Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1
Correspondence
Howard Bussey
howard.bussey{at}mcgill.ca
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ABSTRACT |
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INTRODUCTION |
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The Mpk1p MAP kinase cascade begins with the Pkc1p substrate, Bck1p (Costigan et al., 1992; Lee & Levin, 1992
). BCK1 encodes a protein of the MAPKK kinase family. In response to activation by Pkc1p, Bck1p phosphorylates a pair of redundant MAPK kinases Mkk1p/Mkk2p (Irie et al., 1993
). In turn, these MAPK kinases dually phosphorylate the MAP kinase Mpk1p/Slt2p on tyrosine and threonine residues (Lee et al., 1993
). Phosphorylation of Mpk1p has been demonstrated to occur in response to high temperature, exposure to exogenous mating pheromone, or hypo-osmotic shock (Martin et al., 1993
; Buehrer & Errede, 1997
; Zarzov et al., 1996
). Loss of PKC1 function, or that of any of the components of the MAP kinase cascade under its control, results in a cell lysis defect. This defect is attributable, at least in part, to a defect in the transcription of a variety of genes involved in cell wall biosynthesis (Igual et al., 1996
). Regulation of the PKC1MPK1 cell integrity pathway is dependent on the Rho1p GTPase. GTP-bound Rho1p has been shown to physically associate with Pkc1p, resulting in its activation (Nonaka et al., 1995
; Kamada et al., 1996
). Essential for viability in S. cerevisiae, Rho1p localizes to sites of polarized growth and is implicated in cytoskeletal organization (Evangelista et al., 1997
). In addition to its role in the cell integrity pathway, Rho1p in its GTP bound form activates the 1,3-
-glucan synthase complex through Fks1p (Qadota et al., 1996
). Several regulators of Rho1 activity have been identified: Bem2p (Peterson et al., 1994
) and Sac7p (Schmidt et al., 1997
) are GTPase activating proteins, while Rom1p and Rom2p act as GDPGTP exchange factors for Rho1p (Ozaki et al., 1996
).
In a body of work, links have been made between the cell surface and the PKC1MPK1 cell integrity pathway (Gray et al., 1997; Verna et al., 1997
; Jacoby et al., 1998
; Ketela et al., 1999
; Rajavel et al., 1999
; Philip & Levin, 2001
; Sekiya-Kawasaki et al., 2002
). Acting as sensors for the cell wall, a number of type I membrane proteins respond to vegetative and stress-related growth requirements by activating the PKC1MPK1 cell integrity pathway. Two families of cell-surface sensors have been described. The first, the WSC family of four related genes, appears to be necessary for vegetative growth. The archetype member Wsc1p functions upstream of the cell integrity pathway, with wsc1
mutants showing defective PKC pathway phenotypes. Like Wsc1p, both Mid2p and its functional homologue, Mtl1p, appear to be upstream activators of the cell integrity pathway (Ketela et al., 1999
). Both Mid2p and Mtl1p show structural similarity to the WSC family, but show little amino acid sequence identity. All contain a single membrane-spanning domain, an N-terminal signal sequence, an extracellular domain rich in serine/threonine residues, and a short cytoplasmic tail. Identified as a gene necessary for survival upon exposure to mating pheromone (Ono et al., 1994
), Mid2p is a type I integral plasma membrane protein. Null mutants of mid2 show normal vegetative growth, although some strains have a temperature-sensitive lysis defect (Rajavel et al., 1999
). In addition to the mating-induced death phenotype, mid2
mutants are sensitive to caffeine, and resistant to the chitin-binding dye calcofluor white, while overexpression of MID2 leads to an increase in chitin levels and calcofluor white hypersensitivity (Ketela et al., 1999
). Both Wsc1p and Mid2p are required for the activation of the cell integrity pathway (Gray et al., 1997
; Ketela et al., 1999
; Rajavel et al., 1999
), and physically interact with Rom2p and increase GEF activity toward Rho1p (Philip & Levin, 2001
). Despite these common functions, recent work indicates that Wsc1p is involved in Rho1p-dependent activation of the glucan synthase component, Fks1p, while Mid2p is not (Sekiya-Kawasaki et al., 2002
).
