![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 11, 4483-4492, November 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018
Submitted May 17, 2007;
Revised August 14, 2007;
Accepted August 17, 2007
Monitoring Editor: Tom U. Martin
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Guo et al., 2000; Waters and Hughson, 2000; Whyte and Munro, 2002), is mediated by the exocyst, an evolutionarily conserved octameric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (for review, see Guo et al., 2000; Hsu et al., 2004; Munson and Novick, 2006; Wang and Hsu, 2006). In budding yeast, the exocyst components localize to the growing end of the daughter cell ("bud"), where active exocytosis and membrane addition take place (TerBush and Novick, 1995; Finger et al., 1998, Guo et al., 1999). This localization pattern contrasts that of the membrane fusion machine, the t-SNAREs, which are evenly distributed along both the mother and daughter cell membrane (Brennwald et al., 1994). In mammalian cells, the exocyst components were found in the cytosol, recycling endosomes and trans-Golgi network (Yeaman et al., 2001; Fölsch et al., 2003; Ang et al., 2004; Langevin et al., 2005). However, they are recruited to the PM during a number of cellular processes. For example, in epithelial cells, the exocyst is recruited to the adherens junction region upon cell–cell contact, where it mediates protein and membrane addition at the basolateral domain (Grindstaff et al., 1998; Yeaman et al., 2001); in developing neurons, the exocyst is localized to the growing neurites, where it mediates membrane expansion (Hazuka et al., 1999; Vega and Hsu, 2001); during cell migration, the exocyst is recruited to the leading edges of the PM (Rosse et al., 2006; Zuo et al., 2006).
The mechanism by which the exocyst mediates vesicle tethering to the PM is unclear. One key question yet to be resolved is how the exocyst itself associates with the PM. Using fluorescence recovery after photobleaching (FRAP) analyses and immunoelectron microscopy, Boyd et al. (2004) have shown that Exo70 is stably localized to the yeast bud tip membrane and remains polarized even when the actin cables are disrupted, suggesting that Exo70 is a candidate in this complex involved in membrane targeting of the exocyst. In Madin-Darby canine kidney (MDCK) cells, extragenically expressed GFP-tagged Exo70 is localized to the PM near cell–cell contacts, suggesting that Exo70 may mediate PM association independent of the rest exocyst components in these cells (Matern et al., 2001). Recent structural studies have revealed that Exo70 contains a number of conserved basic residues that cluster on a surface patch at the C-terminal end of the tertiary structure that may directly bind to the PM (Dong et al., 2005; Hamburger et al., 2006; Moore et al., 2007). In fact, the C-terminal sequence of Exo70 is the most evolutionarily conserved region of this protein.
Here, we report that mammalian Exo70 directly interacts with PI(4,5)P2 in the PM via the positively charged residues at its C-terminus. We have also identified key residues in Exo70 that are important for this interaction. Finally, using the ts045 vesicular stomatitis virus glycoprotein (VSV-G) trafficking assay, we found that the Exo70-lipid interaction is critical for PM stages of exocytosis, but not for the trafficking steps through endoplasmic reticulum (ER) and Golgi. Our study revealed a molecular mechanism by which the exocyst directly interacts with the PM that is critical for vesicle tethering and exocytosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture and RNA Interference Experiments
HeLa cells were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO2 incubator. For RNA interference (RNAi) experiments, cells were grown to 50% confluence and transfected with small interfering RNA (siRNA) duplexes using Oligofectamine (Invitrogen, Carlsbad, CA). The human Exo70 siRNA target sequence is 5'-GGTTAAAGGTGACTGATTA-3'. The control Luciferase GL2 siRNA target sequence is 5'-AACGTACGCGGAATACTTCGA-3'. The efficiency of Exo70 knockdown was determined by Western blot.
Confocal Microscopy
Transfected HeLa cells were grown on coverslips, washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 12 min, washed, permeabilized for 5 min with PBST (PBS-Tween), and blocked for 10 min with 2% bovine serum albumin in PBST. The coverslips were incubated sequentially with primary and secondary antibodies for fluorescence observation using the Leica TCS SL laser-scanning confocal microscope (63x objective; Deerfield, IL). Images were processed with Adobe Photoshop (Adobe Systems, San Jose, CA; version 7.0).
