Structure-Function Study of the N-terminal Domain of
Exocyst Subunit Sec3*□
Kyuwon Baek‡1, Andreas Kno ¨dler§1,2, Sung Haeng Lee‡3, Xiaoyu Zhang§4, Kelly Orlando§, Jian Zhang§,
Trevor J. Foskett‡, Wei Guo§5, and Roberto Dominguez‡6
subunit of the exocyst acts as a spatial landmark for exocytosis
through its ability to bind phospholipids and small GTPases.
The structure of the N-terminal domain of Sec3 (Sec3N) was
determined ab initio and defines a new subclass of pleckstrin
homology (PH) domains along with a new family of proteins
that mediate dimerization through domain swapping. The
structure identifies residues responsible for phospholipid bind-
ing, which when mutated in cells impair the localization of exo-
exocytosis. Through its ability to bind the small GTPase Cdc42
and phospholipids, the PH domain of Sec3 functions as a coin-
cidence detector at the plasma membrane.
complex composed of subunits Sec3, Sec5, Sec6, Sec8, Sec10,
Sec15, Exo70, and Exo84. This complex was first identified by
genetic and biochemical methods in the budding yeast Saccha-
romyces cerevisiae (1, 2). A homologous complex was subse-
quently discovered in mammalian cells (3). The exocyst medi-
ates initial tethering of post-Golgi secretory vesicles to the
plasma membrane, a step that precedes SNARE7-driven mem-
brane fusion (4, 5). The exocyst is regulated by numerous cel-
lular factors, and in particular small GTPases, which are pri-
marily responsible for the spatiotemporal control of exocytosis
Recent studies have provided insights into the molecular
architecture and function of tethering proteins. Crystal struc-
tures of nearly full-length Exo70 (7–9) and large fragments of
Sec6 (10), Sec15 (11), and Exo84 (7) have been determined.
Despite the lack of sequence similarity, these structures all
reveal a similar fold, consisting of elongated tandem repeats of
helical bundles, which are predicted to pack against one
another during assembly of the exocyst complex (4). The
recently determined structure of the yeast Dsl1p complex
implicated in Golgi-to-endoplasmic reticulum transport pro-
sisting of helical bundles similar to those of exocyst subunits
(12). The structure suggested a similar architecture, and possi-
bly a common origin, among multisubunit tethering com-
ing domains of the mammalian exocyst subunits Sec5 (13) and
Exo84 (14), which display immunoglobulin-like and pleckstrin
homology (PH) folds, respectively. However, these two do-
mains are missing in the yeast complex and are not considered
part of the conserved core of the exocyst (4).
Studies in yeast suggest that subunit Sec3 plays a pivotal role
in exocyst function and vesicle tethering. Sec3 localizes,
together with Exo70, to the growing end of the daughter cell
(known as the “bud tip”). Although the localization of other
exocyst components relies on the actin cables that serve as
tracks for motor-driven vesicle transport to the daughter cell,
bly (15–17). Genetic analyses and live cell imaging have shown
for localization of the exocyst to the plasma membrane and
exocytosis (18). This region has also been implicated in the
binding of small Rho-family GTPases and phosphatidylinositol
As an important step toward understanding exocyst-medi-
ated vesicle tethering, we identified the precise domain of Sec3
involved in plasma membrane and Cdc42 binding, determined
Grants R01-GM073791 (to R. D.) and R01-GM64690 (to W. G.). Use of the
Industrial Macromolecular Crystallography Association-Collaborative
Macromolecular Crystallography Association through a contract with
Hauptman-Woodward Medical Research Institute. The Advanced Photon
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
supplemental Figs. S1–S4 and Movie S1.
1Both authors contributed equally to this work.
2Supported by a fellowship from the Deutsche Forschungsgemeinschaft.
3Present address: Chosun University School of Medicine, 375 Seosuk-dong,
Dong-gu, Gwangju, Korea 501-759.
4Supported by a Scientist Development Grant from the American Heart
Pennsylvania, Philadelphia, PA 19104. E-mail: firstname.lastname@example.org.