To extend this idea, we performed a screen for genes synthetically interacting with MID2, and found that in the absence of Mid2p, components activated by Wsc1p and Rom2p are essential. To examine the signalling role of Mid2p, we performed a two-hybrid screen with the essential cytoplasmic tail of Mid2p, and identified ZEO1 (YOL109w). We show that ZEO1 encodes a 12 kDa protein that primarily localizes to the cell periphery; that zeo1 mutants are resistant to calcofluor white; and that Zeo1p is necessary for calcofluor white hypersensitivity on high-copy expression of MID2. We also provide genetic and biochemical evidence linking MID2 and ZEO1 with the PKC1MPK1 cell integrity pathway.
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METHODS |
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ZEO1 was amplified from BY4741 genomic DNA using Expand DNA polymerase and the primer pair 9 and 10. A 2·5 kb fragment was excised with SacII and XhoI and cloned into the corresponding sites on pRS316 and pRS426. Fidelity of the PCR was confirmed by DNA sequencing.
Zeo1pHA was created by insertion of a single copy of the haemagglutinin (HA) epitope (YPTDVPDYA) directly upstream of the stop codon between residues 338 and 339 via site-directed mutagenesis (Kunkel et al., 1987) on single-stranded ZEO1 contained on pRS316. Correct insertion of the HA epitope was confirmed by DNA sequencing. A 2·5 kb SstIIXhoI fragment containing ZEO1HA was excised and cloned into pRS426. Zeo1pHA was shown to be functional by the observation that pRS316ZEO1HA could fully complement ZEO1 in a zeo1
rom2
mutant.
Synthetic lethal mutant screen.
Systematic genetic array analysis (SGA) was used to identify genes essential in a mid2 background, as described by Tong et al. (2001)
. Briefly, HAB976 (Table 1
) was obtained in four steps. First, the mid2
: : KanMX4 from YD5241 (Table 1
) was switched to mid2
: : NatMX4 by PCR-based transformation. Second, the NatR transformants were mated to Y3084 (Table 1
) and the MATa/
diploids were transferred onto sporulation medium. MAT
meiotic progeny were then selected on synthetic medium lacking leucine and arginine but containing canavanine. The mating type was confirmed by PCR, according to Huxley et al. (1990)
. Third, cells were replica plated onto medium containing nourseothricin to select for the deletion mutants. Fourth, cells were replica plated onto medium lacking lysine to identify lys2
derivatives. From three SGA screens, 194 potential positives were identified; 11 were confirmed by tetrad analysis.
Localization of Zeo1p.
A ZEO1GFP fusion was generated by inserting a BamHI and NotI site over the start codon via site-directed mutagenesis (Kunkel et al., 1987) with primer 11, using single-stranded ZEO1 contained on pRS316 as template. Clones positive for incorporation of the restriction sites were confirmed by DNA sequencing. Generation of an in-frame GFPZEO1 fusion (GFP F64L S65T; kindly provided by U. Stochaj) was accomplished via a GFP BamHBamHI and ZEO1 BamHIBamHI ligation. Correct orientation of the GFP construct was verified with a NotI diagnostic digestion. Functionality of the GFPZEO1 construct was demonstrated as described for ZEO1HA. Localization of GFPZEO1 was accomplished by examination of live mid-exponential-phase cells carrying pRS316 GFPZEO1.
Calcofluor white tests.
To test strains for sensitivity to calcofluor white, mid-exponential-phase cells were diluted and spotted either onto YEPD agar plates with the indicated amount of calcofluor white, or onto selective media buffered with 10 g MES l-1 and adjusted to pH 6·2. Plates were incubated in the dark.
Chitin assay.
