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Structure and Biochemical Properties of Fission Yeast Arp2/3
Complex Lacking the Arp2 Subunit
*
□
S
Received for publication, April 3, 2008, and in revised form, June 17, 2008 Published, JBC Papers in Press, July 18, 2008, DOI 10.1074/jbc.M802607200
Brad J. Nolen
1
and Thomas D. Pollard
From the Departments of Molecular, Cellular and Developmental Biology, Cell Biology, and Molecular Biophysics and
Biochemistry, Yale University, New Haven, Connecticut 06520-8103
Arp2/3 (actin-related protein 2/3) complex is a seven-subunit
complex that nucleates branched actin filaments in response to
cellular signals. Nucleation-promoting factors such as WASp/
Scar family proteins activate the complex by facilitating the acti-
vating conformational change and recruiting the first actin
monomer for the daughter branch. Here we address the role of
the Arp2 subunit in the function of Arp2/3 complex by isolating
a version of the complex lacking Arp2 (Arp2⌬Arp2/3 complex)
from fission yeast. An x-ray crystal structure of the ⌬Arp2
Arp2/3 complex showed that the rest of the complex is unper-
turbed by the loss of Arp2. However, the Arp2⌬Arp2/3 complex
was inactive in actin nucleation assays, indicating that Arp2 is
essential to form a branch. A fluorescence anisotropy assay
showed that Arp2 does not contribute to the affinity of the com-
plex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-
promoting factor protein. Fluorescence resonance energy trans-
fer experiments showed that the loss of Arp2 does not prevent
VCA from recruiting an actin monomer to the complex. Trun-
cation of the N terminus of ARPC5, the smallest subunit in the
complex, increased the yield of Arp2⌬Arp2/3 complex during
purification but did not compromise nucleation activity of the
full Arp2/3 complex.
Dynamic rearrangements of the actin cytoskeleton are essen-
tial for cellular responses to the environment, and cells employ
a host of proteins to control nucleation, polymerization, cap-
ping, severing, bundling, cross-linking, and depolymerization
of actin (1). Fission yeast have two well characterized nucleators
of actin filaments, formins (Cdc12 and For3) and Arp2/3 com-
plex (consisting of seven-subunits, Arp3, Arp2, and ARPC1–5)
(2). Formins assemble unbranched filaments present both in
cables that span the length of interphase cells and in the con-
tractile ring, which constricts during cytokinesis (3, 4). Arp2/3
complex nucleates branched filaments in cortical structures
called actin patches, which are sites of endocytosis located at
cell poles during interphase and the cleavage furrow during
cytokinesis (5–7).
Arp2/3 complex nucleates actin filament branches on the
sides of pre-existing (mother) filaments (8). The new (daughter)
filament grows at an angle of 78° on the side of the mother
filament (9). Arp2/3 complex from most species is intrinsically
inactive but is stimulated to form an actin filament branch
through interactions with proteins called nucleation promoting
factors (NPFs),
2
ATP, an actin monomer, and the side of a
mother filament (2). WASp/Scar family proteins, the prototyp-
ical NPFs, contain a C-terminal region termed VCA (verprolin
homology, central, acidic), which is the minimal fragment
required to activate nucleation by Arp2/3 complex. Crystallo-
graphic and electron microscopic data suggest that a conforma-
tional change reorients the two actin-related subunits, Arp2
and Arp3, like two successive actin subunits along the short
pitch helix of an actin filament to create the nucleus for polym-
erization of the daughter filament (9, 10). A model built by
fitting crystal structures into reconstructions of electron tomo-
grams of branch junctions shows that all seven subunits of
Arp2/3 complex contact the mother filament and that the
barbed ends of Arp2 and Arp3 interact with the pointed end of
the daughter filament (9).