Large Unilamellar Vesicle Sedimentation Assay
Large unilamellar vesicle (LUV) sedimentation assay was performed as previously described (Hokanson and Ostap, 2006). Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). LUVs with a 100-nm diameter were prepared by size extrusion. Various lipids were mixed at different molar ratios, dried with nitrogen stream, and resuspended at a concentration of 2 mM in a buffer containing 12 mM HEPES, pH 7.0, and 176 mM sucrose. The mixed lipids were subjected to five cycles of freeze-thaw and a 1-min bath sonication before being passed through 100-nm filters using a mini-extruder. LUVs were dialyzed overnight in the HNa100 buffer (10 mM HEPES, pH 7.0, 100 mM NaCl, 1 mM EGTA, and 1 mM dithiothreitol [DTT]). The percentages of phosphotidylserine (PS), PI(3)P, PI(4)P, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3 indicated in the text are the molar percentages of total PS and PIPs with the remainder being phosphatidylcholine (PC). Lipid concentrations are given as total lipid. The binding of Exo70 to LUVs was determined by sedimentation assays in 200 µl total volume using TLA-100 rotor (Beckman Coulter, Fullerton, CA). Sucrose-loaded LUVs were precipitated at 150,000 x g for 30 min at 25°C. The supernatants and pellets were subjected to 10% SDS-PAGE and stained with SYPRORed (Invitrogen) for quantification of free and bound materials with the Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Membrane Fractionation
HeLa cells were plated in 10-cm dishes at 1.5 x 106 cells per dish. The next day cells were transfected with DNA by FuGene6 reagent and incubated at 37°C overnight. Homogenization and subcellular fractionation of the cells to isolate the PM fraction, cytosol, the low-density microsomal fraction (LDM), and the high-density microsomal fraction (HDM) were performed basically as previously described (Weber et al., 1988). All steps were performed at 4°C in the presence of a protease inhibitor cocktail. The distribution of Exo70 and Sec8 were detected by monoclonal antibodies (kind gifts of Dr. Shu-Chan Hsu, Rutgers University).
GST Pulldown Assay
HeLa cells were transfected with GFP-tagged Exo70 or exo70-1, and the cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors. Cell lysates were incubated overnight with glutathione-Sepharose conjugated with GST or GST-TC10 (Q75L) at 4°C. After incubation, the beads were washed five times with the lysis buffer, and the bound proteins were analyzed by Western blot using an anti-GFP antibody. To detect the interaction of Exo70 and Sec8 in the cell, HeLa cells were transfected with GST-tagged Exo70 or exo70-1, and cell lysates were incubated overnight with glutathione-Sepharose beads at 4°C. After incubation, the beads were washed, and the bound proteins were detected by Western blot using anti-GST or anti-Sec8 monoclonal antibodies.
VSV-G Trafficking Assay
HeLa cells were transfected with EXO70 siRNA. luciferase siRNA was used as the negative control. After 24 h of the siRNA treatment, HeLa cells were transfected with VSV-G-45ts-GFP mutant and immediately placed at 40°C. After overnight growth, the cells were shifted to 32°C for 0, 15, 30, 60, and 90 min in the presence of cycloheximide (100 µg/ml). The cells were then fixed for GFP observation or immunofluorescence. The 8G5 mAb against the extracellular domain of VSV-G was kindly provided by Dr. Douglas Lyles (Wake Forest University). No detergent was used in the immunofluorescence procedure. Cells with surface VSV-Gs were quantified, and statistical analyses were performed using Student's t test. In some cases, HeLa cells were transfected with VSV-G-myc and GST-Exo70 or GST-exo70-1 after 24 h of the EXO70 siRNA treatment. The cells were divided into two sets based on their treatments. For Set I, the cells were fixed, permeabilized, and stained with anti-myc mAb (9E10) and anti-GST polyclonal antibody to test the intracellular traffic of VSV-G and to detect the expression of Exo70 or exo70-1 at 0-, 30-, 60-, and 90-min points. For Set II, cells of the 90-min point group were first stained with the 8G5 antibody, then permeabilized, and stained with anti-GST polyclonal antibody. Anti-mouse Alexa488 and anti-rabbit Alexa594 were used as secondary antibodies for the above experiments. For the quantification of surface VSV-G signals at various points, boundary of the cell surface was outlined, and average fluorescence intensity of surface VSV-G signal was quantified using ImageJ 1.73v software and then divided by the perimeter of the cell surface. For the quantification of VSV-G in different membrane compartments, boundaries of the whole cell, the Golgi, and the cell periphery were outlined, and VSV-G fluorescence in these areas was then quantified using ImageJ 1.73v software after subtraction of background outside the cell using the following equations:
 |
 |
 |
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Xu et al., 2005; Zuo et al., 2006). We next mapped the region of Exo70 that is required for its association with the PM by examining the localization of a series of GFP-tagged Exo70 truncates in HeLa cells. As shown in Figure 1B, Exo70 with its C-terminal domain deleted (amino acids 1-408; Exo70-NT) was cytosolic, whereas the C-terminus of Exo70 (amino acids 403-653; Exo70-CT) was enriched at the PM (Figure 1B), suggesting a critical role of Exo70 C-terminus in membrane association. The crystal structure of yeast Exo70 revealed that this protein is a long rod composed mainly of 
-helices that fold into four domains (named domains A, B, C, and D; Dong et al., 2005; Hamburger et al., 2006). Domain D (the C-terminal 114 amino acids) is the most evolutionarily conserved domain in Exo70 that contains a number of basic residues that cluster into an electro-positive patch on the surface of the C-terminus. Because in many cases the association of proteins with the PM involves interactions with the negatively charged phospholipids in membrane via clusters of basic residues (for reviews, see McLaughlin et al., 2002; Balla, 2005), it is likely that those positively charged residues at the C-terminus of mammalian Exo70 are directly involved in membrane association. We sought to examine whether domain D is required for the PM localization of Exo70. We generated truncations of Exo70, in which the last 95 amino acids (aa 559-653) or the last 45 amino acids (aa 609-653) were deleted. The resulting Exo70 fragments, Exo70 (aa 1-558) and Exo70 (aa 1-608), were both cytosolic (Figure 1B), indicating that domain D of mammalian Exo70, especially the last 45 amino acids, is required for the association of Exo70 with the PM. We have also tested the localization of Exo70 (aa 403-558) and Exo70 (aa 403-608), which are C-terminal fragments of Exo70 with the last 95 amino acids (aa 559-653) or the last 45 amino acids (aa 609-653) deleted, and found that both of the fragments were cytosolic (Figure 1B). We next investigated whether the C-terminal fragments of Exo70 containing aa 403-653 or aa 540-653 (domain D) were able to associate with the PM. As shown in Figure 1B, both fragments were localized to the PM, suggesting that domain D of Exo70 is sufficient for its PM localization. A portion of these fragments was also detected in the nucleus, probably resulting from nonspecific retention of the GFP fusion. Collectively, these data indicate that the C-terminus of Exo70 is both necessary and sufficient for the PM targeting of Exo70.
|
; Hokanson and Ostap, 2006). PI(4,5)P2 is the major phosphatidylinositol species, and constitutes 1–5% of the total lipids in the PM (for review, see McLaughlin et al., 2002). Binding experiments were performed with LUVs composed of the neutral PC and PI(4,5)P2 at 5% or the acidic phospholipid PS in molar percentages of 20, 40, and 60%. Because the recombinant Exo70 protein could only access the outer leaflet of the reconstituted LUV vesicles, the effective PI(4,5)P2 and PS in the reaction were only half of the total. As shown in Figure 2A, Exo70 bound to LUVs containing 5% PI(4,5)P2, but not to LUVs containing 100% neutral PC. Exo70 also bound to PS; however, the binding was weak unless the molar ratio of PS in the LUVs was raised to 60%. As a control, GST did not bind to LUVs with any lipid composition.
|
|
). PI(4,5)P2 comprises more than 95% of the bis-phosphoinositides in mammals (Bonangelino et al., 2002). PI(3)P and PI(3,5)P2 are mainly localized to the endosomal compartments (for reviews, see Behnia and Munro, 2005; Di Paolo and De Camilli, 2006). PI(3,4,5)P3 is present in negligible amount in mammalian cells at the "resting" state, but is up-regulated at specific domains of the PM in response to certain stimuli (Insall and Weiner, 2001). Recently, it was found that PI(3,4,5)P3 is also localized to the basolateral domain of MDCK cells (Gassama-Diagne et al., 2006). As shown in Figure 2C, Exo70 barely binds to PI(3)P; the affinity of Exo70 for PI(4,5)P2 (Kd = 13.92 ± 3.0 µM) is 
4–5-fold higher than PI(3,5)P2 (Kd = 58.34 ± 9.2 µM) and more than 10-fold higher than PI(4)P (Kd = 173.12 ± 15.5 µM). The affinity of Exo70 for PI(3,4,5)P3 (Kd = 15.55 ± 4.6 µM) is comparable to that of PI(4,5)P2.