Total cellular chitin was measured as described by Bulawa et al. (1986), and outlined by Ketela et al. (1999)
. In brief, washed cells (approx. 25 mg wet cells) were resuspended in 500 µl 6 % (v/v) potassium hydroxide and incubated at 80 °C for 90 min. After cooling at room temperature, 50 µl glacial acetic acid was added. Insoluble material was washed twice with water and resuspended in 250 µl 50 mM sodium phosphate (pH 6·3); 2 mg Streptomyces griseus chitinase (Sigma) was added, and incubated at 25 °C for 2 h. Tubes were centrifuged at 15 000 g for 5 min; 250 µl of the supernatant was transferred to a fresh tube and 1 mg Helix pomatia
-glucuronidase (Sigma) was added. Tubes were incubated at 37 °C for 2 h and then assayed for N-acetylglucosamine content.
Western blot and solubilization tests.
For solubilization tests, total cell extracts were prepared from mid-exponential-phase cells grown in selective medium, by vigorous vortexing in lysis buffer [50 mM Tris/HCl (pH 7·5), 1 mM EDTA, 5 % (v/v) glycerol] in the presence of glass beads and protease inhibitors (Complete protease inhibitor cocktail; Boehringer Mannheim). The resulting slurry was clarified by centrifugation at 3500 g for 10 min at 4 °C to remove unbroken cells and cell walls. The resulting supernatant was divided and one aliquot subjected to centrifugation at 50 000 g at 4 °C for 30 min. The supernatant was withdrawn, and the pellet resuspended in an equal volume of lysis buffer. Samples were resolved by SDS-PAGE and then subjected to Western blotting. Immunodetection of Zeo1pHA was achieved by using anti-HA monoclonal antibody HA11 (Babco) at 1 : 1000 dilution and horseradish-peroxidase-conjugated secondary antibody (Amersham Life Sciences) at a 1 : 2000 dilution. Bands were visualized by enhanced chemiluminescence (Amersham Life Sciences).
Measurement of Mpk1p phosphorylation.
The detection of Mpk1p/Slt2p phosphorylation was carried out as described by Martin et al. (2000). In brief, cells were grown overnight in selective medium at room temperature. For heat-shocked cells, overnight cultures were diluted and grown for 3 h at 37 °C. A 25 ml sample of mid-exponential-phase cells was collected in an equal volume of ice-cold water and centrifuged at 4 °C for 4 min at 1623 g. The cell pellet was briefly washed in 1 ml ice-cold water and transferred to a pre-chilled 1·5 ml collection tube. Cells were repelleted for 20 s at 15 000 g. The supernatant was immediately removed and the pellet resuspended in 300 µl ice-cold lysis buffer [50 mM Tris/HCl pH 7·5, 10 % (v/v) glycerol, 1 % (v/v) Triton X-100, 0·1 % (w/v) SDS, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 50 mM
-glycerol phosphate, 5 mM sodium pyrophosphate], in the presence of glass beads and protease inhibitors (Complete protease inhibitor cocktail; Boehringer Mannheim). Equal concentrations of protein were fractionated by 8 % (v/v) SDS-PAGE. Membranes were probed with phospho-p44/42 MAPK (Thr202/Tyr204) antibody (New England Biolabs) at 1 : 500 dilution to detect dually phosphorylated Mpk1p. Control of sample loading was monitored using anti-HA, as described above.
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RESULTS |
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The cytoplasmic tail of Mid2p is essential for function
As Mid2p shows signalling distinct from Wsc1p, we examined how this signalling specificity was mediated. To act as a stress sensor this plasma-membrane-spanning protein must be able both to detect stress and to relay this information intracellularly. The large highly O-mannosylated extracellular domain presumably involved in signal detection is essential for function (Ketela et al., 1999; and see Fig. 1
). To test if the cytoplasmic tail of Mid2p is required for this intracellular relay, we constructed a Mid2p mutant with a deletion of the C-terminal cytoplasmic domain (amino acid residues 252376) from MID2, generating MID2
CYT under the control of the MID2 promoter. Although this mutant localized normally to the cell periphery (not shown), it was unable to confer sensitivity to calcofluor white when expressed at multicopy levels (Fig. 1
). Philip & Levin (2001)
demonstrated that a deletion of this domain also fails to complement the mating-induced death phenotype of mid2
mutants. These results indicate that the cytoplasmic domain is required for a fully functional Mid2p.