Numerous questions remain about the mechanism of
branching nucleation despite many structural and kinetic stud-
ies. Nucleotide binding favors a conformation of Arp3 that may
contribute to activation (11, 12). The V region of NPFs binds an
actin monomer (13, 14), recruiting it to the branch point,
whereas the C and A regions bind to Arp2/3 complex and are
thought to facilitate conformational changes required for
nucleation (14, 15). Cross-linking, radiation footprinting, and
NMR experiments have implicated all but two subunits
(ARPC2 and ARPC4) in interactions with VCA (16 –19), but no
high resolution structural information on VCA binding is avail-
able. A model based on small angle x-ray scattering of Arp2/3
complex bound to actin and VCA has the actin monomer
located at the barbed end of Arp2 (20). Expression of recombi-
nant human Arp2/3 complex subunits in insect cells demon-
strated that ARPC2 and ARPC4 are essential for the integrity of
the complex and that both of these subunits and Arp3 are nec-
essary to assemble a complex that can nucleate actin filaments
*This work was supported, in whole or in part, by National Institutes of Health
Grant GM066311. This work was also supported by National Institutes of
Health Ruth Kirschstein postdoctoral fellowship GM074374-02. The costs
of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 3DWL) have been deposited
in the Protein Data Bank, Research Collaboratory for Structural Bioinformat-
ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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The on-line version of this article (available at http://www.jbc.org) contains
supplemental text and Figs. S1–S4.
1
To whom correspondence should be addressed: Tel.: 203-432-3194; Fax:
203-432-6161; E-mail: bradley.nolen@yale.edu.
2
The abbreviations used are: NPF, nucleation promoting factor; DTT, dithio-
threitol; GST, glutathione S-transferase; OG-actin, Oregon Green-actin;
FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent pro-
tein; CFP, cyan fluorescent protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 39, pp. 26490–26498, September 26, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
26490 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 39•SEPTEMBER 26, 2008
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(21). This study did not address the role of Arp2 in the stability
and nucleation activity of Arp2/3 complex.
Here, we show that a complex lacking the Arp2 subunit
(⌬Arp2 Arp2/3 complex) can be isolated from fission yeast.
Except for the absence of Arp2, the loss of Arp2 did not perturb
the crystal structure of Arp2/3 complex. ⌬Arp2 Arp2/3 com-
plex bound Wsp1-VCA but did not nucleate actin filaments, so
Arp2 is essential to initiate a branch. Mutational analysis
showed that branching nucleation does not require the N ter-
minus of ARPC5 to anchor Arp2 as proposed in one model of
activation (22).
EXPERIMENTAL PROCEDURES
Purification of S. pombe Arp2/3 Complex—We purified
native Arp2/3 complex from Schizosaccharomyces pombe
strain TM011 typically starting with 500 g of wet cells. Ten
milliliters of a turbid culture of cells was inoculated per liter of
media made from 35 g/liter YES (Q-biogene) and grown with
vigorous shaking overnight at 30 °C. In the morning, an addi-
tional 70 g/liter YES was added, and the cultures were grown to
an optical density of ⬃6.0 at 600 nm. Cells were then pelleted
and washed in lysis buffer (20 mMTris, pH 8.0, 50 mMNaCl, and
1m
MEDTA). Pellets of cells were resuspended in 1 ml of lysis
buffer per gram of wet cells and stored at ⫺80 °C until lysis.