Mutations in Domain D Disrupt the Plasma Membrane Association of Exo70
The direct binding of Exo70 with PI(4,5)P2 suggests a critical role for the domain D basic residues in the PM association of Exo70. To identify the responsible residues that are essential for Exo70 to bind to the PM, the conserved basic residues in domain D of Exo70 (Figure 3A) were targeted for mutagenesis and the resulting mutants were tested for their cellular localization (summarized in Supplementary Table 1). Among the 10 mutants we tested, 6 failed to associate with the PM. The other four mutations had no effect on Exo70 localization, suggesting that not all of the basic residues are involved in PI(4,5)P2 binding. We focused on one of the mutants, named exo70-1, in which residues K632 and K635 have been mutated to alanine. As shown in Figure 3B, GFP-tagged wild-type Exo70 (GFP-Exo70) was enriched at the PM, whereas GFP-tagged exo70-1 was distributed diffusely throughout the intracellular regions. These results indicate that K632 and K635 are required for the PM localization of Exo70.
|
Next, we examined the interaction of the mutant exo70-1 protein with phospholipids in the cosedimentation assay. As shown in Figure 4 and Table 1, the interaction between exo70-1 and 5% PI(4,5)P2 was almost abolished, indicating that K632 and K635 are critical residues for the binding of Exo70 to PI(4,5)P2. Interestingly, the binding of exo70-1 for PS was only partially affected, suggesting that these two residues confer certain degree of specificity for PI(4,5)P2.
|
). We then tested whether mutations in exo70-1 affect this interaction. Cell lysates expressing GFP-tagged Exo70 or exo70-1 were incubated with GST-TC10 (Q67L) conjugated to the glutathione-Sepharose for binding reaction. Exo70 or the exo70 mutant bound to the Sepharose was detected by immunoblotting using an anti-GFP antibody. The amount of exo70-1 bound to the TC10 beads was comparable to that of the wild-type Exo70 (Supplementary Figure 1A). As a control, neither the wild-type Exo70 nor exo70-1 bound to the GST beads. The result indicates that mutating the two key basic residues in exo70-1 does not affect its interaction with TC10. Therefore, the loss of PM association of the exo70 mutant is unlikely resulted from impaired interaction with TC10. The binding results strongly suggest that Exo70 associates with the PM via its direct interaction with PI(4,5)P2. In addition to TC10, we have also tested the binding of exo70-1 with another exocyst component Sec8. We found that mutations on exo70-1 did not affect its interaction with Sec8 in the cell (Supplementary Figure 1B). This result is consistent with the previous reports in mammalian and yeast cells that the interaction of Exo70 with the other exocyst components for complex assembly is mediated by the N-terminal domains of Exo70 (Inoue et al., 2003; Dong et al., 2005).
Exo70 Recruits Sec8 to the Plasma Membrane
We next asked whether Exo70 is involved in recruiting other members of the exocyst to the membrane. It has been shown that the exocyst components were mostly located in the cytoplasm in cultured HeLa cells (Zuo et al., 2006). We then examined whether an increase of Exo70 at the PM would recruit Sec8 to the membrane. GFP-Exo70 and GFP-exo70-1 were expressed in HeLa cells, and Sec8 in these cells was detected by immunofluorescence staining using the anti-Sec8 mAb 2E12. As shown in Figure 5, both GFP-Exo70 and Sec8 were detected at the PM. On the contrary, in cells expressing GFP-exo70-1 that is deficient in binding PI(4,5)P2, Sec8 was found in the cytoplasm and intracellular membrane structures. This result suggests that the wild type, but not the mutant Exo70, is able to recruit the other exocyst components to the PM. Because exo70-1 maintains its ability to interact with Sec8, the loss of PM association of Sec8 in cells expressing GFP-exo70-1 is unlikely resulted from a defect in exocyst complex assembly.
