Correspondence to Wei Guo: guowei{at}sas.upenn.edu
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Abbreviations used in this paper: Lgl, lethal giant larvae; SC, synthetic complete media.
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Introduction |
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The yeast homologues of LglSro7p and Sro77pwere originally identified as high-copy suppressors of Rho3p mutants, which is important for actin organization and polarized exocytosis (Kagami et al., 1998). Sro7p and Sro77p are homologous in sequence and share overlapping functions (Kagami et al., 1998; Larsson et al., 1998). Recent studies suggest that Lgl proteins regulate the late stage of exocytosis (Lehman et al., 1999; Musch et al., 2002; Widberg et al., 2003). For example, in epithelial cells, Lgl associates with syntaxin 4, the t-SNARE protein that mediates vesicle fusion at the basolateral domain of the plasma membrane (Musch et al., 2002). In yeast, Sro7p and Sro77p directly interact with the t-SNARE protein Sec9p, and double deletion of SRO7 and SRO77 genes results in exocytosis defects (Lehman et al., 1999). Based on the findings above, it is speculated that the role of Lgl in cell polarization may be attributed to its function in exocytosis, and it may promote targeted vesicle fusion at specific areas of the plasma membrane (Bilder et al., 2000; Musch et al., 2002). However, Lgl proteins themselves may not be able to confer the targeting information, as its distribution in the cell is not very restrictive. For example, in fly cells, Lgl is localized to both the cytosol and cell cortex (Bilder et al., 2000; Ohshiro et al., 2000; Peng et al., 2000). In epithelial cells, Lgl is distributed in the cytosol and is recruited to the lateral membrane after cellcell contactinitiated polarization (Musch et al., 2002). Although aPKC phosphorylation may help to limit the function of Lgl to the basolateral domain (Betschinger et al., 2003), there is evidence that vesicle targeting may be restricted to the region of the lateral membrane close to the junctional complex. (Louvard, 1980; Kreitzer et al., 2003). In yeast, Sro7p is distributed in the cytosol and along the entire plasma membrane (Larsson et al., 1998; Lehman et al., 1999). However, exocytosis is restricted to the bud tips in growing daughter cells. Therefore, there must be additional mechanisms that spatially restrict and/or kinetically promote the action of Lgl in exocytosis at specific areas of the plasma membrane.
The exocyst is an evolutionally conserved octameric protein complex consisting of Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p. This complex is localized to specific sites of the plasma membrane, where it tethers secretory vesicles for subsequent SNARE assembly and exocytosis (Lipschutz and Mostov, 2002; Novick and Guo, 2002; Hsu et al., 2004). How the exocyst, as a tethering complex (Whyte and Munro, 2002), communicates with and activates SNAREs is unknown. The exocyst is polarized to sites of active exocytosis and cell surface expansion. In epithelial cells, the exocyst localizes to trans-Golgi and recycling endosomes (Yeaman et al., 2001; Fölsch et al., 2003) and is recruited to the vicinity of adherens junctions upon the generation of apicalbasolateral asymmetry, where it mediates exocytosis exclusively at the basolateral membrane (Grindstaff et al., 1998). In yeast, exocyst proteins localize to the bud tips and motherdaughter junctions, which are sites of active exocytosis and cell growth (TerBush and Novick, 1995; Finger et al., 1998, Guo et al., 1999a). This pattern of localization is in contrast to that of t-SNAREs (Sso1p and Sso2p), which are distributed along the entire cell membrane (Brennwald et al., 1994). The yeast exocyst is a direct downstream effector of the Rab protein Sec4p (Guo et al., 1999b). In addition, the yeast exocyst is under the control of the Rho family of small GTPases, which coordinates actin organization and the membrane traffic for polarized cell growth (Adamo et al., 1999, 2001; Guo et al., 1999b; Robinson et al., 1999; Zhang et al., 2001).
In this study, we report that the yeast Lgl proteins Sro7/77p directly interact with a component of the exocyst, Exo84p, and disruption of this interaction leads to defects in exocytosis. Genetic analyses using an array of yeast mutants demonstrate a pathway in which the exocyst and Sro7 mediate signaling from the Rho3p and Rab protein Sec4p to control SNARE assembly. We also demonstrate that overexpression of Lgl and t-SNAREs not only helps improve exocytosis kinetically in exocyst mutants but also rescues the polarity defects in these cells. Our results suggest that the interaction of Lgl with the exocyst and upstream small GTPases at specific domains of the plasma membrane is important for localized SNARE assembly and polarized exocytosis. Conversely, up-regulation of Lgl and downstream t-SNARE functions may reenforce cell polarity. Altogether, our studies shed light on the molecular function of Lgl in exocytosis and cell polarization.