Medicine, University of Pennsylvania, A507 Richard Building, 3700 Hamil-
ton Walk, Philadelphia, PA 19104. Tel.: 215-573-4559; Fax: 215-573-5851;
7The abbreviations used are: SNARE, soluble NSF attachment protein recep-
green fluorescent protein; Bgl2, endo-?1,3-glucanase; 5-FOA, 5-fluoroo-
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10424–10433, April 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
10424 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 14•APRIL 2, 2010
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its crystal structure, and studied its function in cells. We show
that the core of this domain, consisting of residues 71–241 and
referred to as Sec3N, defines a new subclass of PH domains.
the PH domain through domain swapping. A phosphate ion
and the C terminus of a neighboring molecule in the crystal
bind in the predicted phospholipid-binding pockets at the dis-
tions. Mutations of positively charged residues identified by
these interactions impair the polarized localization of exocyst
and secretion defects. Based on this structure, a new family of
proteins was identified containing Sec3-like PH domains,
including amisyn, a protein implicated in the regulation of
SNARE complex assembly.
Protein Preparation—The cDNA encoding for yeast Sec3N
(residues 71–241) was amplified by PCR and cloned into the
vector comprises a glutathione S-transferase affinity purifica-
tion tag and a thrombin-cleavage site. BL21(DE3) competent
cells (Invitrogen) were transformed with this construct and
grown in LB medium at 37 °C to A600? 0.6. Expression was
side and carried out overnight at 20 °C. Cells were harvested by
centrifugation, resuspended in phosphate-buffered saline (140
mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4(pH
Corp.). Affinity purification on a glutathione-Sepharose col-
umn (Amersham Biosciences) was done according to the man-
ufacturer’s protocol. Sec3N-glutathione S-transferase was
eluted with 50 mM Tris-HCl (pH 7.5), 10 mM glutathione and
dialyzed against 20 mM Tris-HCl (pH 7.5), 20 mM NaCl, 1 mM
of 1:1000 (Hematologic Technologies Inc.). Further purifica-
sham Biosciences) in 20 mM Tris-HCl, (pH 7.5), 1 mM dithio-
threitol, and 1 M NaCl gradient. Selenomethionine-substituted
Sec3N was obtained by a similar procedure, growing cells in
SelenoMet medium (Athena Enzyme Systems), supplemented
with 70 mg/ml selenomethionine (Acros Organics).
(pH 7.5), 50 mM NaCl, 5 mM dithiothreitol and concentrated to
Biotech). Crystals were obtained using the hanging drop,
vapor-diffusion method. A typical setup consisted of a 1:1
(v/v) mixture of protein solution and a well solution contain-
ing 100 mM Bicine (pH 9.5), 14% polyethylene glycol
monomethyl ether 5000, 1 mM dithiothreitol at 20 °C. Crys-
tal quality was improved with addition of 10 mM of CdCl2or
BaCl2. Crystals were flash-frozen in liquid nitrogen using
Paratone-N (Hampton Research) as cryoprotectant.
Data Collection, Structure Determination, and Analysis—X-
crystals (Table 1) using beamline 17-BM at the Industrial Mac-
Team facility of the Advance Photon Source (Argonne, IL).
Data indexation and scaling were carried out with the program
HKL2000 (HKL Research Inc.). The structure was determined
using the multiwavelength anomalous dispersion method at
Sec3N monomer in the asymmetric unit) were found using the
program SnB (23). The positions of the selenium atoms were
refined, and phases were calculated with the program Phenix
2.0-Å resolution, and subsequent model building and refine-
ment iterations were performed with the programs Coot and
Phenix. Illustrations were prepared with the program PyMOL
(DeLano Scientific LLC.). The solvent-accessible area buried at
the dimer interface, defined as the locus of the center of a sol-
vent probe of radius 1.4 Å as it rolls over the van der Waals
surface of the protein, was calculated with the CCP4 program
Plasmids and Yeast Strains—Sec3 mutations were generated
with the QuikChange site-directed mutagenesis kit (Agilent
Technologies) using a CEN-LEU2 plasmid (pG1273) contain-
ing the full-length Sec3 gene as a template. To generate exo70
sec3 double mutants, yeast cells bearing the sec3?N mutation,
exo70::HIS3 deletion, and carrying an EXO70 balancer plasmid
(CEN-URA3) and plasmids with exo70–45 or exo70-47 were
transformed with various sec3 mutant variants. The transfor-
mants were tested for growth upon losing the EXO70 balancer
on 5-fluoroorotic acid (5-FOA) plates as described previously
Bgl2 Secretion Assay—Wild-type, exo70-47, and exo70-47
sec3-303 cells were grown to early log phase at 25 °C. sec10-2
mutant cells were grown at 25 °C and moved for 1 h to 37 °C.