|
). As shown in Figure 6B, in luciferase siRNA-treated cells, VSV-G-GFP was retained in the ER before the cells were shifted from 40 to 32°C (0 min). After the shift, the protein was translocated from ER to the Golgi apparatus around 30 min and then to the cell surface at 90 min. The exocytosis of VSV-G-GFP at the PM at 90 min was clearly detected by immunostaining of nonpermeabilized cells with the 8G5 antibody (Figure 6B). In EXO70 siRNA-treated cells, the translocation of VSV-G-GFP from the ER to the Golgi apparatus and from the Golgi to the PM followed time courses similar to that in control siRNA-treated cells. However, at 90 min, the extracellular exposure of VSV-G-GFP was barely detectable in nonpermeabilized cells; instead, it could only be detected after 180 min (Figure 6C), suggesting that the fusion of the exocytic vesicles with the PM was severely delayed. Quantification of the GFP signal in different intracellular compartments indicates that the cellular distributions of VSV-G-GFP at various time points after the temperature shift were similar in EXO70 siRNA and luciferase siRNA-treated cells (Figure 6, D and E): at 0 min, a majority of VSV-G-GFP was located at the ER (88% for luciferase siRNA treatment and 84% for EXO70 siRNA treatment); at 30 min, most of the VSV-G-GFP was located at the Golgi apparatus (91% for luciferase siRNA and 92% for EXO70 siRNA treatment); and at 90 min, VSV-G-GFP was located at the cell periphery (88% for luciferase siRNA and 74% for EXO70 siRNA treatment). In contrast, quantification of the surface VSV-G signal revealed a different pattern in EXO70 siRNA and luciferase siRNA-treated cells. As shown in Figure 6F, at 0-, 15-, and 30-min points, fluorescence intensity of exposed VSV-G was negligible both in control and EXO70 siRNA-treated cells, which suggests that VSV-G-GFP was translocating in the intracellular compartments; at 60 and 90 min, fluorescence intensity of exposed VSV-G was much less in EXO70 siRNA-treated cells than in control siRNA-treated cells; at 180 min, fluorescence intensity of surface VSV-G in EXO70 siRNA-treated cells began to appear at the cell surface. These results suggest that the fusion of the exocytic VSV-G vesicles with the PM was delayed in cells treated with EXO70 siRNA. These results demonstrated that Exo70 is probably not required for the trafficking of VSV-G from the intracellular compartments to the cell periphery, but is essential for the efficient tethering or subsequent docking/fusion of VSV-G–containing exocytic vesicles with the PM.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
PI(4,5)P2 and PS are the major negatively charged lipids in the PM. The fact that Exo70 binds to both 5% PI(4,5)P2 and 60% PS indicates that the interaction of Exo70 with the phospholipids is electrostatic in nature. This type of interaction has been found in a number of proteins, such as N-WASP and MARCKS (McLaughlin and Murray, 2005). Comparing the charges of PI(4,5)P2 versus PS at the physiological pH, LUVs composed of 5% PI(4,5)P2 have approximately the same amount of effective charges as LUVs composed of 15–20% PS. However, the interaction of Exo70 with LUVs composed of 20% PS is much weaker. Mutations on exo70-1 that eliminate some of the positive charges in the Exo70 C-terminus nearly abolished the ability of Exo70 to bind PI(4,5)P2; however, the ability of this mutant to bind PS at high concentrations (
60%) was only partially affected. These data suggest that Exo70 has significant binding specificity for PI(4,5)P2 over PS. We have also examined the interaction of Exo70 with other phosphoinositides and observed its selectivity for PI(4,5)P2 over the stereoisomeric PI(3,5)P2 and other monophosphorylated phosphoinositides. We have also found that Exo70 binds PI(3,4,5)P3 with an affinity that is comparable to that for PI(4,5)P2. PI(3,4,5)P3 has recently been found to be localized to the basolateral domain in MDCK cells (Gassama-Diagne et al., 2006), and the exocyst has been implicated in basolateral vesicle targeting (Grindstaff et al., 1998). In other types of mammalian cells, although the concentration of PI(3,4,5)P3 is low at the PM in resting cells, it can be rapidly up-regulated in response to extracellular stimuli. It is therefore possible that Exo70 binds to PI(3,4,5)P3 under certain physiological circumstances or in certain cell types.