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Results |
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The two-hybrid analysis (Fig. 4 D) also suggests that the mutant exo84-202 protein still maintains a weak interaction with Sro7p. Moreover, its interaction with Sro77p was 43% of the wild-type level. These residual interactions may explain why the secretion defect in the exo84-202 mutant is not as significant as those observed for other sec genes. Sro7p and Sro77p are functionally redundant in yeast genome, as the deletion of only one of them does not cause any secretion defects (Lehman et al., 1999). We speculated that further disruption of the interaction between the exo84-202 and Sro proteins by the deletion of Sro7p (or especially Sro77p) in the exo84-202 strain may aggravate secretion defects in the cells. Therefore, we generated exo84-202 sro7
and exo84-202 sro77
strains and examined their secretion defects by EM. As shown in Fig. 5 A, exo84-202 cells accumulate secretory vesicles (average, 206 ± 60.6 vesicles/section) at 37°C. Further deletion of SRO7 in the exo84-202 background resulted in an increased accumulation of vesicles (262 ± 44.5 vesicles/section). In exo84-202 sro77
cells, there was a much larger increase in the number of secretory vesicles (460 ± 76.7 vesicles/section), indicating severe secretion defects. The severity of secretion defects in these mutants strongly correlated with the degree of binding disruption. We have also examined the growth of these cells on plates. As shown in Fig. 5 C, exo84-202 sro7
grows poorly compared with exo84-202 at 34°C (a semipermissive temperature for exo84-202). exo84-202 sro77
cells were barely able to grow under the same condition. These results suggest that further disruption of the Sro7/77pExo84p interaction in the exo84-202 background aggravates secretion and growth defects in the cells. The most likely explanation for the observed results is that Exo84p interacts with both Sro7p and Sro77p in the cells, forming parallel pathways for the regulation of exocytosis (Fig. 5 D). Deletion of SRO7 or SRO77 in the exo84-202 background removes one of the two parallel pathways, thus aggravating the exocytosis defects.
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Genetic analyses of the regulatory pathway involving the exocyst and Sro7/77p
Making use of our collection of yeast mutants, we next performed genetic experiments that were aimed at understanding the contexts of the Exo84pSro7/77p interaction. We found that SRO7 overexpression rescued exo84-202 growth at the restrictive temperature (Table II). SRO7 also suppressed several other exocyst mutants, including sec3-2, sec5-19, sec8-9, sec10-2, sec15-1, and exo70-38, which is similar to the results obtained by Lehman et al. (1999). We also found that overexpression of SRO7 can rescue SEC3 and EXO70 deletion (Table II and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502055/DC1), suggesting that the regulatory function of SRO7 is very likely downstream of the exocyst. However, overexpression of SRO7 cannot bypass EXO84 deletion. This observation and the results reported by Wiederkehr et al. (2004) suggest that Exo84p and several other exocyst components (Sec6p, Sec8p, Sec10p, and Sec15p) form the core tethering machinery for exocytosis. Sro7p, as a regulator in this process, cannot bypass the core vesicle-tethering proteins.
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SRO7 and SSO2 rescue the polarity defects of exocyst mutants
The above genetic analyses have shown that overexpression of SRO7 rescues several exocyst mutants. Then, we asked whether the Lgl function in kinetic regulation of exocytosis spatially contributes to cell polarization. The exocyst component Sec3p demarcates the subdomains of the plasma membrane for efficient and precise targeting of the secretory vesicles for exocytosis (Finger et al., 1998; Wiederkehr et al., 2003). Although vesicles can still be transported to daughter cells in the absence of Sec3p, they cannot find the appropriate sites within the buds. This results in isotropic membrane fusion along the entire daughter cell membrane rather than at the tip of the daughter membrane (the bud tip). After generations of growth, the sec3 cells are "rounder" in shape for both mother and daughter cells (lower cell length/width ratio), which is different from the normally ellipsoid shapes of wild-type yeast cells (Wiederkehr et al., 2003). Despite secretion and polarity defects, sec3
cells can survive at 25°C, allowing us to investigate the role of SRO7 by using genetics and cell biological methods. As shown in Fig. 7 A, sec3
cells grow poorly at 25°C and can barely survive at 34°C. However, cells that were transformed with 2µ plasmids containing SRO7 or SSO2 have much-improved growth at 25°C and become viable at 34°C. We have also compared the cell doubling times in liquid culture at 25°C. As shown in Fig. 7 B, doubling time of the sec3
cells (202 min) was more than two times longer than that of the wild-type cells (80 min). However, overexpression of SRO7 or SSO2 accelerated the growth rate of sec3
cells near the wild-type level. We measured invertase secretion in these cells at 25°C and 34°C. As shown in Fig. 7 C, sec3
cells accumulate 25% of the produced invertase. Overexpression of SRO7 or SSO2 greatly reduced the amounts of invertase in the cells, and growth and secretion properties correlated with each other.
