 30 °C for 30 min to allow the hydrolysis by invertase of sucrose into glucose and fructose. Activity was determined as production of reducing sugars (Jue & Lipke, 1985).

Calcofluor White (CFW) susceptibility.
CFW plates were prepared as described by Ram et al. (1994). Cells were grown overnight in YEPD and dilutions of 106, 105, 104 and 103 cells ml–1 were made. Three microlitres of each dilution series was then spotted onto a series of Petri dishes containing CFW. The growth of respective strains was determined after 2 days at room temperature.

ELISA.
ELISA (Reen, 1994) were carried out as described previously (Wojciechowicz & Lipke, 1989). Microtitre plate wells were coated with Concanavalin A (ConA) (10 µg ml–1 in Buffer A: PBS, pH 7·5, 20 µM CaCl2, 20 µM MgSO4), 50 µl per well, overnight at 4 °C. The wells were blocked by overlaying ConA with 1 % BSA in PBS, 100 µl per well, at room temperature for 30 min. The plates were washed three times with PBS-Tween 20 (0·5 %); the samples were then added in sets of three wells per sample and incubated for 2 h at room temperature. After washing, wells were blocked with 1 % yeast invertase (Sigma), 100 µl per well in buffer A, and incubated for 2 h at room temperature. The plates were washed and antibody against {alpha}-agglutinin was added at a 1 : 1000 dilution, 50 µl per well, and incubated overnight at 4 °C (Chen et al., 1995). After washing, the secondary antibody, anti-rabbit IgG-alkaline phosphatase conjugate, was added at a dilution of 1 : 1000, 50 µl per well, and the plates were incubated for 2 h at room temperature, then washed extensively. The substrate p-nitrophenyl phosphate in diethanolamine was added, 50 µl per well, and OD405 was read in an ELISA plate spectrophotometer after 10–30 min incubation.

Immunoblots.
Blots were carried out to detect {alpha}-agglutinin and carboxypeptidase Y in culture supernatants and cell extracts. The antibodies were a specific polyclonal antibody for {alpha}-agglutinin (Wojciechowicz & Lipke, 1989), followed by a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Sigma) and an anti-carboxypeptidase Y-HRP conjugate from Research Diagnostics. All incubations were carried out in PBS supplemented with 0·5 % Tween 20 as well as 0·2 M methyl-{alpha}-mannopyranoside and 1 mg S. cerevisiae invertase ml–1 (Sigma) to adsorb anti-mannan antibodies. Blots were developed with luminol reagent (Pierce SuperSignal West).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phenotypes of AC59
We have previously reported on a genetic screen which identified W303-1B-derived cells that had temperature-sensitive cell surface localization of the cell wall mannoprotein adhesin {alpha}-agglutinin. Many of these mutants had defects in synthesis of GPI anchors or their transfer to peptide chains (Benachour et al., 1999; Benghezal et al., 1995; Flury et al., 2000; Richard et al., 2002; Umemura et al., 2003). A second set of strains derived from this screen had wild-type levels of intermediates in GPI synthesis and were able to transfer the GPI to nascent polypeptides. Therefore, the failure of these strains to display cell surface {alpha}-agglutinin implied a defect in secretion of cell wall components or in assembly of the wall itself. We tested for cell wall assembly defects among this latter set of strains. Of those that were non-agglutinable due to a defect in localization of {alpha}-agglutinin at the restrictive temperature, most showed additional cell wall-related defects (data not shown).

Strain AC59 showed marked and consistent cell wall defects at permissive (23 °C) and semi-permissive temperatures (30 °C). AC59 was hypersensitive to CFW, a fluorescent dye that prevents assembly of fibrous polysaccharides (Albani et al., 2000). The dye (5 µg ml–1) killed AC59, whereas parental strain W303-1B was resistant (Fig. 1). Such sensitivity is characteristic of cells with defects in cell wall synthesis and assembly (Lussier et al., 1997; Neiman et al., 1997; Ram et al., 1994; Vossen et al., 1997).



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Fig. 1. CFW sensitivity. Tenfold serial dilutions of the indicated cells were grown at 23 °C on YEPD without or with CFW.

 
Lysis rate assays measure the overall integrity of the wall in yeast cells and can compare characteristics of related strains (De Groot et al., 2003; Ovalle et al., 1998, 1999). AC59 grown at 23 °C was hypersensitive to lysis with Zymolyase, a lytic mixture of protease and 1,3-{beta}-glucanase (Kitamura & Yamamoto, 1972) (Fig. 2). The lag time, an indication of the integrity of the outer mannoprotein layer, was only 4·8 min, about half the time for the wild-type. Subsequently, the cells lysed at more than twice the rate of the parental strain W303-1B, itself a relatively sensitive strain (Fig. 2) (Ovalle et al., 1999). Indeed, some AC59 cells lysed after prolonged incubation in hypotonic medium in the absence of added enzyme. These results also were consistent with alteration in cell wall structure in AC59 cells.