NaF and NaN3were added to a concentration of 10 mM. An
amount of cells equivalent to 10 A600/ml was collected and
washed three times with washing buffer (20 mM Tris-HCl, pH
7.5, 10 mM NaN3, and 10 mM NaF). Cells were resuspended in
300 ?l of spheroplast solution (50 mM Tris-HCl, pH 7.5, 1.4 M
sorbitol, 10 mM NaF, 10 mM NaN3, 30 mM 2-mercaptoethanol,
and 0.2 mg/ml zymolyase), and incubated for 30 min at 37 °C.
The resulting spheroplasts were pelleted at 2000 rpm, and
supernatants (external pools) were collected. After washing,
7.5, 100 mM NaCl, 2 mM MgCl2, 0.5% Triton X-100, and prote-
ase inhibitor) on ice for 10 min. The samples were centrifuged
blot using a rabbit anti-Bgl2 polyclonal antibody.
Microscopy—Chromosomal GFP tagging of exocyst compo-
nents was performed as described (15). Cells were grown to
using a Leica DM IRB microscope equipped with a 100? oil
immersion objective and a high resolution charge-coupled
device camera (ORCA-ER, Hamamatsu Photonics). The fluo-
rescence distribution in cell images was analyzed with the pro-
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gram ImageJ (rsb.info.nih.gov/ij). A straight line was drawn
used to determine the gray value of each point along the line, a
measure of the fluorescence intensity.
Sec3N Folds into a PH Domain—We had previously mapped
the GTPase and membrane-binding activities of Sec3 to an
N-terminal fragment comprising residues 1–320 (18, 19).
Sequence analysis suggested that Sec3-(1–320) contained N-
and C-terminal disordered, non-conserved regions. The frag-
ment 71–250 appeared to form an independent globular
ordered regions. Our genetic analysis also indicated that dele-
in cells (supplemental Fig. S1). Attempts to crystallize Sec3-
(71–250) yielded needle-like crystals of poor quality that could
not be improved. Analysis of protein samples from old crystal-
lization setups showed partial protein degradation, a condition
known to affect crystallization. A stable fragment of slightly
smaller mass (residues 71–241, referred to here as Sec3N) was
identified using a combination of mass spectrometry and
N-terminal sequencing. This fragment was subcloned and
yielded excellent quality crystals, diffracting the x-rays to
?2.0-Å resolution (Table 1 and “Experimental Procedures”).
shown below, accounts for the plasma membrane-binding
activity of Sec3.
the Protein Data Bank. Therefore, the structure was deter-
of selenomethionine-substituted protein. Sec3N has a single
methionine residue in 171 amino acids, resulting in a weak
anomalous signal. However, the crystal contained four Sec3N
molecules in the asymmetric unit, and the implementation of
4-fold non-crystallographic symmetry averaging improved sig-
was built and refined to 2.0-Å resolution (Table 1).
During model building, the core domain of the Sec3N mon-
omer was recognized as a PH domain. The PH domain belongs
to a structural superfamily that includes the phosphotyrosine-
binding, Ena/VASP homology, and Ran-binding domains (27).
These domains lack sequence similarity, but all share a core
structure of 100–120 amino acids consisting of a seven-
stranded, semi-open, antiparallel ?-barrel (strands ?1 to ?7),
capped at one end by a C-terminal ?-helix (?1). At the other
end of the barrel three inter-strand variable loops (VL1, VL2,
Sec3N shares this core structure, although it lacks statistically
significant sequence identity with classic PH domains. For
instance, the PH domain of Sec3 and that of the prototypical
PH-domain-containing protein phospholipase C? (28) (PDB
code: 1mai) superimpose with r.m.s.d. of 2.15 Å for 87 equiva-
lent C? positions, whereas the two proteins share only 7%
sequence identity (Fig. 1A). The PH fold appears to be con-
(supplemental Fig. S3), including human Sec3, which shares
16.4% sequence identity (37.4% sequence similarity) with yeast
to be involved in phospholipid binding are well conserved (dis-
In addition to Sec3-related sequences from different species,
a BLAST search identified proteins previously unsuspected to
contain PH domains (examples are given in Fig. 1 and
supplemental Fig. S3). Most of these proteins are still unchar-
acterized. However, an interesting example is the protein ami-
syn (also known as syntaxin-binding protein 6 or STXBP6), a
210-amino acid polypeptide implicated in the regulation of
SNARE complex assembly and exocytosis through its C-termi-
nal coiled-coil, a vesicle-SNARE-homology domain (29, 30).