In yeast, we have found that the amount of the exocyst complex associated with the PM was much lower in the temperature-sensitive mss4 mutant cells, in which the PI(4,5)P2 level in the PM was reduced (data not shown), indicating that PI(4,5)P2 mediates the membrane targeting of the exocyst. Moreover, the structural analysis of Exo70 provided important insights into the potential mechanism of membrane association of Exo70 (Dong et al., 2005; Hamburger et al., 2006). On the basis of the crystal structure information of yeast Exo70, we have made mutations on the rat Exo70 residues K632 and K635 (exo70-1), which are positively charged amino acids well conserved in the yeast Exo70 sequence. Our in vitro vesicle sedimentation experiments demonstrated that these mutations disrupted the PI(4,5)P2-binding. Furthermore, the VSV-G trafficking analysis further demonstrated its functional importance in exocytosis at the PM. While we were preparing this article, Moore et al. (2007) resolved the crystal structure of mouse Exo70. Analysis of the structure indicates that the point mutations on exo70-1 are localized on the loop between Helix 18 and 19 on the surface of the mouse Exo70, which is almost identical to that of the yeast Exo70.
Taking advantage of the VSV-G trafficking assay, we were able to analyze the role of Exo70 in various stages of membrane traffic. More importantly, using the exo70-1 mutant in the EXO70 RNAi knockdown cells, we were able to specifically examine the functional significance of Exo70- PI(4,5)P2 interaction in VSV-G exocytosis. The exocyst has been found in various cellular compartments, including Golgi and recycling endosomes, in addition to the PM (Yeaman et al., 2001; Fölsch et al., 2003; Ang et al., 2004; Langevin et al., 2005). Here we found that the Exo70-PI(4,5)P2 interaction is not involved in the early stages of VSV-G trafficking through the endoplasmic reticulum and Golgi. Rather, it is critical for the PM events such as vesicle tethering and fusion. When the Exo70-PI(4,5)P2 interaction was disrupted, the transport of VSV-G to the PM was barely changed. However, the incorporation of VSV-G into the PM was significantly affected, as revealed by the 8G5 antibody specifically recognizing the extracellular domain of VSV-G. Similarly the exocyst has been implicated in tethering and fusion of Glut4-containing vesicles in 3T3-L1 adipocytes (Inoue et al., 2003; Ewart et al., 2005; Tsuboi et al., 2005). It is possible that other exocyst components also interact with phospholipids (Moskalenko et al., 2003). However, specific disruption of Exo70-PM interaction is sufficient to block exocytosis in mammalian cells.
The association of the exocyst with the PM is an important step in vesicle tethering. When and where this interaction takes place may regulate the kinetics and location of exocytosis. In the budding yeast S. cerevisiae, the exocyst complex is specifically localized to the growing tip of the daughter cell (the "bud tip"), which is the site of active exocytosis and cell surface expansion. Moreover, Exo70 primarily functions at the early stages of the yeast cell cycle, suggesting a temporal control of Exo70 function (He et al., 2007). In mammalian cells, growth factor signaling involving small GTPases may mediate the subunits assembly, translocation of the exocyst from intracellular compartments to, or activation of the exocyst at, the PM (Sugihara et al., 2002; Moskalenko et al., 2002, 2003; Inoue et al., 2003; Takaya et al., 2004; Zuo et al., 2006). Future work will be focused on the identification and characterization of proteins that temporally and/or spatially regulate Exo70 and other exocyst components using different eukaryotic systems.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
* These authors contributed equally to this work.
Address correspondence to: Wei Guo (guowei{at}sas.upenn.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Balla, T. (2005). Inositol-lipid binding motifs: signal integrators through protein-lipid and protein-protein interactions. J. Cell Sci 118, 2093–2104.
Behnia, R., and Munro, S. (2005). Organelle identity and the signposts for membrane traffic. Nature 438, 597–604.[CrossRef][Medline]
Bonangelino, C. J., Nau, J. J., Duex, J. E., Brinkman, M., Wurmser, A. E., Gary, J. D., Emr, S. D., and Weisman, L. S. (2002). Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p. J. Cell Biol 156, (6), 1015–1028.
Boyd, C., Hughes, T., Pypaert, M., and Novick, P. (2004). Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol 167, (5), 889–901.