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Fig. 2. Zymolyase-induced spheroplast lysis of strains AC59 and W303-1B. Cells were grown at 23 °C to mid-exponential phase and washed. Lysis was monitored and quantified as described in Methods (Ovalle et al., 1999). Symbols: filled inverted triangle, W303-1B without added Zymolyase; open inverted triangle, W303-1B with added Zymolyase; filled circle, AC59 without added Zymolyase; open circle, AC59 in presence of Zymolyase. For W303-1B the lag time in the presence of Zymolyase was 9·1 min and the maximal lysis rate (MLR) was 0·013. For AC59, the lag time was 4·8 min and the MLR was 0·029.

 
AC59 Hyper-excretion of {alpha}-agglutinin
MAT{alpha} strains with cell wall defects can fail to cross-link {alpha}-agglutinin into the cell wall and instead excrete it into the growth medium (Lu et al., 1995). AC59 cells were only slightly agglutinable after growth at the permissive temperature, implying a cell wall assembly defect (data not shown). Therefore, we tested growth medium from AC59 for increased amounts of the mannoprotein. Growth medium from cultures incubated at 23 and 37 °C was collected, dialysed and concentrated by lyophilization and resuspension. Bioassays of {alpha}-agglutinin activity in the culture supernatants showed that at permissive temperature, AC59 and W303-1B excreted comparable amounts of {alpha}-agglutinin. The parental strain excreted 55 % as much activity at 37 °C as at 23 °C. In contrast, AC59 excreted 40 % more activity at 37 °C than at 23 °C, and 2·5-fold more than W303-1B at 37 °C (Table 2). Similar results were seen in immunoblots and in ELISAs (Wojciechowicz & Lipke, 1989) of the concentrated culture supernatants (data not shown), confirming the temperature sensitivity of the cross-linking defect.


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Table 2. Excretion of {alpha}-agglutinin by strains AC59 and W303-1B

AC59 and W303-1B were grown in YEPD at 23 °C to early exponential phase, then divided into two flasks and growth was continued at 23 or 37 °C for 3 h. The growth media were then lyophilized, resuspended and the {alpha}-agglutinin quantified in an agglutination assay. Mean and standard deviation from triplicate tubes are reported.

 
{alpha}-Agglutinin release into the medium did not appear to be a result of spontaneous lysis of mutant cells in culture conditions. Little invertase was released into the medium after derepression (Table 3). Immunoblots for carboxypeptidase Y showed that the enzyme was undetectable in the 37 °C culture supernatants under conditions that would have detected 0·03 % of the amount present in extracts of the intact cells (data not shown).


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Table 3. Secretion of invertase to cell surface and medium in AC59

AC59 was grown in YEPD to exponential phase, then harvested, resuspended in YP with 0·1 % glucose for 35 min at 23 °C to derepress invertase, then split into two cultures and incubated for 2 h at 23 or 37 °C. After the incubation, cell densities were determined and the washed cells were assayed for invertase activity. The culture supernatants were concentrated fourfold and also assayed for activity. Results are means of duplicate tubes.

 
BET1 complementation of AC59
To identify genes that complement the defects in AC59, we transformed the strain with a low-copy-number YCp50-based S. cerevisiae gene library, selecting for transformants that grew at 37 °C. Several transformants grew at the restrictive temperature, and a complementing plasmid was isolated from one colony and amplified in E. coli. This plasmid allowed AC59 to grow at 37 °C on retransformation. Restriction mapping and partial sequencing revealed that the insert contained 3 ORFs on chromosome IX, including the BET1 gene. pAN101, a plasmid specifically containing this gene (Newman et al., 1990), complemented AC59 for growth at 37 °C, although not completely (Fig. 3).



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Fig. 3. pAN101 complementation of AC59 temperature sensitivity. Three strains were streaked on YEPD and grown at 37 °C for 4 days.

 
The bet1-1 mutant RSY944 is recessive (Wuestehube et al., 1996) and we confirmed that RSY944xW303-1B diploids were not temperature-sensitive. Similarly, diploids from an AC59 X N435-1A cross grew well at 37 °C, showing that the ts– phenotype in AC59 was also recessive. As expected, the temperature sensitivities of AC59 and RSY944 failed to complement, because none of the diploids derived from a cross grew at 37 °C, and all grew at 23 °C. Therefore, the temperature sensitivity of the AC59xRSY944 diploids implied that they were homozygous for bet1.