Although its characterization is still limited, amisyn has been
proposed to form part of a vesicle-docking complex and is
its relationship with Sec3N (Fig. 1B), we now predict that the
presence of an N-terminal PH domain (residues 1–153).
Dimerization of Sec3N—The Sec3N fragment crystallized
here is 50–70 amino acids longer than the canonical PH
domain, and the structure includes an additional ?-helix at the
Crystallographic data, phasing, and refinement statistics
Values in parentheses correspond to the highest resolution shell.
Unit cell a/b/c, Å
Unit cell ?/?/?, °
Root mean square bonds, Å
Root mean square angles, °
B-factor protein, Å2
B-factor solvent, Å2
aRmerge? ?hkl(I ? ?I?)/?I, where I and ?I? are the observed and mean intensities of all observations of reflection hkl, including its symmetry-related equivalents.
bRfactor? ?hkl?Fobs? ? ?Fcalc?/??Fobs?, where Fobsand Fcalcare the observed and calculated structure factors of reflection hkl.
cRfreeand Rfactorwere calculated for a randomly selected subset of the reflections (5%) that were omitted during refinement.
10426 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 14•APRIL 2, 2010
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N terminus (?0) and two ?-strands at the C terminus (?8 and
?9). To our surprise, the four Sec3N molecules in the asym-
antiparallel dimers (Fig. 2). Dimerization involves domain
swapping of strands ?8 and ?9 of each monomer, which
overlap in “crossed arms” arrangement (supplemental
movie S1). The dimer interface is extensive, burying 4074 Å2
(2037 Å2per monomer) of solvent-accessible surface area
(“Experimental Procedures”). This value is much larger than
the average crystal packing contact (570 Å2) or protein-pro-
tein complex interface (1910 Å2), and even greater than the
average homodimer interface (3880 Å2) (31–33). The dimer-
ization interface is hydrophobic in character and involves
residues from strands ?8 and ?9 and helix ?1 (Tyr-217, Ile-
218, Phe-221, Val-229, Trp-231, Phe-236, and Leu-238).
Additional stabilization of the dimer interface results from
incorporation of strands ?8 and ?9 from two different
monomers into extended antiparallel ?-sheets with strands
?5, ?6, and ?7 of each of the PH domains (Fig. 2).
The dimerization of several PH domains has been observed
in crystal structures, but never confirmed in solution (34–38).
ical size-exclusion chromatography, analytical ultracentrifuga-
tion, and multiangle light scattering (supplemental Fig. S4),
tration. However, we cannot totally exclude the possibility that
of phospholipase C? (28) (PDB code: 1MAI). Phospholipase C? (PLCD1) is colored blue and Sec3N is colored green (helices), yellow (?-strands in front), red
equivalent region of human Sec3, and the Sec3N-related proteins amisyn and maize roothairless (or rth1). PLCD1 was also aligned based on a structure
and colored according to A. Residues predicted to be important for phosphoinositide binding are highlighted (red contour). Uniprot accession codes:
SEC3_YEAST (P33332), SEC3_HUMAN (Q9NV70), AMISYN_HUMAN (Q8NFX7), rth1_MAIZE (Q5YLM3), and PLCD1_RAT (P10688).
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the region C-terminal to the PH domain contributes to dimer-
tallographically independent) dimers in the asymmetric unit
(r.m.s.d. of 0.85 Å between equivalent C? atoms) suggests that
dimerization is not a serendipitous crystallization artifact.
human sequence (39). Finally, recruitment at the plasma mem-
brane may increase the local concentration of Sec3 and facili-
PH domains (34–36).
Lipid-binding Pocket—By analogy with other PH domains,
the predicted phospholipid-binding site in Sec3 consists of a
large, positively charged pocket, surrounded by VL1–3 (Fig.
3A). Two such pockets are symmetrically disposed at the distal
ends of the Sec3N dimer (Fig. 2 and supplemental movie S1).