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Morphology of exo84-202 mutant cells
Although overexpression of SRO7 can rescue the cell polarity defects of sec3, we examined whether the exo84-202 mutant, in which the physical interaction between the exocyst and Sro7p is disrupted, has polarity defects. Examination of the morphology of exo84-202 cells indicated that this mutant, like sec3
, is indeed rounder in shape (Fig. 9 A). This rounder shape can be observed even at 25°C and becomes more obvious at elevated temperatures. Also, like sec3
, Sec4p and Myo2p are less restricted in daughter cells (Fig. 9 B). Actin, although slightly diffused, remains polarized to the daughter cells (unpublished data), which is similar to observations made by Lehman et al. (1999) in sro7
sro77
cells. We have also examined whether exo84-202 cells form normal mating projections ("shmoos") in response to
-factor treatment. As shown in Fig. 9 C (right), in contrast to the "sharper" shmoos formed in wild-type cells, exo84-202 cells project rounder shmoos. Because SSO2 is a strong suppressor for exo84-202 (Table III), we examined whether SSO2 can rescue the polarity defects of exo84-202. As shown in Fig. 9, SSO2 overexpression greatly improved the polarity of exo84-202 cells.
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Discussion |
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The Sro7/77pSNARE interaction is a critical positive regulatory event for SNARE assembly and membrane fusion, and our biochemical and genetic analyses raise the possibility that the exocyst and upstream small GTPases provide the activation signal or spatial restriction for Sro7/77p function. It was shown that t-SNARE proteins and Sro7/77p are evenly distributed along the entire plasma membrane (Brennwald et al., 1994; Larsson et al., 1998; Lehman et al., 1999), whereas the exocyst is concentrated to the bud tip (TerBush and Novick, 1995; Finger et al., 1998; Guo et al., 1999a). Sro7/77pexocyst interaction may be important for restricting exocytosis to specific sites of the plasma membrane for polarized cell growth. Although the in vitro binding affinity for Exo84pSro7p interaction is high (Kd = 46 ± 6 nM), their in vivo interaction may be restricted to the bud tip, and this interaction may be modulated by several regulators such as Kin1/2p (Elbert et al., 2005). Overall, our data suggest a model in which Exo84p interacts with Sro7/77p, which then promotes SNARE-mediated membrane fusion at specific regions of the plasma membrane. Although the yeast Lgl proteins themselves are not polarized in distribution, they may achieve their function in polarized exocytosis kinetically through their localized interaction with the exocyst.
Does the function of Lgl in exocytosis contribute to cell polarization? Taking advantage of the simple morphology of budding yeast, we examined the effects of SRO7 and t-SNARE SSO2 overexpression on the exocyst mutant with polarity defects. The sec3 and exo84-202 cells are rounder in shape. These phenotypes resemble those of the polarisome mutants that play important regulatory roles during cell polarization in yeast (Sheu et al., 2000). We found that overexpression of SRO7 and t-SNARE SSO2 not only helped to rescue the secretion defects but also significantly improved the polarization of these cells. Neither Sro7p nor Sso2p has a polarized distribution pattern in the cells. Particularly, Sso2p, which is a yeast t-SNARE protein responsible for the fusion reaction of lipid bilayers, does not contain any targeting information in its sequence. The rescuing effects on cell polarity from Sro7p and Sso2p, therefore, probably contributed through their positive roles in promoting secretion kinetically in the cells. Consistent with this observation, it was recently shown that overexpression of Sec1p, a protein that interacts with t-SNARE, also rescues the polarity defect of sec3
cells (Wiederkehr et al., 2004). Our data suggest that, although the localized activation of Lgl may be controlled by the exocyst and small GTPases, increased Lgl and SNARE functions may conversely contribute to cell polarization. This reciprocal regulation may "fine tune" or reenforce cell polarity, leading to tightly restricted secretion at the tip of the bud. In this regard, it is interesting to note that a previous study demonstrated that delivery of the polarisome component Bud6p/Aip3p to the bud relies on the functional secretory pathway (Jin and Amberg, 2000). We do not exclude the possibility that other yet unidentified functions of Lgl may also contribute to this rescuing effect. Future identification and characterization of Lgl-interacting proteins may help to elucidate the role(s) of Lgl in cell polarization.