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A zeo1 mutant is resistant to calcofluor white, and MID2-induced hypersensitivity to calcofluor white is Zeo1p-dependent
Chitin is vital to maintain cell wall integrity. Calcofluor white is a fluorescent dye that intercalates with nascent chitin chains, preventing microfibril assembly (Elorza et al., 1983) and inhibiting growth. Previously, we demonstrated that mid2
cells are resistant to calcofluor white and that MID2 is required for synthesis of supplemental chitin under conditions of cell stress (Ketela et al., 1999
). If Zeo1p provides a structural link in Mid2p-mediated signalling, then zeo1
cells should also display a calcofluor white resistance phenotype. The results in Fig. 4
(a) show that at a calcofluor white concentration of 15 µg ml-1 on rich medium, zeo1
cells are more resistant to calcofluor white than wild-type cells. Cells harbouring MID2 on a 2 µm plasmid show enhanced sensitivity to calcofluor white, and we tested if ZEO1 was involved in this hypersensitivity. Fig. 4(b)
shows that zeo1
mutants overexpressing MID2 were less sensitive to calcofluor white than a corresponding wild-type. This attenuation of hypersensitivity to calcofluor white was partial, suggesting the involvement of additional proteins in this process. Resistance to calcofluor white is often associated with defects in chitin synthesis, and this is the case for mid2
-associated calcofluor white resistance and MID2-induced hypersensitivity (Ketela et al., 1999
). Overexpression of MID2 causes hypersensitivity to calcofluor white by increasing total cell wall chitin levels (Ketela et al., 1999
). Since zeo1
mutants are able to attenuate MID2-mediated hypersensitivity to calcofluor white, we tested if this was through a reduction in the MID2-associated increase in chitin. However, we found that this increase in chitin was independent of ZEO1.
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DISCUSSION |
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Zeo1p is peripherally associated with the plasma membrane
Zeo1p is present in extracts in both soluble and insoluble forms and localizes largely to the cell periphery. Although it lacks a transmembrane domain, Zeo1p localizes to the cell surface, presumably through proteinprotein interactions or interactions with the inner surface of the plasma membrane. As Zeo1p localization is not dependent on the presence of either Mid2p or Mtl1p alone, it may bind to both of these plasma membrane proteins, and possibly to others. Null mutants of ZEO1 resemble mid2 mutants in being resistant to calcofluor white, with the mid2
zeo1
double mutant being no more resistant to calcofluor white than either single mutant, consistent with their operating in a single pathway. The ZEO1 gene is required for the high-copy MID2-induced hypersensitivity to calcofluor white, providing further evidence of a biological link between Zeo1p and Mid2p. Since high-copy expression of ZEO1 does not induce hypersensitivity to calcofluor white, it appears that Mid2p requires Zeo1p, but that Zeo1p itself cannot transduce this signal. As the suppression of MID2-induced hypersensitivity to calcofluor white by zeo1
is only partial, there may be other proteins involved in transducing this signal.
Previous work has reported that zeo1 mutants grow slowly on galactose, and that high-copy ZEO1 expression leads to Zeocin resistance (MIPS code: HRB113). Zeocin is a copper-chelated glycopeptide antibiotic of the bleomycin family. Although bleomycin antibiotics perturb the plasma membrane of cells, their activity is believed to be primarily due to their ability to bind and degrade DNA. The ble gene isolated from the bacterium Streptoalloteichus hindustanus encodes a 14 kDa protein that has the ability to bind Zeocin and inhibit its DNA strand cleavage activity (Drocourt et al., 1990
; Calmels et al., 1991
). Zeo1p has no similarity to the ble gene product and it is unclear how ZEO1 confers resistance to Zeocin in Saccharomyces cerevisiae. A number of genomic expression studies show ZEO1 transcriptional activity to be frequently altered under cell stress (Gasch et al., 2000
; Posas et al., 2000
). For example, ZEO1 transcription is increased fourfold in HOG1-deleted cells exposed to 0·4 M NaCl for 10 min (Posas et al., 2000
). These experiments implicate ZEO1 in the response of S. cerevisiae to cellular stress.