Despite multiple attempts, Sec3N
could not be co-crystallized with
bound phosphoinositides. Further-
more, crystals of the unliganded
protein soaked with various phos-
phoinositides dissolved immedi-
tides bound Sec3N in the crystals,
but their binding was incompatible
with crystal packing contacts.
It has been previously shown that
the locations of inorganic sulfate
and phosphate ions in phosphoino-
sitide-free structures of PH do-
mains, such as those of Grp1 and
Dapp1, coincide with the locations
of the phosphate groups of phos-
phoinositides in the structures of
is very informative that one of the
Sec3N dimers contains a phosphate
ion bound in each of the phospho-
lipid-binding pockets at the distal
phates, which are distinguishable
from solvent atoms at 2.0-Å resolu-
tion, present B-factors of ?40 Å2,
comparable to that of the neighbor-
ing residues, and were probably
incorporated during purification, as
cation buffer. What is more, the C
terminus and the carboxylic group
of Glu-240 from a neighboring
molecule in the crystal also bind in
the phospholipid-binding pocket
near the phosphate ion (Fig. 3B).
Although only one of the Sec3N
dimers shows this arrangement,
nearly identical interactions occur
Because the molecules of the
dimer are crystallographically independent, this corre-
sponds to two independent observations of the same set of
interactions. The presence of these crystal contacts probably
explains why the crystals could not withstand phosphoino-
sitide soaking. More importantly, these contacts probably
mimic protein-phosphoinositide interactions. We thus
attempted to model inositol 1,4,5-trisphosphate in the
phospholipid-binding pocket, so as to position the three
phosphate groups of the phosphoinositide on top of the
phosphate ion, the C terminus and the carboxylic group of
Glu-240 from the neighboring molecule (Fig. 3C). Although
the resulting model is unlikely to reflect the actual binding
orientation or phosphoinositide specificity of the Sec3N
pocket, it points to the residues most likely to be involved in
protein-phosphoinositide interactions (Arg-137, Lys-155,
Arg-157, Arg-168, Lys-194, and possibly Lys-135).
while the other is colored blue. The phosphate ions that bind in the predicted phosphoinositide-binding
FIGURE 3. Phosphoinositide-binding pocket. A, electrostatic surface representation of Sec3N near the pre-
the area corresponding to the yellow square in A. The C terminus of a symmetry-related molecule binds in the
phosphoinositide-binding pocket (green backbone and red electron density map). A phosphate ion, the C
model identifies some of the amino acids most likely to participate in protein-phosphoinositide interactions.
10428 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 14•APRIL 2, 2010
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Structural studies have identified the determinants of PH
domain-phosphoinositide interaction and specificity (28, 35,
40–43). Classic PH domains present a basic signature motif,
the C terminus of strand ?1, the (R/K)XR sequence near the N
terminus of strand ?2, and a tyrosine (or asparagine) residue in
indiscriminate phosphoinositide binding but not specificity.
Specificity appears to depend on additional interactions with
non-conserved residues in the VLs, which for this reason are
also known as the specificity determining regions.
Sec3 shares some of these features with classic PH domains
of PH domains. Thus, the position corresponding to the lysine
amisyn, and RTH1 (Fig. 1 and supplemental Fig. S3). This sub-
stitution of a deeply buried residue of the signature sequence is
likely to affect the orientation of the bound phosphoinositide.
Note that the overall orientation of bound phosphoinositides
varies significantly among PH domains (40). A compensatory
Sec3), corresponding to the location of the canonical tyrosine
of lysine 155 in Sec3 (Fig. 3) occupies a position very similar to
that of the amino group of the conserved lysine residue in
strand ?1 of classic PH domains. Despite these important dif-
ferences Sec3 shares with classic PH domains the (R/K)XR
This sequence corresponds to the basic patch (Lys-134 to Arg-
membrane binding and localization (18). In addition, other
could play a role in determining phosphoinositide specificity,
including Arg-157, Arg-168, and Lys-194. These positions are
highly conserved within the Sec3 PH domain subclass, but not
among classic PH domains. It thus appears that the Sec3 sub-
class of PH domains has highly conserved basic amino acids,
suitably positioned in the structure to allow for the specific
binding of phosphoinositides. A more detailed knowledge of
the specific lipid interactions will require the determination of
phosphoinositide-bound structures of this subclass of PH
domains. However, the information obtained here was invalu-
able in designing mutagenesis experiments to address the role
of lipid binding in vivo.