As a result of the identification of a bet1 allele being the basis for the cell wall phenotypes in AC59, we obtained ANY114, a temperature-sensitive bet1-1 mutant. Like AC59, ANY114 excreted {alpha}-agglutinin into the medium and the excretion was increased 2- to 2·5-fold at restrictive temperature (data not shown). Therefore, the excretion phenotype was present in an independently derived bet1 mutant.

Secretion of invertase
bet1-1 confers temperature sensitivity for protein secretion as well as for growth (Newman & Ferro-Novick, 1987). In bet1-1 mutants incubated at restrictive temperature, invertase secretion is blocked between the ER to the Golgi and the enzyme is not transported to the cell surface (Newman et al., 1990). Therefore, we assayed AC59 for invertase secretion to the cell wall and into the supernatant after derepression at 23 or 37 °C. The activity in the wall and in the growth medium was significantly reduced at 37 °C relative to 23 °C (Table 3). Therefore, the bet1 allele in AC59 blocked invertase secretion, as expected.

BET1 complementation of wall phenotypes
Plasmid-borne BET1 complemented the CFW sensitivity of AC59 (Fig. 1). To determine if BET1 would correct the hyper-excretion of {alpha}-agglutinin, we transformed AC59 with pAN101 containing BET1 and with YCp50 without an insert. These transformed cells were incubated at restrictive or permissive temperature and the culture supernatants were isolated. The dialysed and concentrated supernatants were assayed for {alpha}-agglutinin. pAN101 transformation greatly reduced the amount of {alpha}-agglutinin excreted into the medium at 37 °C and reduced excretion to a lesser extent at 23 °C (Table 4). Therefore, BET1 complemented AC59 for hyper-excretion of {alpha}-agglutinin at both the permissive and restrictive temperatures.


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Table 4. Effect of plasmid-borne BET1 on {alpha}-agglutinin excretion in AC59

Cultures of AC59, transformed by pAN101 (BET1) or by YCp50 were grown in supplemented YNB medium to exponential phase. The respective cultures were pelleted, resuspended in fresh medium and incubated at 23 or 37 °C for 2 h. The growth medium was collected, lyophilized and resuspended to 1/10 of the original volume. Excreted {alpha}-agglutinin was quantified in an agglutination assay.

 
The BET1-bearing plasmid also promoted {alpha}-agglutinin cross-linking to the cell wall. We tested agglutinability of YCp50- and pAN101-transformed AC59. These strains, and W303-1A (MATa) and W303-1B (MAT{alpha}), were grown at 23 °C and agglutinated with the standard tester strain X2180-1A (MATa) in a qualitative agglutination assay (Lipke et al., 1989). pAN101 increased the agglutinability of AC59 to wild-type levels, while the YCp50-transformed AC59 cells were poorly agglutinable. This pAN101-mediated increase in agglutinability implied that the presence of the BET1 allele allowed cross-linking of the functional {alpha}-agglutinin into the wall.

Paradox of increased excretion of {alpha}-agglutinin at the restrictive temperature
The bet1-dependent increase in excretion of {alpha}-agglutinin at 37 °C (Table 2) contrasted with the block in invertase secretion. The apparent paradox of hyper-excretion in a pathway blocked at the ER had two possible explanations. One possibility was that the source of the excreted material was a post-ER pool of {alpha}-agglutinin present in the cell prior to being transferred to 37 °C. Another possibility was that {alpha}-agglutinin was secreted to the cell wall via an alternative BET1-independent secretory pathway. We designed experiments to distinguish between these possibilities.

Secretion of {alpha}-agglutinin after inhibition of synthesis
There is evidence of a pool of {alpha}-agglutinin in wild-type cells (Terrance, 1983). To determine if a pool of {alpha}-agglutinin existed in the parental strain W303-1B, we assayed production of {alpha}-agglutinin after blocking protein synthesis. Cells were treated with or without cycloheximide (10 µg ml–1, a concentration causing growth arrest) and with or without the added pheromone a-factor, then assayed for cell surface {alpha}-agglutinin. When cycloheximide and a-factor were added to the culture at the same time, {alpha}-agglutinin continued to be secreted to the cell wall (Fig. 4, column 1 vs 2), though in lesser amounts than in cells induced without cycloheximide (column 3). That cycloheximide inhibited {alpha}-agglutinin synthesis was shown by its effectiveness when the cells were pre-incubated with the drug (column 4). Thus in W303-1B, {alpha}-agglutinin reserves can be localized to the cell surface for at least 30 min after addition of cycloheximide.



