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
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ABSTRACT |
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INTRODUCTION |
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-Agglutinin is a wall mannoprotein that facilitates mating by mediating specific and kinetically irreversible adhesion of mating type
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-
-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 -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
-agglutinin to the cell wall is also defective in kre mutants, which have defects in synthesis of 1,6-
-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-
-glucan-deficient strains have disorganized walls as well as defects in mannoprotein incorporation. Analysis of rho1 mutants defective in synthesis of 1,3-
-glucan reveals that this glucan is essential for synthesis and anchorage of 1,6-
-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-
-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 -agglutinin. These mutants were isolated in a screen for temperature-sensitive growth and failure to cross-link
-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.
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METHODS |
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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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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
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 ml1. 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 ml1 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 ml1 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
-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 1030 min incubation.
Immunoblots.
Blots were carried out to detect -agglutinin and carboxypeptidase Y in culture supernatants and cell extracts. The antibodies were a specific polyclonal antibody for
-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-
-mannopyranoside and 1 mg S. cerevisiae invertase ml1 (Sigma) to adsorb anti-mannan antibodies. Blots were developed with luminol reagent (Pierce SuperSignal West).
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RESULTS |
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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 ml1) 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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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 -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
-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
-agglutinin. pAN101 transformation greatly reduced the amount of
-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
-agglutinin at both the permissive and restrictive temperatures.
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Paradox of increased excretion of -agglutinin at the restrictive temperature
The bet1-dependent increase in excretion of -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
-agglutinin present in the cell prior to being transferred to 37 °C. Another possibility was that
-agglutinin was secreted to the cell wall via an alternative BET1-independent secretory pathway. We designed experiments to distinguish between these possibilities.
Secretion of -agglutinin after inhibition of synthesis
There is evidence of a pool of -agglutinin in wild-type cells (Terrance, 1983
). To determine if a pool of
-agglutinin existed in the parental strain W303-1B, we assayed production of
-agglutinin after blocking protein synthesis. Cells were treated with or without cycloheximide (10 µg ml1, a concentration causing growth arrest) and with or without the added pheromone a-factor, then assayed for cell surface
-agglutinin. When cycloheximide and a-factor were added to the culture at the same time,
-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
-agglutinin synthesis was shown by its effectiveness when the cells were pre-incubated with the drug (column 4). Thus in W303-1B,
-agglutinin reserves can be localized to the cell surface for at least 30 min after addition of cycloheximide.
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Limited size of the -agglutinin pool
The concept of a post-ER pool of -agglutinin suggests a finite reserve and eventual depletion of the pool. To demonstrate these attributes we assayed AC59 for excretion of
-agglutinin into the supernatant during two successive rounds of incubation. At 23 °C,
-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
-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
-agglutinin. At the restrictive temperature, the pool can be excreted, but not replenished.
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DISCUSSION |
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The identification of a bet1 mutation as the source of cell wall defects in AC59 led to a paradoxical phenotype. The mutant excreted active -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
-agglutinin that is localized to compartments later in the secretion pathway than the BET1-dependent step. The failure of the mutant to cross-link
-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
-agglutinin secretion were BET1-independent, the secretion block caused by the mutation would have no effect, and transport of newly synthesized
-agglutinin from the ER to the Golgi and beyond would continue at the restrictive temperature. Therefore, more
-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
-agglutinin export were BET1-independent, AC59 would be able to replenish the pool of
-agglutinin at restrictive temperature. However, the pool is depleted at restrictive temperature, as expected if replenishment is BET1-dependent (Fig. 6
).
-Agglutinin in intracellular compartments
Our results also confirm the presence of a substantial pool of active -agglutinin localized in compartments that are after the Bet1p-dependent step in secretion (Terrance & Lipke, 1981
). There was secretion and cross-linking of active
-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 -agglutinin in a discrete secretory compartment, or it may be a by-product of the slow processing of this mannoprotein.
-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
). Pulsechase analysis shows that wall incorporation is maximal 45 min after pheromone treatment (Lu et al., 1994
). In that study,
-agglutinin with Golgi-like glycosylation was maximally labelled within 5 min and plasma membrane-bound forms in 1520 min. These results implied that transport to the Golgi was rapid, so the majority of the
-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
-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 -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
-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 -agglutinin into the wall, presumably because it is missing a component essential for the cross-linking. That essential component is not
-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
-agglutinin into walls during a cycloheximide block (Fig. 4
, column 2). The defect was not due to changes in levels of 1,6-
-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 -agglutinin from a pool that is distal to the BET1-dependent steps in secretion. Because there is
-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.
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ACKNOWLEDGEMENTS |
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Received 25 March 2004;
revised 28 June 2004;
accepted 13 July 2004.
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