Synthetic Growth Defects of sec3 Mutants with exo70-47—
The exocyst subunits Exo70 and Sec3 display some functional
but lethal when combined (18, 44, 45). For this reason, mutant
sec3?N (deletion of residues 1–320) lacks any detectable exo-
and even lethality, when combined with exo70 mutations.
Therefore, to understand the functions of positively charged
residues identified by the crystal structure to form part of the
phosphoinositide-binding pocket, we studied sec3 mutants in
viously studied by us, the point mutation Arg-595 3 Ala in
causes a synthetic growth defect with sec3?N (44). Therefore,
we generated various combinations of mutants of Sec3 target-
ing residues Lys-134, Lys-135, Lys-136, Arg-137, Lys-155, Arg-
exo70-47 sec3-303, containing Sec3 mutations Lys-135 3
Glu and Arg-137 3 Glu, showed reduced growth on 5-FOA
plates in which the wild-type exo70 balancer was removed (Fig.
4A). Moreover, single mutations of Sec3 residues Lys-135 or
Arg-137 (alleles sec3-301 and sec3-300, respectively) also led to
growth defects in the exo70-47 mutant background. In con-
trast, the double mutant exo70-47 sec3-302, containing Sec3
mutations Lys-134 3 Glu and Arg-136 3 Glu displayed nor-
mal growth. This is all consistent with the crystal structure in
which the side chains of residues Lys-135 and Arg-137 are ori-
ented toward the phosphoinositide-binding pocket (Fig. 3),
site direction. Alleles sec3-306 (carrying mutations Lys-135 3
Glu, Arg-137 3 Glu, Lys-155 3 Glu, and Arg-157 3 Glu) and
sec3-307 (mutations Lys-135 3 Glu, Arg-137 3 Glu, and Arg-
168 3 Glu) both resulted in lethality in the exo70-47 back-
an important role for these residues in lipid binding (Fig. 3).
Taken together, the results suggest that residues Lys-135,
Arg-137, Lys-155, Arg-157, and Arg-168, which are solvent-
exposed and clustered together in a basic pocket in the crystal
structure (Fig. 3), play an important role in Sec3 function in
yeast cells, most likely through their involvement in phospho-
The Double Mutant exo70-47 sec3-303 Is Defective in Bgl2
Secretion—We next asked whether the double mutant
exo70-47 sec3-303 displays exocytosis defects. This mutant
was selected, because it showed growth defects, but was still
viable (Fig. 4A), thus allowing us to perform secretion assays
and fluorescence microscopy analyses. We examined the
secretion of the cell wall modification enzyme endo-?1,3-
glucanase (Bgl2), a widely used marker of polarized secretion
at the daughter cell membrane. Wild-type cells did not accu-
mulate Bgl2 (Fig. 4B), whereas cells expressing the single
mutant allele exo70-47 showed moderate accumulation of
Characterization of the sec3-exo70-47 double mutants
sec3-202 Lys-134, Lys-135, Lys-136, Arg-137
sec3-302 Lys-134, Lys-136
sec3-304 Lys-155, Arg-157
sec3-306Lys-135, Arg-137, Lys-155, Arg-157
sec3-307 Lys-135, Arg-137, Arg-168
sec3-308Lys-155, Arg-157, Arg-168
sec3-309 Arg-137, Lys-155
sec3-310 Arg-137, Lys-155, Arg-168
sec3-311 Arg-137, Arg-157
sec3-312 Arg-137, Arg-157, Arg-168
aCell viability is indicated by symbols ranging from normal growth (? ? ? ?) to
? ? ? ?
? ? ?
? ? ?
? ? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
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Bgl2. In contrast, cells containing the double mutant
exo70-47 sec3-303 displayed pronounced intracellular accu-
mulation of Bgl2. As a positive control, we confirmed that
mutant sec10-2, a temperature-sensitive allele of the exocyst
subunit Sec10, accumulated Bgl2 in cells at the restrictive
temperature of 37 °C (Fig. 4B).