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Fig. 4. Effect of cycloheximide and a-factor on cell wall display of {alpha}-agglutinin in W303-1B. Cells were grown in synthetic complete medium at 30 °C to an OD660 of 0·3, then pelleted and resuspended in one-half volume of fresh medium. The culture was divided into four samples and BSA (0·2 µg ml–1) was added to each tube to facilitate solubility of the pheromone (Terrance & Lipke, 1981). All tubes were incubated at 30 °C for 1·5 h with shaking at 150 r.p.m. Cycloheximide (10 µg ml–1) and/or a-factor were added at the marked times. The cells were then pelleted and assayed in the quantitative agglutination assay.

 
Source of {alpha}-agglutinin excreted from AC59
Similar experiments in AC59 also implied the existence of a secretable pool of {alpha}-agglutinin (Fig. 5). AC59 cultures were incubated for 1 h at the restrictive temperature with or without cycloheximide and with induction with a-factor (or {alpha}-factor as an inactive control; note that pheromones were added at the same time as cycloheximide, corresponding to the timing in column 2 in Fig. 4 for W303-1B). The culture supernatants were concentrated and assayed for {alpha}-agglutinin activity as before. The amount of {alpha}-agglutinin excreted was similar whether or not cells were induced with a-factor and whether or not cycloheximide had been added (Fig. 5). Thus, cycloheximide had no significant effect on {alpha}-agglutinin excretion when the secretory pathway was blocked by the temperature sensitivity of Bet1p.



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Fig. 5. Effect of cycloheximide and a-factor on secretion of {alpha}-agglutinin into growth medium by AC59. AC59 cells were grown at 23 °C in YEPD to an OD660 of 0·3. The cells were pelleted and resuspended in one-half volume of synthetic complete medium, then divided into four samples. a-Factor, {alpha}-factor, and/or cycloheximide (10 µg ml–1) was added as marked. The tubes were incubated at 37 °C for 1 h with shaking at 150 r.p.m. The cells were pelleted and the supernatants were saved, lyophilized and resuspended to 1/10 of the original volume. {alpha}-Agglutinin concentration was determined by the quantitative agglutination assay.

 
The results in Fig. 5 support the idea of a post-ER pool of secreted {alpha}-agglutinin and appear to rule out secretion of the glycoprotein through a bet1-independent secretion pathway. If there were such a pathway, cycloheximide would reduce the excretion, a result that we did not observe. Also, the amount of excreted {alpha}-agglutinin was similar with or without a-factor treatment in the absence of cycloheximide (Fig. 5, column 3 vs 4). Therefore, the {alpha}-agglutinin synthesized in response to a-factor could not be secreted, so secretion was prevented by the bet1 block itself. These results argue that the excretion to the medium represented material that had already passed the Bet1p-dependent steps of the secretion pathway.

Limited size of the {alpha}-agglutinin pool
The concept of a post-ER pool of {alpha}-agglutinin suggests a finite reserve and eventual depletion of the pool. To demonstrate these attributes we assayed AC59 for excretion of {alpha}-agglutinin into the supernatant during two successive rounds of incubation. At 23 °C, {alpha}-agglutinin was efficiently excreted into the medium during both rounds (Fig. 6; columns 1 and 2, and data not shown). In contrast, at the restrictive temperature, excretion of {alpha}-agglutinin during the first round (column 3) was much greater than during the second round (column 4). These results are consistent with the existence of a finite post-ER pool of {alpha}-agglutinin. At the restrictive temperature, the pool can be excreted, but not replenished.



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Fig. 6. Excretion of {alpha}-agglutinin in successive incubations. AC59 cells were grown at 25 °C to mid-exponential phase, pelleted, resuspended in fresh medium and divided into two flasks. The respective cultures were incubated at 25 or 37 °C for 2 h, then pelleted and the supernatants were saved (Round 1). The pellets were washed three times in medium warmed to the culture temperature, then resuspended in medium pre-warmed to the same temperature and incubated for a second 2 h period at 25 or 37 °C. The cells were pelleted and the supernatants saved (Round 2). The respective supernatants were lyophilized and resuspended in water at 13-fold concentration. The amount of {alpha}-agglutinin excreted into the supernatant was determined in an ELISA.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our results expand the known effects of Bet1p, a v-SNARE required for ER-Golgi transport, showing that a temperature-sensitive allele of bet1 in AC59 caused cell wall defects in addition to secretion inhibition and growth arrest (Newman & Ferro-Novick, 1987; Newman et al., 1990). The identification of the defect in AC59 as an allele of BET1 is based on three observations. The growth arrest of AC59 was not complemented in a cross with a bet1-1 strain; both the growth arrest and cell wall phenotypes were complemented by a plasmid-borne BET1; and, furthermore, another bet1 strain had a cell wall cross-linking defect.