Brennwald, P., Kearns, B., Champion, K., Keranen, S., Bankaitis, V., and Novick, P. (1994). Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 79, (2), 245–258.[CrossRef][Medline]
Di Paolo, G. P., and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443, (7112), 651–657.[CrossRef][Medline]
Dong, G., Hutagalung, A. H., Fu, C., Novick, P., and Reinisch, K. M. (2005). The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol 12, 1094–1100.[CrossRef][Medline]
Finger, F. P., Hughes, T. E., and Novick, P. (1998). Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, (4), 559–571.[CrossRef][Medline]
Ewart, M. A., Clarke, M., Kane, S., Chamberlain, L. H., and Gould, G. W. (2005). Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in 3T3–L1 adipocytes. J. Biol. Chem 280, (5), 3812–3816.
Fölsch, H., Pypaert, M., Maday, S., Pelletier, L., and Mellman, I. (2003). The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. J. Cell Biol 163, 351–362.
Gassama-Diagne, A., Yu, W., ter Beest, M., Martin-Belmonte, F., Kierbel, A., Engel, J., and Mostov, K. (2006). Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat. Cell Biol 8, (9), 963–970.[CrossRef][Medline]
Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, E., Scheller, R. H., and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93, (5), 731–740.[CrossRef][Medline]
Guo, W., Roth, D., Walch-Solimena, C., and Novick, P. (1999). The exocyst is an effector for Sec4p targeting secretory vesicles to sites of exocytosis. EMBO J 18, (4), 1071–1080.[CrossRef][Medline]
Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S., and Novick, P. (2000). Protein complexes in transport vesicle targeting. Trends Cell Biol 10, (6), 251–255.[CrossRef][Medline]
Hamburger, Z. A., Hamburger, A. E., West, A. P., Jr, and Weis, W. I. (2006). Crystal structure of the S. cerevisiae exocyst component Exo70p. J. Mol. Biol 356, (1), 9–21.[CrossRef][Medline]
He, B., Xi, F., Zhang, J., TerBush, D., Zhang, X., and Guo, W. (2007). Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth. J. Cell Biol 176, (6), 771–777.
Hokanson, D., and Ostap, M. (2006). Myo1c binds tightly and specifically to phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. Proc. Natl. Acad. Sci. USA 103, (9), 3118–3123.
Hazuka, C. D., Foletti, D. L., Hsu, S. C., Kee, Y., Hopf, F. W., and Scheller, R. H. (1999). The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains. J. Neurosci 19, (4), 1324–1334.
Hsu, S. C., TerBush, D., Abraham, M., and Guo, W. (2004). The exocyst complex in polarized exocytosis. Int. Rev. Cytol 233, 243–265.[Medline]
Inoue, M., Chang, L., Hwang, J., Chiang, S. H., and Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422, (6932), 629–633.[CrossRef][Medline]
Insall, R. H., and Weiner, O. D. (2001). PIP3, PIP2, and cell movement—similar messages, different meanings? Dev. Cell 1, (6), 743–747.
Langevin, J, Morgan, M. J., Sibarita, J. B., Aresta, S., Murthy, M., Schwarz, T., Camonis, J., and Bellaiche, Y. (2005). Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev. Cell 9, (3), 355–376.[Medline]
Lefrancois, L., and Lyles, D. S. (1982). The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. II. Monoclonal antibodies of nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes. Virology 121, (1), 168–174.[CrossRef][Medline]
Matern, H. T., Yeaman, C., Nelson, W. J., and Scheller, R. H. (2001). The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells. Proc. Natl. Acad. Sci. USA 17, 9648–9653.
McLaughlin, S., Wang, J., Gambhir, A., and Murray, D. (2002). PIP2 and proteins: interactions, organization, and information flow. Annu. Rev. Biophys. Biomol. Struct 31, 151–175.[CrossRef][Medline]
McLaughlin, S., and Murray, D. (2005). Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, (7068), 605–611.[CrossRef][Medline]
Moore, B. A., Robinson, H. H., and Xu, Z. (2007). The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst. J. Mol. Biol 371, (2), 410–421.[CrossRef][Medline]
Moskalenko, S., Henry, D. O., Rosse, C., Mirey, G., Camonis, J. H., and White, M. A. (2002). The exocyst is a Ral effector complex. Nat. Cell Biol 1, 66–72.
Moskalenko, S., Tong, C., Rosse, C., Mirey, G, Formstecher, E., Daviet, L., Camonis, J., and White, M. A. (2003). Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem 278, (51), 51743–51748.