The core mechanisms for the generation and maintenance of cell polarity are evolutionally conserved. The sequences and many functions of the exocyst and Lgl proteins are also conserved. Previous analysis shows that both fly and mammalian Lgl can partially rescue the yeast sro7 sro77
mutant (Kagami et al., 1998; Larsson et al., 1998). It will be interesting to extend this study to higher eukaryotes to examine whether Lgl and the exocyst carry out their functions in targeting regulators of cell morphogenesis for the establishment and reenforcement of cell polarity.
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Materials and methods |
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Yeast two-hybrid assays
The cDNAs encoding exocyst components and Sro7p and Sro77p were subcloned in pACTII and pOBD8 vectors, respectively, and were expressed in Y190 cells. Interactions were detected by measuring whole cell ß-galactosidase activities as previously described (Guo et al., 1999a).
Recombinant proteins
For purification of recombinant Sro7p, a protein A sequence was engineered at the NH2 terminus of Sro7p, and the fusion protein was expressed under the control of the ADH1 promoter in yeast. Yeast lysates containing the recombinant protein were incubated with IgGSepharose beads. For in vitro synthesis of radiolabeled Sro7p, Sro7 was subcloned into pcDNA3, the purified plasmid was added to the rabbit reticulocyte lysatecoupled in vitro transcriptiontranslation system under the T7 promoter (Promega) in the presence of [35S]methionine, and the plasmid was processed as described by the manufacturer. The in vitrosynthesized Sro7 is shown in Fig. 2 A as doublets, which either resulted from the degradation or downstream initiation of translation by the translation machinery in the reticulocyte lysate system. For in vitro synthesis of radiolabeled Exo84p, Exo84 cDNA was amplified by PCR and was directly used in the in vitro transcriptiontranslation reaction.
In vitro binding assays
For binding of the radiolabeled Exo84p to protein ASro7p Sepharose beads, 5 µl [35S]methionine-labeled in vitro transcriptiontranslation reaction mixture was diluted to 85 µl in binding buffer (10 mM Hepes/KOH, pH 7.4, 140 mM KCl, 2 mM MgCl2, 0.5% Triton X-100, and protease inhibitors) and was preincubated with Sepharose beads for 15 min on ice followed by centrifugation. The resulting supernatant was used for a 100-µl binding reaction using 2 µM immobilized protein ASro7p. The reaction mixture was incubated for 90 min at 4°C, and the supernatants were separated from the pellets by centrifugation. A similar procedure was used to test the binding between radiolabeled Sro7p and GST-Exo84N conjugated to glutathioneSepharose beads, except that the binding reaction took place in 500 µl of binding buffer (20 mM Hepes-NaOH, 140 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT, and 1 mM PMSF) for 4 h at 4°C. The samples were subjected to SDS-PAGE and autoradiography. Quantitation was performed using a phosphoimager (model Storm; Molecular Dynamics) with ImageQuant software (Molecular Dynamics).
To obtain the dissociation constant of the Sro7pExo84p interaction, various amounts of purified GST-Exo84N were incubated with 10 nM protein ASro7p conjugated to IgGSepharose beads in 1,000 µl of binding buffer. The reaction mixtures were incubated at RT for 2 h followed by washing. The protein samples were subjected to SDS-PAGE, and the gels were stained by Coomassie blue. The bound and free GST-Exo84N were quantified with ImageQuant and were plotted with a single rectangular hyperbola equation (B = BmaxC/[Kd + C]) using the SigmaPlot software (Systat Software, Inc.). The dissociation constant was calculated from each plot by nonlinear regression.
Coprecipitation assays
Yeast cells expressing GST or GST-Sro7p in a CEN plasmid under the TEF promoter were grown to early log phase, and cell lysates were prepared by disrupting the cell wall using glass beads in buffer A (20 mM Hepes, 150 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, and protease inhibitors). The lysates were incubated with glutathioneSepharose 4B for 1 h at 4°C. The beads were washed, and proteins that were precipitated with the GST fusion proteins from lysates were analyzed by Western blot. For immunoprecipitation of Sro7p and Exo84p at their endogenous levels, Exo84p was tagged with the myc epitope by chromosomal integration, and detergent extracts were prepared by using buffer A. A rabbit anti-Sro7p antibody was used at a 1:300 dilution in the lysates for precipitation, and precipitated Sro7p and Exo84p were detected by Western blot analysis. As controls, rabbit anti-GFP and anti-GST pAbs (Convance, Inc.) were used in the same procedure.