The identification of a bet1 mutation as the source of cell wall defects in AC59 led to a paradoxical phenotype. The mutant excreted active {alpha}-agglutinin into the medium, whereas in wild-type cells this GPI-linked mannoprotein is cross-linked into the cell wall matrix through its GPI-derived glycan. On the other hand, bet1 mutations block secretion of invertase and other proteins between the ER and the Golgi apparatus, early in the secretion pathway (Newman & Ferro-Novick, 1987) (Table 3). The resolution of this paradox lies in the finding of a substantial pool of {alpha}-agglutinin that is localized to compartments later in the secretion pathway than the BET1-dependent step. The failure of the mutant to cross-link {alpha}-agglutinin into the wall must then be due to a bet1-dependent failure to properly localize another component necessary to the wall cross-linking process.

Cellular role of Bet1p
Bet1p, Bos1p and Sec23p are v-SNAREs on ER-derived vesicles. These proteins interact with the Golgi t-SNARE Sed5p to mediate vesicle fusion with the Golgi complex (Lian & Ferro-Novick, 1993; Newman et al., 1990; Tsui & Banfield, 2000). BET1 is essential, and bet1 mutations block secretion and proper localization of proteins including invertase, carboxypeptidase Y and acid phosphatase in a pre-Golgi compartment (Newman & Ferro-Novick, 1987).

Role of Bet1p in secretion of GPI-anchored proteins
GPI-anchored proteins leave the ER in vesicles different from those carrying an integral transport protein (Muniz et al., 2001). The proper sorting of GPI-anchored proteins is dependent on the v-SNAREs Bet1p, Bos1p and Sec23-3p, and at 37 °C, a bet1-1, bos1-1 or sec23-3 mutation blocked Gas1p from reaching the plasma membrane (Morsomme et al., 2003). Two of our experiments are consistent with this BET1-dependent secretion of GPI-anchored cell wall proteins. First, if {alpha}-agglutinin secretion were BET1-independent, the secretion block caused by the mutation would have no effect, and transport of newly synthesized {alpha}-agglutinin from the ER to the Golgi and beyond would continue at the restrictive temperature. Therefore, more {alpha}-agglutinin would be excreted at 37 °C in the absence of cycloheximide than in its presence. Instead, inactivation of Bet1p prevented excretion as efficiently as did cycloheximide (Fig. 5). Second, if {alpha}-agglutinin export were BET1-independent, AC59 would be able to replenish the pool of {alpha}-agglutinin at restrictive temperature. However, the pool is depleted at restrictive temperature, as expected if replenishment is BET1-dependent (Fig. 6).

{alpha}-Agglutinin in intracellular compartments
Our results also confirm the presence of a substantial pool of active {alpha}-agglutinin localized in compartments that are after the Bet1p-dependent step in secretion (Terrance & Lipke, 1981). There was secretion and cross-linking of active {alpha}-agglutinin to the cell surface after a cycloheximide-mediated block in protein synthesis in W303-1B (Fig. 4) and X2180-1B (Terrance, 1983), and after a bet1-mediated block in secretion (Figs 5 and 6). In each strain the pool was depleted in a 90 min incubation with cycloheximide (Figs 4 and 6) (Terrance, 1983).

The pool may result from sequestration of {alpha}-agglutinin in a discrete secretory compartment, or it may be a by-product of the slow processing of this mannoprotein. {alpha}-Agglutinin is constitutively transcribed and translated, and the levels of mRNA and surface protein are up-regulated in response to the sex pheromone a-factor (Hauser & Tanner, 1989; Sijmons et al., 1987; Wojciechowicz & Lipke, 1989). Although transcript levels rise within a few minutes after pheromone treatment (Lipke et al., 1989), surface expression increases for 90 min, a considerably longer time than other cell surface proteins (Roh et al., 2002; Roy et al., 1991; Sentandreu et al., 1983; Terrance & Lipke, 1981). Pulse–chase analysis shows that wall incorporation is maximal 45 min after pheromone treatment (Lu et al., 1994). In that study, {alpha}-agglutinin with Golgi-like glycosylation was maximally labelled within 5 min and plasma membrane-bound forms in 15–20 min. These results implied that transport to the Golgi was rapid, so the majority of the {alpha}-agglutinin available for cross-linking into the wall would be stored in later post-Golgi and/or plasma membrane compartments. Thus, there is a demonstrable reservoir of {alpha}-agglutinin in secretory compartments before pheromone treatment. This material in the reservoir could be secreted into the medium if there were a failure in cell wall cross-linking reactions. AC59 exhibited such a failure at restrictive temperature, due to the absence of functional Bet1p.