After thawing, an additional 0.4 ml of lysis buffer plus DTT (to
1m
M) and Complete protease inhibitor tablets (Roche Applied
Science, 1 tablet per 50 ml) were added per gram of cells. All
subsequent steps were at 0–4 °C. Cells were lysed using a
Microfluidizer (Microfluidics, model 110EH). Phenylmethyl-
sulfonyl fluoride was added to 1 mM, and the lysate was centri-
fuged at 30,000 ⫻gfor 20 min. The supernatant was centri-
fuged a second time at 125,000 ⫻gfor 1 h, filtered through
cheesecloth, and loaded onto a 150-ml Q-Sepharose column
equilibrated with lysis buffer. The column was washed with 1.5
column volumes of lysis buffer containing 1 mMDTT, and pro-
tein eluted with 20 mMTris, pH 8.0, 300 mMNaCl, 1 mMEDTA,
1m
MDTT. Protein was precipitated from the eluted fractions
with 40% ammonium sulfate, pelleted, and resuspended in 50
ml of PKME (25 mMPIPES, pH 7.0, 50 mMKCl, 1 mMEGTA, 3
mMMgCl
2
,1mMDTT, and 0.1 mMATP) and dialyzed over-
night against the same buffer. The sample was then loaded onto
an 8-ml column of glutathione-Sepharose 4B (GE Healthcare)
pre-charged with GST-N-WASp-VCA and equilibrated with
PKME. GST-N-WASp-VCA was purified as described for
GST-WASp-VCA (23). Arp2/3 complex was eluted with a lin-
ear 60-ml gradient of 50–1000 mMNaCl. Peak fractions were
pooled and dialyzed against 20 mMTris, pH 8.0, 100 mMNaCl,
1m
MEGTA, 1 mMDTT, and 1 mMMgCl
2
and fractionated on
a 0.5- ⫻5-cm column of Mono Q (GE Healthcare) equilibrated
with 20 mMTris, pH 8.0, 100 mMNaCl, 1 mMEGTA, 1 mM
DTT, and 1 mMMgCl
2
and eluted with a linear 20-ml gradient
of 100–400 mMNaCl. Pooled fractions were concentrated to
1.0 ml with Amicon Ultra-15 concentrators (Millipore) and
loaded onto a Superdex 200 HR16/60 gel-filtration column (GE
Healthcare) equilibrated with 20 mMTris, pH 8.0, 100 mM
NaCl, and 1 mMDTT. Peak fractions were pooled and diluted
2-fold with 20 mMTris, pH 8.0, and 1 mMDTT before concen-
trating and flash freezing in liquid nitrogen. Protein concentra-
tion was determined by measuring absorbance at 280 nm with
extinction coefficients determined by the Von Hippel method
(24).
Purification of S. pombe Wsp1-VCA Constructs—pGV67-
Wsp1-VCA was constructed by amplifying S. pombe Wsp1 res-
idues 497–574 with 5⬘-BamHI and 3⬘-EcoRI restriction sites
and cloning into pGV67, a plasmid derived from p21d (Nova-
gen) and containing an N-terminal glutathione S-transferase
(GST) tag followed by a tobacco etch virus cleavage site.
pGV67-Wsp1-Cys-VCA was constructed using the same pro-
cedure, with a single cysteine inserted 5⬘to the 497–574
fragment.
Escherichia coli strain BL21(DE3) was transformed with
either pGV67-Wsp1-VCA or pGV67-Wsp1-Cys-VCA, grown
to A
600 nm
of 0.8, and induced with 0.4 mMisopropyl 1-thio-

-
D-galactopyranoside for overnight expression at 22 °C. Cells
from an 8-liter culture were harvested and lysed in 300 ml of 20
mMTris, pH 8.0, 25 mMNaCl, 2 mMDTT, and 1 mMphenyl-
methylsulfonyl fluoride containing four protease inhibitor tab-
lets (Roche Applied Science) per 50 ml. Cells were lysed with a
Branson sonicator, and the lysate was cleared by centrifugation
at 100,000 ⫻gfor 30 min. Supernatant was loaded onto a 35-ml
column DEAE-Sepharose equilibrated in lysis buffer and eluted
with a linear 300-ml gradient of 25–700 mMNaCl. Fractions
containing recombinant protein were pooled and diluted with
lysis buffer lacking NaCl to reduce the salt to 140 mM. The
sample was then loaded onto a 10-ml column of glutathione-
Sepharose 4B equilibrated with 20 mMTris, pH 8.0, 140 mM
NaCl, 2 mMEDTA, and 2 mMDTT (binding buffer) and washed
with 5 column volumes of the same buffer. The wash buffer was
allowed to drain to the top of the column bed, and 30
lof75
Mtobacco etch virus protease (purified from E. coli by expres-
sion from the pRK1043 tobacco etch virus vector kindly pro-
vided by D. Waugh (25)) was mixed into the glutathione-Sepha-
rose slurry. Protein was released from the column during
incubation with gentle rocking overnight at 4 °C. Eluted protein
was pooled and loaded onto a 0.5- ⫻5-cm Mono Q column
equilibrated with 20 mMTris, pH 8.0, 100 mMNaCl, and 2 mM
DTT. Protein was eluted with a 20-ml linear gradient of 100–
800 mMNaCl, concentrated, and purified by size-exclusion
chromatography onto a Superdex 75 HR16/60 column (GE
Healthcare) in 20 mMTris, pH 8.0, 100 mMNaCl, and 1 mM
DTT before re-concentrating and flash freezing in liquid
nitrogen.