Localization of Exocyst Components in the Double Mutant
exo70-47 sec3-303—To examine the localization of exocyst
components in mutant cells, we tagged subunits Sec5, Sec6,
Sec8, Exo84, and wild-type and mutant Sec3 (sec3-303) with
GFP by chromosomal integration in cells bearing the single
exo70-47 mutation or double exo70-47 sec3-303 mutations.
Cells were then analyzed using fluorescence microscopy. In
the exo70-47 single mutant, GFP-tagged Sec3, Sec5, Sec8, or
Exo84 localized normally to the daughter cell plasma mem-
at the bud tip (Fig. 5A). In the double mutant exo70-47 sec3-
303, GFP-tagged Sec5, Sec8, and Exo84 remained polarized
to the daughter cell, but were diffused throughout the bud,
and not specifically associated with the bud tip membrane.
On the other hand, the sec3-303-GFP mutant was diffused
throughout the cell in the exo70-47 mutant background. Fig.
5 (B and C) shows representative plots of the fluorescence
intensity along a line drawn through the middle of the bud
and into the cell body in single and double mutants (express-
ing either Sec3-GFP, sec3-303-GFP, or Exo84-GFP). In
exo70-47 cells, Sec3-GFP and Exo84-GFP showed localized
staining at the bud tip, as indicated by a sharp peak of fluo-
rescence coinciding with the location of the plasma mem-
brane. In contrast, in sec3-303 exo70-47 cells, sec3-303-GFP
was diffused throughout the cell, and there was no fluores-
cence peak associated with the plasma membrane (Fig. 5B).
Exo84-GFP was polarized in sec3-303 exo70-47 cells, but the
fluorescence distribution was diffused (Fig. 5C).
These results are consistent with the model of exocyst
targeting proposed by Novick and colleagues (16), wherein
most exocyst components associate with secretory vesicles
traveling toward the bud tip along actin cables, whereas Sec3
and Exo70 bind to the bud tip membrane and act as land-
marks for vesicle tethering. Thus, in the exo70-47 single
the membrane presumably though their interaction with Sec3.
In the exo70-47 sec3-303 double mutant, the sec3-303 mutant
protein is unable to bind directly to the bud tip membrane and
is diffused throughout the cell. On the other hand, the other
exocyst components are still able to enter the daughter cell by
traveling with secretory vesicles along actin cables, but are no
are both defective. Interestingly, GFP-Sec6 was distributed
exocyst subunit. Taken together, our results demonstrate that
mutations of residues Lys-135 and Arg-137 in the phosphoin-
exocyst complex, which in turn results in impaired secretion
could be expected that further disrupting the phosphoinosi-
mutant strain, in which the chromosomal copy of EXO70 was deleted and
replaced by the HIS3 gene. The exo70-47 mutant was expressed under the
EXO70 promoter in a CEN TRP1 plasmid, and the cells were supplemented
on 5-FOA-containing medium, which triggers the elimination of the EXO70
balancer plasmids and subsequent generation of the exo70-47 sec3 double
mutants (right panel). Controls were grown on medium without 5-FOA (left
panel). sec3-303 and several other mutants showed different degrees of syn-
lethal with exo70-47. B, EXO70, exo70-47, and exo70-47 sec3-303 cells were
grown to early log phase. The sec10-2 mutant cells were grown to early log
sec10-2 mutants showed defects in Bgl2 secretion. The sec3-303 exo70-47
mutant. Ponceau S staining is shown to indicate equal protein loading.
10430 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 14•APRIL 2, 2010
by guest on October 26, 2015
tide-binding pocket of Sec3, for instance by introducing
shown in Fig. 4).
To understand the function and regulation of the exocyst
is targeted to the plasma membrane. Recent work has shown
that Sec3 and Exo70 interact with phospholipids and small
GTPases and play key roles in recruiting the exocyst to specific
on Exo70 was aided by knowledge of its crystal structure (7). In
the case of Sec3, the interactions with phospholipids and the
minal ?320 amino acids (18, 19), but in the absence of struc-
tural information these results could not be rationalized. Here,
we determined the structure of the N-terminal domain of Sec3
(Sec3N). The structure defines a new subclass of PH domains
binding. Genetic analysis and cell imaging further demon-
strated that membrane binding through these residues is
membrane and exocytosis in yeast.
lipids. However, it is becoming increasingly clear that only a
ing number of PH domains have been implicated in protein-
protein interactions (27, 48). The interactions of various PH
domains with protein targets have been visualized crystallo-
graphically, including complexes with small GTPases (14,
49–51) (PDB codes: 1rrp, 1zc3, 2fju, and 2w2x), and heterotri-
meric G proteins subunits (56, 57) (PDB codes: 1omw and
2rgn). These structures suggest that the interactions of the PH
domain with protein targets are highly variable and difficult to
predict, because they can involve virtually any interface of the
domain with phosphoinositides, which are constrained to the
pocket formed by loops VL1–3.