In S. cerevisiae an intracellular pool of Chs3p, the catalytic subunit of chitin synthase III, shows similar features. Like {alpha}-agglutinin, Chs3p is synthesized continuously and is temporarily sequestered in endosomal vesicles (chitosomes) (Lagorce et al., 2002; Ziman et al., 1996). Chitosomes may represent a pool of chitin synthase enzymes, ready to be mobilized for chitin ring formation, and regulated by the cell cycle (Ziman et al., 1996, 1998). Transport of Ch3p to the site of formation of the chitin ring is mediated by a secretory pathway involving vesicular transport from the endosome to the plasma membrane (Chuang & Schekman, 1996; Ziman et al., 1996). The internal stores of Chs3p are also rapidly shifted to the plasma membrane under conditions of cell stress (Valdivia & Schekman, 2003). Like Chs3p, an urgent need to up-regulate {alpha}-agglutinin surface expression for the mating reaction may be facilitated by the existence of a pool of active protein ready to be cross-linked into the wall.

A role for BET1 in GPI-dependent cross-linking?
AC59 fails to cross-link {alpha}-agglutinin into the wall, presumably because it is missing a component essential for the cross-linking. That essential component is not {alpha}-agglutinin itself, because it is abundantly available from the pool. There must be a required component that depends on Bet1p activity, presumably in ER-Golgi transport. An intriguing observation implies that the missing component is not a required enzyme or other newly synthesized protein: the parental strain W303-1B cross-links {alpha}-agglutinin into walls during a cycloheximide block (Fig. 4, column 2). The defect was not due to changes in levels of 1,6-{beta}-glucans; there was no significant difference in incorporation of this polysaccharide between AC59 bet1 and BET1 strains (Claudia Abeijon, personal communication). Therefore the defect is not apparent in current models of cell wall assembly.

In summary, at restrictive temperature the bet1 mutation in AC59 results in excretion of soluble {alpha}-agglutinin from a pool that is distal to the BET1-dependent steps in secretion. Because there is {alpha}-agglutinin available, the failure to cross-link it into the wall must be due to a requirement for another component whose availability or activity depends on functional Bet1p. Such a component presumably originates in the ER and might be an enzyme that has a short lifetime, or another component that must be newly synthesized and secreted.


   ACKNOWLEDGEMENTS
 
We thank Dr Claudia Abeijon for the cell wall incorporation analysis. Drs Jeanne Hirsch, Susan Ferro-Novick and Randy Schekman kindly provided essential materials. Dr Anne Dranginis contributed many helpful discussions. This work was supported by NIGMS-SCORE grant S06 60654.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Albani, J. R., Sillen, A., Plancke, Y. D., Coddeville, B. & Engelborghs, Y. (2000). Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study. Carbohydr Res 327, 333–340.[CrossRef][Medline]

Benachour, A., Sipos, G., Flury, I., Reggiori, F., Canivenc-Gansel, E., Vionnet, C., Conzelmann, A. & Benghezal, M. (1999). Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity. J Biol Chem 274, 15251–15261.[Abstract/Free Full Text]

Benghezal, M., Lipke, P. N. & Conzelmann, A. (1995). Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae. J Cell Biol 130, 1333–1344.[Abstract]

Chen, M. H., Shen, Z. M., Bobin, S., Kahn, P. C. & Lipke, P. N. (1995). Structure of Saccharomyces cerevisiae alpha-agglutinin. Evidence for a yeast cell wall protein with multiple immunoglobulin-like domains with atypical disulfides. J Biol Chem 270, 26168–26177.[Abstract/Free Full Text]

Chuang, J. S. & Schekman, R. W. (1996). Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p. J Cell Biol 135, 597–610.[Abstract]

Costello, L. C. & Orlean, P. (1992). Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A. J Biol Chem 267, 8599–8603.[Abstract/Free Full Text]

De Groot, P. W., Hellingwerf, K. J. & Klis, F. M. (2003). Genome-wide identification of fungal GPI proteins. Yeast 20, 781–796.[CrossRef][Medline]

Flury, I., Benachour, A. & Conzelmann, A. (2000). YLL031c belongs to a novel family of membrane proteins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast. J Biol Chem 275, 24458–24465.[Abstract/Free Full Text]

Gaynor, E. C., Mondesert, G., Grimme, S. J., Reed, S. I., Orlean, P. & Emr, S. D. (1999). MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast. Mol Biol Cell 10, 627–648.[Abstract/Free Full Text]

Hamada, K., Terashima, H., Arisawa, M. & Kitada, K. (1998). Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae. J Biol Chem 273, 26946–26953.[Abstract/Free Full Text]

Hauser, K. & Tanner, W. (1989). Purification of the inducible alpha-agglutinin of S. cerevisiae and molecular cloning of the gene. FEBS Lett 255, 290–294.[CrossRef][Medline]

Hirsch, J. P. & Cross, F. R. (1993). The pheromone receptors inhibit the pheromone response pathway in Saccharomyces cerevisiae by a process that is independent of their associated G alpha protein. Genetics 135, 943–953.[Abstract/Free Full Text]