Generation of exo84 mutants
For generation of the exo84-202 mutant, mutagenesis focusing on the NH2 terminus of Exo84p was performed by using error-prone PCR as previously described (Zhang et al., 2001). The forward primer covers 300 bp upstream of the starting codon, and the reverse primer covers the Exo84 ORF bp 1900 region. Mutagenized PCR products were mixed with linearized CEN LEU2 plasmid containing the Exo84 promoter and bp 9002260 tagged with a 12Xmyc sequence at the 3' end. Then, this mixture was transformed into GY1264, in which the endogenous EXO84 gene was disrupted by HIS3 and supplemented with a CEN URA3 EXO84 plasmid to allow gap repair between the PCR products and the linearized plasmid. Transformants were selected on synthetic complete media (SC) LeuHisUra plates at 25°C and were replicated onto SC LeuHis plates containing 1 mg/ml 5-fluoroorotic acid to select for the loss of the CEN URA3 EXO84 plasmid. The selected colonies were replicated onto three sets of SC LeuHis plates and were incubated at 18°C, 25°C, and 37°C, respectively, to allow the identification of temperature-sensitive mutants. Possible mutants were confirmed by the retransformation of isolated plasmids into the GY1264 host strain. In addition to the exo84-202 strain, we also generated strains with the SRO7 or SRO77 gene deleted in the exo84-202 background (exo84-202 sro7 and exo84-202 sro77
, respectively) by replacement of the chromosomal copy of SRO genes with KanM cassette. The desired strains were selected by 0.2 mg/ml G418 on SC plates and were confirmed by PCR. Besides exo84-202, we have also generated other exo84 mutants by random mutagenesis of the whole Exo84 ORF by using a similar error-prone PCR strategy (Zhang et al., 2005). One such mutant, exo84-121, was used in this study.
Light microscopy
Immunofluorescence staining of yeast cells was performed as previously described (Walch-Solimena et al., 1997). Rabbit anti-Sec4p and anti-Myo2p pAbs were used at a 1:1,000 dilution followed by a secondary AlexaFluor594-conjugated goat antirabbit IgG antibody (Molecular Probes). The digital images were captured by a fluorescence microscope (model DM IRB; Leica) using a 100x objective and a high resolution CCD camera (model ORCA-ER; Hamamatsu Photonics). Immunofluorescence signals were quantified as pixels by using OpenLab 3.1.4 software (Improvision). For morphological analysis, the length and width of yeast cells were measured on differential interference contrast images using OpenLab software, which is basically similar to that used in the Saccharomyces cerevisiae Morphological Database (http://scmd.gi.k.u-tokyo.ac.jp/). The roundness of the cells was calculated as the axial ratio (length/width) in the mother cells. To observe shmoo formation, Mat a yeast cells were cultured to OD600 = 0.2 followed by 1 mg/ml -factor treatment. Light microscopy images were taken 6 h after
-factor addition. The curvature radius of the shmoo tips was analyzed by using the OpenLab 3.1.4 program.
EM
Cells were collected by vacuum filtration using a 0.45-µm nitrocellulose membrane and were fixed for 1 h at RT in 0.1 M cacodylate, pH 7.4, 3% formaldehyde, 1 mM MgCl2, and 1 mM CaCl2. The cells were spheroplasted and fixed with 1% glutaraldehyde (in PBS, pH 7.4) at 4°C overnight. The spheroplasts were washed in 0.1 M cacodylate buffer and were postfixed twice with ice-cold 0.5% OsO4 and 0.8% potassium for 10 min each. After dehydration and embedding in Spurr's epoxy resin (Polysciences, Inc.), thin sections were cut and transferred onto 600 mesh uncoated copper grids (Ernest Fullam, Inc) and were poststained with uranyl acetate and lead citrate. Cells were observed on a transmission electron microscope (model 1010; JEOL) at 100,000x.
Online supplemental material
Fig. S1 shows that SRO7 overexpression rescues exo70. The supplemental materials and methods paragraph explains the methods used for the rescuing experiment. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200502055/DC1.
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Acknowledgments |
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This work is supported by grants from the National Institutes of Health (RO1-GM64690), American Cancer Society, and the Pew Scholar Program in Biomedical Sciences (to W. Guo).
Submitted: 9 February 2005
Accepted: 10 June 2005
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