Some PH domains share both properties, by binding phos-
phoinositides and specific protein targets, in particular small
GTPases. In such cases, the PH domain is thought to function
as a coincidence detector (48, 52). It is proposed that such PH
brane-associated protein target, so as to recruit a PH-contain-
both the lipid and the protein target. Such a mechanism, pro-
viding both added affinity and increased selectivity for certain
membrane loci, has been implicated in specific Golgi targeting
of certain PH domains (53, 54). As shown here and in previous
work (18, 19), Sec3 binds lipid membranes and GTPases, and
both activities reside within the Sec3N fragment studied here.
and the exo70-47 sec3-303 double mutant cells were GFP-tagged by chromosomal integration. GFP-tagged Sec3, Sec5, Sec8, and Exo84 were localized to the
bud tip in the single mutants. In the double mutants, sec3-303-GFP was diffused throughout the cell, while the rest of the GFP-tagged exocyst proteins were
depicting the fluorescence intensity along the bud and into the cell body (along the white line) in single (exo70-47) and double (exo70-47 sec3-303) mutants
expressing Sec3-GFP or sec3-303-GFP (B) or Exo84-GFP (C) (see insets).
APRIL 2, 2010•VOLUME 285•NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10431
by guest on October 26, 2015
We have now shown that a PH domain mediates both these
interactions. Thus, Sec3, and probably other members of its
structural subclass, join the growing number of PH domain-
containing proteins that function as coincidence detectors at
Yeast Sec3 is a large protein of 1336 amino acids. The region
C-terminal to the PH domain is predicted to be predominantly
helical in structure. Residues 320–470 within this region are
predicted with high probability to form a coiled-coil structure.
Fold recognition programs further suggest that the 727-amino
acid region starting from residue 610 to the C terminus of the
protein consists of a series of long helices separated by short
loops. This is the signature pattern of helical bundles, charac-
teristic of other subunits of the exocyst (4, 10, 39). Indeed, the
crystal structures of four other subunits of the exocyst show a
related fold consisting of consecutively stacked helical bundles
(7–11). Each bundle typically contains three helices. The third
helix of each bundle is usually longer and contributes its C-ter-
minal half to the next bundle, helping to stabilize bundle-bun-
dle interactions, a fold somewhat related to that of the spectrin
with yeast Sec3 but appears to share a similar domain organi-
zation, including an N-terminal PH domain (residues 1–140),
followed by a predicted coiled-coil domain (residues 155–255)
and a series of C-terminal helical bundles (residues 258–894).
As we have shown here, residues implicated in phospholipid
binding in yeast Sec3N are also well conserved in the human
sequence, as well as in other eukaryotes. This fact was previ-
ously underappreciated, as the N terminus of Sec3 shows rela-
tively low sequence similarity across species. Our preliminary
results indicate that the N terminus of human Sec3 also binds
phospholipids8; further cell biological studies are being carried
out to investigate the function of this domain in mammalian
The structure of Sec3N also identifies a number of proteins
that carries this new subclass of PH domains. One of these
proteins is amisyn, implicated in binding to the t-SNARE pro-
tein syntaxin in neuronal cells and known to partially co-sedi-
ment with membranes (30). Future studies will examine
whether the PH domain of amisyn binds phospholipids and
whether this interaction is implicated in the recruitment of
amisyn to the plasma membrane and regulation of SNARE
function during vesicle docking and fusion.
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Roberto Dominguez Download full-text
J. Biol. Chem.
Zhang, Trevor J. Foskett, Wei Guo and
Lee, Xiaoyu Zhang, Kelly Orlando, Jian
Kyuwon Baek, Andreas Knödler, Sung Haeng
Domain of Exocyst Subunit Sec3
Structure-Function Study of the N-terminal
doi: 10.1074/jbc.M109.096966 originally published online February 5, 2010
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