Hoffman, C. S. & Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272.[CrossRef][Medline]

Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153, 163–168.[Medline]

Jue, C. K. & Lipke, P. N. (1985). Determination of reducing sugars in the nanomole range with tetrazolium blue. J Biochem Biophys Methods 11, 109–115.[CrossRef][Medline]

Kapteyn, J. C., Montijn, R. C., Vink, E., de la Cruz, J., Llobell, A., Douwes, J. E., Shimoi, H., Lipke, P. N. & Klis, F. M. (1996). Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 6, 337–345.[Abstract]

Kapteyn, J. C., Van Den Ende, H. & Klis, F. M. (1999). The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1426, 373–383.[Medline]

Kitamura, K. & Yamamoto, Y. (1972). Purification and properties of an enzyme, zymolyase, which lyses viable yeast cells. Arch Biochem Biophys 153, 403–406.[Medline]

Kollar, R., Petrakova, E., Ashwell, G., Robbins, P. W. & Cabib, E. (1995). Architecture of the yeast cell wall. The linkage between chitin and beta(1->3)-glucan. J Biol Chem 270, 1170–1178.[Abstract/Free Full Text]

Kollar, R., Reinhold, B. B., Petrakova, E., Yeh, H. J., Ashwell, G., Drgonova, J., Kapteyn, J. C., Klis, F. M. & Cabib, E. (1997). Architecture of the yeast cell wall. Beta(1->6)-glucan interconnects mannoprotein, beta(1->3)-glucan, and chitin. J Biol Chem 272, 17762–17775.[Abstract/Free Full Text]

Kostova, Z., Yan, B. C., Vainauskas, S., Schwartz, R., Menon, A. K. & Orlean, P. (2003). Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly. Eur J Biochem 270, 4507–4514.[Abstract/Free Full Text]

Kwon-Chung, K. J., Hicks, J. B. & Lipke, P. N. (1990). Evidence that Candida stellatoidea type II is a mutant of Candida albicans that does not express sucrose-inhibitable alpha-glucosidase. Infect Immun 58, 2804–2808.[Medline]

Lagorce, A., Le Berre-Anton, V., Aguilar-Uscanga, B., Martin-Yken, H., Dagkessamanskaia, A. & Francois, J. (2002). Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae. Eur J Biochem 269, 1697–1707.[Abstract/Free Full Text]

Leidich, S. D., Kostova, Z., Latek, R. R., Costello, L. C., Drapp, D. A., Gray, W., Fassler, J. S. & Orlean, P. (1995). Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene. J Biol Chem 270, 13029–13035.[Abstract/Free Full Text]

Lian, J. P. & Ferro-Novick, S. (1993). Bos1p, an integral membrane protein of the endoplasmic reticulum to Golgi transport vesicles, is required for their fusion competence. Cell 73, 735–745.[Medline]

Lipke, P. N. & Ovalle, R. (1998). Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180, 3735–3740.[Free Full Text]

Lipke, P. N., Terrance, K. & Wu, Y. S. (1987). Interaction of alpha-agglutinin with Saccharomyces cerevisiae a cells. J Bacteriol 169, 483–488.[Medline]

Lipke, P. N., Wojciechowicz, D. & Kurjan, J. (1989). AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell–cell interactions during mating. Mol Cell Biol 9, 3155–3165.[Medline]

Lu, C. F., Kurjan, J. & Lipke, P. N. (1994). A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol Cell Biol 14, 4825–4833.[Abstract]

Lu, C. F., Montijn, R. C., Brown, J. L., Klis, F., Kurjan, J., Bussey, H. & Lipke, P. N. (1995). Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol 128, 333–340.[Abstract]

Lussier, M., White, A. M., Sheraton, J. & 17 other authors (1997). Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147, 435–450.[Abstract/Free Full Text]

Montijn, R. C., Vink, E., Muller, W. H., Verkleij, A. J., Van Den Ende, H., Henrissat, B. & Klis, F. M. (1999). Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae. J Bacteriol 181, 7414–7420.[Abstract/Free Full Text]

Morsomme, P., Prescianotto-Baschong, C. & Riezman, H. (2003). The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER. J Cell Biol 162, 403–412.[Abstract/Free Full Text]

Muniz, M., Morsomme, P. & Riezman, H. (2001). Protein sorting upon exit from the endoplasmic reticulum. Cell 104, 313–320.[Medline]

Neiman, A. M., Mhaiskar, V., Manus, V., Galibert, F. & Dean, N. (1997). Saccharomyces cerevisiae HOC1, a suppressor of pkc1, encodes a putative glycosyltransferase. Genetics 145, 637–645.[Abstract/Free Full Text]

Newman, A. P. & Ferro-Novick, S. (1987). Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection. J Cell Biol 105, 1587–1594.[Abstract]

