Molecular Biology of the Cell
Vol. 18, 4483–4492, November 2007
Phosphatidylinositol 4,5-Bisphosphate Mediates the
Targeting of the Exocyst to the Plasma Membrane for
Exocytosis in Mammalian Cells□
Jianglan Liu,* Xiaofeng Zuo,* Peng Yue, and Wei Guo
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
The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the
plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the
exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst
subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the
interaction of Exo70 with PI(4,5)P2. Disrupting Exo70-PI(4,5)P2interaction abolished the membrane association of Exo70.
We have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting
other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we
demonstrate that Exo70-PI(4,5)P2interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not
for their transport to the PM.
Exocytosis is important for a variety of cellular functions,
ranging from the release of hormones to the incorporation of
membrane proteins for cell growth and morphogenesis. The
late stage of exocytosis is a multistep process that includes
directional transport, tethering, docking, and fusion of post-
Golgi secretory vesicles with the plasma membrane (PM).
The tethering step, defined as the initial contact of secretory
vesicles with the PM before SNARE-mediated docking and
fusion (Pfeffer, 1999; 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 mem-
brane addition take place (TerBush and Novick, 1995; Finger
et al., 1998, Guo et al., 1999). This localization pattern con-
trasts 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 mam-
malian cells, the exocyst components were found in the
cytosol, recycling endosomes and trans-Golgi network (Yea-
man et al., 2001; Fo ¨lsch 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 mem-
brane 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)P2in 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
This article was published online ahead of print in MBC in Press
on August 29, 2007.
DThe 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 (email@example.com).
© 2007 by The American Society for Cell Biology 4483
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
DNA Plasmid Construction
Wild-type rat Exo70 (rExo70) cDNA and various truncates of Exo70 were
cloned in-frame into pEGFP-C1 for expression as green fluorescent protein
(GFP) fusions or in pEBG for expression as glutathione S-transferase (GST)
fusion in mammalian cells. rExo70 was also cloned into pGEX-KG (a modified
form of pGEX-2T, from Amersham Biosciences, Piscataway, NJ) for expres-
sion as GST fusion in bacteria. The Exo70 mutants were generated using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All the
constructs were confirmed by nucleotide sequencing.
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 ?g/ml streptomycin in a 5%
CO2incubator. For RNA interference (RNAi) experiments, cells were grown
to 50% confluence and transfected with small interfering RNA (siRNA) du-
plexes using Oligofectamine (Invitrogen, Carlsbad, CA). The human Exo70
siRNA target sequence is 5?-GGTTAAAGGTGACTGATTA-3?. The control
Luciferase GL2 siRNA target sequence is 5?-AACGTACGCGGAATACT-
TCGA-3?. The efficiency of Exo70 knockdown was determined by Western
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 flu-
orescence observation using the Leica TCS SL laser-scanning confocal micro-
scope (63? 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 pre-
viously described (Hokanson and Ostap, 2006). Phospholipids were pur-
chased from Avanti Polar Lipids (Alabaster, AL). LUVs with a 100-nm diam-
eter 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 ?l total volume using TLA-100 rotor (Beckman Coulter, Fuller-
ton, CA). Sucrose-loaded LUVs were precipitated at 150,000 ? 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,
HeLa cells were plated in 10-cm dishes at 1.5 ? 106cells 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
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 ?g/ml). The cells were then fixed
for GFP observation or immunofluorescence. The 8G5 mAb against the ex-
tracellular 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 trans-
fected 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 intracel-
lular 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 quantifica-
tion 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:
Golgi (%) ? FluorescenceGolgi/Fluorescencetotal
ER ? Cytoplasm (%) ? FluorescenceER ? cytoplasm/Fluorescencetotal
Cell periphery (%) ? (Fluorescencetotal? Fluorescencecytoplasm
Association of Exo70 with the Plasma Membrane in HeLa
We have examined the localization of GFP-tagged rat Exo70
in HeLa cells. As shown in Figure 1A, GFP-Exo70 was
clearly detected at the PM as revealed by serial optical
sectioning from different axis (Figure 1A). In addition, cells
expressing GFP-Exo70 displayed filopodia-like structures as
previously reported (Wang et al., 2004; 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 cytoso-
lic, 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 pro-
tein 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-termi-
nus. 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-termi-
nus of mammalian Exo70 are directly involved in membrane
association. We sought to examine whether domain D is
J. Liu et al.
Molecular Biology of the Cell 4484
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.
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.
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.
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.
Lefrancois, L., and Lyles, D. S. (1982). The interaction of antibody with the
major surface glycoprotein of vesicular stomatitis virus. II. Monoclonal anti-
bodies of nonneutralizing and cross-reactive epitopes of Indiana and New
Jersey serotypes. Virology 121(1), 168–174.
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). PIP2and
proteins: interactions, organization, and information flow. Annu. Rev. Bio-
phys. Biomol. Struct. 31, 151–175.
McLaughlin, S., and Murray, D. (2005). Plasma membrane phosphoinositide
organization by protein electrostatics. Nature 438(7068), 605–611.
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.
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.
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.
Pfeffer, S. R. (1999) Transport-vesicle targeting: tethers before SNAREs. Nat.
Cell Biol. 1(1), E17–E22.
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 filopo-
dia formation. Nat. Cell Biol. 4, 73–78.
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.
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.
Waters, M. G., and Hughson, F. M. (2000) Membrane tethering and fusion in
the secretory and endocytic pathways. Traffic 1(8):588–597.
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-ex-
change protein, with exocyst protein Exo70. Proc. Natl. Acad. Sci. USA 102,
Yeaman, C., Grindstaff, K. K., Wright, J. R., 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.
J. Liu et al.
Molecular Biology of the Cell4492