Zeo1p affects Mpk1p phosphorylation in a Mid2p-dependent manner
Phosphorylation of Mpk1p in response to stress is partially dependent on Mid2p (Ketela et al., 1999; Rajavel et al., 1999
). Although zeo1
mutants fail to show a reduction in Mpk1p phosphorylation on heat stress, they do show a constitutive activation of Mpk1p at 22 °C. Thus, Zeo1p may act as a negative regulator for this signalling pathway, with its absence leading to an elevated basal Mpk1p phosphorylation that is Mid2p-dependent. Since the influence of Zeo1p on the calcofluor white phenotype of Mid2p is only partial, other proteins may also modulate these Mid2p responses.
Mid2p and Zeo1p signal the cell integrity pathway in a ROM2-independent manner
In our work several lines of evidence indicate that Mid2pZeo1p signalling to the cell integrity pathway can be independent of Rom2p. The pattern of synthetic interactions of MID2 with WSC1 and its network with ROM2 suggests a non-overlapping function for Mid2p, a finding strengthened by the lack of synthetic lethality between MID2 with BCK1 or MPK1, and indicating that Mid2p acts directly through Rho1p to this cell integrity pathway. In a second line of evidence, the death of mid2 cells on exposure to mating pheromone appears to be due to insufficient activation of Rho1p, since this phenotype is largely suppressed by over expression of Rho1p (Ketela et al., 1999
). Therefore, if activation of Rho1p requires Rom2p, then rom2
mutants should also be sensitive to mating pheromone. Although rom2
mutants do exhibit morphological defects in response to mating pheromone, they remain viable (Manning et al., 1997
). Further, overexpression of ROM2 is unable to suppress the pheromone-induced death phenotype of a mid2
mutant. Finally, high-copy expression of MID2 is able to suppress the high-temperature growth phenotype of a rom2
mutant. Since this phenotype is due to a reduced Rho1p GEF activity, Mid2p can thus signal to Rho1p in a Rom2p-independent manner.
Mid2p and Zeo1p appear to play opposing roles in the regulation of Rho1p. Rom2p has been shown to have a major role in activating Rho1p (Ozaki et al., 1996). Indeed, like MID2, RHO1 is capable of suppressing the temperature sensitivity of a rom2 mutant. Further, Rom2p (Ozaki et al., 1996
) and Mid2p (Phillip & Levin, 2001) stimulate Rho1p-specific GDPGTP exchange activity. Since the slow growth of rom2
mutants is at the level of Rho1p, the synthetic lethality of rom2
mid2
mutants and the suppression of rom2
growth defects in a rom2
zeo1
double mutant suggest that Zeo1p and Mid2p play reciprocal roles in stimulating Rho1p. The idea that Mid2p and Zeo1p signal at the Rho1p level is further supported by genetic interactions of Mid2p with the Rho1p GTP-activating protein, Sac7p, where mid2 mutants can rescue the slow growth phenotype of sac7 mutants at 18 °C and overexpression of MID2 is lethal in a sac7
background. Genetic interactions between MID2, ZEO1 and components of the Rho1p GDPGTP exchange factor and GTP-activating protein suggest that Mid2p acts positively, while Zeo1p acts as a negative regulator of the pathway. For the Mid2p response to calcofluor white, Zeo1p is a critical component of this signalling pathway. Indeed, Philip & Levin (2001)
demonstrated that in response to calcofluor white, GTP loading of Rho1p is increased, suggesting that the Zeo1p control of the Mid2p response to calcofluor white is at the level of the Rho1p GTPase.
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ACKNOWLEDGEMENTS |
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Received 8 May 2003;
revised 17 June 2003;
accepted 19 June 2003.
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