Newman, A. P., Shim, J. & Ferro-Novick, S. (1990). BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol 10, 3405–3414.[Medline]

Orlean, P. (1997). Biogenesis of yeast cell wall and surface components. In Molecular and Cellular Biology of the Yeast Saccharomyces, pp. 229–362. Edited by J. Pringle, J. Broach & E. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Ovalle, R., Lim, S. T., Holder, B., Jue, C. K., Moore, C. W. & Lipke, P. N. (1998). A spheroplast rate assay for determination of cell wall integrity in yeast. Yeast 14, 1159–1166.[CrossRef][Medline]

Ovalle, R., Spencer, M., Thiwanont, M. & Lipke, P. N. (1999). The spheroplast lysis assay for yeast in microtiter plate format. Appl Environ Microbiol 65, 3325–3327.[Abstract/Free Full Text]

Ram, A. F., Wolters, A., Ten Hoopen, R. & Klis, F. M. (1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 1019–1030.[Medline]

Reen, D. J. (1994). Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol 32, 461–466.[Medline]

Richard, M., De Groot, P., Courtin, O., Poulain, D., Klis, F. & Gaillardin, C. (2002). GPI7 affects cell-wall protein anchorage in Saccharomyces cerevisiae and Candida albicans. Microbiology 148, 2125–2133.[Medline]

Roh, D. H., Bowers, B., Riezman, H. & Cabib, E. (2002). Rho1p mutations specific for regulation of beta(1->3)glucan synthesis and the order of assembly of the yeast cell wall. Mol Microbiol 44, 1167–1183.[CrossRef][Medline]

Roy, A., Lu, C. F., Marykwas, D. L., Lipke, P. N. & Kurjan, J. (1991). The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin. Mol Cell Biol 11, 4196–4206.[Medline]

Sentandreu, R., Herrero, E., Elorza, M. V., Rico, H. & Pastor, J. (1983). Synthesis and assembly of wall polymers on regenerating yeast protoplasts. Experientia Suppl 46, 187–195.[Medline]

Shahinian, S. & Bussey, H. (2000). {beta}-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol 35, 477–489.[CrossRef][Medline]

Sijmons, P. C., Nederbragt, A. J., Klis, F. M. & Van den Ende, H. (1987). Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells. Arch Microbiol 148, 208–212.[Medline]

Terrance, K. (1983). Sexual agglutination in Saccharomyces cerevisiae. PhD thesis, City University of New York.

Terrance, K. & Lipke, P. N. (1981). Sexual agglutination in Saccharomyces cerevisiae. J Bacteriol 148, 889–896.[Medline]

Tsui, M. M. & Banfield, D. K. (2000). Yeast Golgi SNARE interactions are promiscuous. J Cell Sci 113, 145–152.[Abstract/Free Full Text]

Tsukahara, K., Hata, K., Nakamoto, K. & 9 other authors (2003). Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly. Mol Microbiol 48, 1029–1042.[CrossRef][Medline]

Umemura, M., Okamoto, M., Nakayama, K., Sagane, K., Tsukahara, K., Hata, K. & Jigami, Y. (2003). GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast. J Biol Chem 278, 23639–23647.[Abstract/Free Full Text]

Valdivia, R. H. & Schekman, R. (2003). The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci U S A 100, 10287–10292.[Abstract/Free Full Text]

Vossen, J. H., Muller, W. H., Lipke, P. N. & Klis, F. M. (1997). Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins. J Bacteriol 179, 2202–2209.[Abstract]

Wojciechowicz, D. & Lipke, P. N. (1989). Alpha-agglutinin expression in Saccharomyces cerevisiae. Biochem Biophys Res Commun 161, 46–51.[Medline]

Wojciechowicz, D., Lu, C. F., Kurjan, J. & Lipke, P. N. (1993). Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily. Mol Cell Biol 13, 2554–2563.[Abstract]

Wuestehube, L. J., Duden, R., Eun, A., Hamamoto, S., Korn, P., Ram, R. & Schekman, R. (1996). New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. Genetics 142, 393–406.[Abstract/Free Full Text]

Zhao, H., Shen, Z. M., Kahn, P. C. & Lipke, P. N. (2001). Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules. J Bacteriol 183, 2874–2880.[Abstract/Free Full Text]

Ziman, M., Chuang, J. S. & Schekman, R. W. (1996). Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway. Mol Biol Cell 7, 1909–1919.[Abstract]

Ziman, M., Chuang, J. S., Tsung, M., Hamamoto, S. & Schekman, R. (1998). Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae. Mol Biol Cell 9, 1565–1576.[Abstract/Free Full Text]

Received 25 March 2004; revised 28 June 2004; accepted 13 July 2004.



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