Munson, M., and Novick, P. (2006). The exocyst defrocked, a framework of rods revealed. Nat. Struct. Mol. Biol 13, (7), 577–581.[CrossRef][Medline]
Papayannopoulos, V., Co, C., Prehoda, K. E., Snapper, S., Taunton, J., and Lim, W. A. (2005). A polybasic motif allows N-WASP to act as a sensor of PIP(2) density. Mol. Cell 17, (2), 181–191.[CrossRef][Medline]
Pfeffer, S. R. (1999). Transport-vesicle targeting: tethers before SNAREs. Nat. Cell Biol 1, (1), E17–E22.[CrossRef][Medline]
Rosse, C., Hatzoglou, A., Parrini, M. C., White, M. A., Chavrier, P., and Camonis, J. (2006). RalB mobilizes the exocyst to drive cell migration. Mol. Cell Biol 26, 727–734.
Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. (2002). The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol 4, 73–78.[CrossRef][Medline]
Takaya, A., Ohba, Y., Kurokawa, K., and Matsuda, M. (2004). RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells. Mol. Biol. Cell 15, 2549–2557.
TerBush, D. R., and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol 130, (2), 299–312.
Tsuboi, T., Ravier, M. A., Xie, H., Ewart, M. A., Gould, G. W., Baldwin, S. A., and Rutter, G. A. (2005). Mammalian exocyst complex is required for the docking step of insulin vesicle exocytosis. J. Biol. Chem 280, (27), 25565–25570.
Vega, I. E., and Hsu, S. C. (2001). The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J. Neurosci 21, (11), 3839–3848.
Wang, S., Liu, Y, Adamson, C. L., Valdez, G., Guo, W., and Hsu, S.-C. (2004). The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization. J. Biol. Chem 279, 35958–35966.
Wang, S., and Hsu, S. C. (2006). The molecular mechanisms of the mammalian exocyst complex in exocytosis. Biochem. Soc. Trans 34, (Pt 5), 687–690.[CrossRef][Medline]
Waters, M. G., and Hughson, F. M. (2000). Membrane tethering and fusion in the secretory and endocytic pathways. Traffic 1, (8), 588–597.[CrossRef][Medline]
Weber, T. M., Joost, H., Simpson, I. A., and Cushman, S. W. (1988). Receptor Biochemistry and Methodology. ed. R. C. Kahn and L. C. Harrison, New York: Alan R. Liss, 171–187.
Whyte, J. R., and Munro, S. (2002). Vesicle tethering complexes in membrane traffic. J. Cell Sci 115, (Pt 13), 2627–2637.
Xu, K. F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J., and Vaughan, M. (2005). Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70. Proc. Natl. Acad. Sci. USA 102, 2784–2789.
Yeaman, C., Grindstaff, K. K., Wright, J. R., and Nelson, W. J. (2001). Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol 155, (4), 593–604.
Zuo, X., Zhang, J., Zhang, Y., Hsu, S. C., Zhou, D., and Guo, W. (2006). Exo70 interacts with the Arp2/3 complex and regulates cell migration. Nat. Cell Biol 8, (12), 1383–1388.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. H. Hutagalung, J. Coleman, M. Pypaert, and P. J. Novick An Internal Domain of Exo70p Is Required for Actin-independent Localization and Mediates Assembly of Specific Exocyst Components Mol. Biol. Cell, January 1, 2009; 20(1): 153 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ischebeck, I. Stenzel, and I. Heilmann Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion PLANT CELL, December 1, 2008; 20(12): 3312 - 3330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Spiczka and C. Yeaman Ral-regulated interaction between Sec5 and paxillin targets Exocyst to focal complexes during cell migration J. Cell Sci., September 1, 2008; 121(17): 2880 - 2891. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Orlando, J. Zhang, X. Zhang, P. Yue, T. Chiang, E. Bi, and W. Guo Regulation of Gic2 Localization and Function by Phosphatidylinositol 4,5-Bisphosphate during the Establishment of Cell Polarity in Budding Yeast J. Biol. Chem., May 23, 2008; 283(21): 14205 - 14212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang, K. Orlando, B. He, F. Xi, J. Zhang, A. Zajac, and W. Guo Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 J. Cell Biol., January 10, 2008; 180(1): 145 - 158. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