Preparation of Rhodamine-labeled Sp-Wsp1-Cys-VCA—Sp-
Wsp1-Cys-VCA was purified as the SpWsp1-VCA construct,
except that DTT was excluded from the final gel filtration
buffer. A fresh 20 mMstock of tetramethylrhodamine-6-male-
imide (Invitrogen) in dimethylformamide was added dropwise
to the pooled peak fractions of Sp-Wsp1-Cys-VCA while stir-
ring on ice. Reaction was stopped after1hbytheaddition of 1
mMDTT. Labeled protein was separated from free dye by ion-
exchange chromatography on a 0.5- ⫻5-cm Mono Q column
and through repeated concentration and dilution in an Amicon
Ultra protein concentrator. Concentration of SpWsp1-Rho-
VCA (Rho-VCA) was determined by measuring absorbance at
552 nm with an extinction coefficient of 44,660 M
⫺1
cm
⫺1
(14).
Arp2/3 Complex Crystal Structures
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Preparation of Labeled and Unlabeled and Actin Monomers—
We purified actin by established procedures: chicken skeletal
muscle actin (26), pyrene-labeled actin (27), and Oregon
Green-actin (OG-actin) (28).
Construction of Mutant Strains of S. pombe—We constructed
mutant strains by PCR-based gene targeting (29). For ARPC5
mutants, cassettes containing a KanMX6 module and the
P3nmt1 thymine-repressible promoter were amplified with
long primers to generate a 5⬘-flanking sequence complimentary
to a region 330 nucleotides upstream from the ARPC5 start
codon. The 3⬘-flanking sequence was complimentary to either
the first 60 nucleotides of the ARPC5 coding region (with codon
3 mutated to change arginine to glutamic acid) or to a region 42
nucleotides downstream from the start codon (for ARPC5⌬N).
Cassettes were transformed into the TM011 strain of S. pombe
using the lithium acetate method (29, 30). Transformed cells
were plated on YES plates and incubated for ⬃18hat30°C
before replica plating onto YES plates containing 100 mg/liter
G418/Geneticin (Invitrogen). Replica plates were incubated for
2–3 days, and large colonies were re-streaked on fresh YES
plates. Genomic DNA was isolated from mutant strains (29),
and the junctions and the entire coding region of ARPC5 were
sequenced to verify the presence of the mutations. ARPC3-YFP
and arp2-CFP strains were provided by Chris Beltzner, and
were constructed by replacing the ARPC3 or arp2 stop codon in
FY527 (h
⫺
leu1–32 ura4-D18 his3-D1 ade6-M216) or FY528
(h
⫹
leu1–32 ura4-D18 his3-D1 ade6-M210) with a pFA6a-
YFP-kanMX6 or pFA6a-CFP-kanMX6 cassette, crossing, and
analyzing tetrads.
Fluorescence Anisotropy Assays—We used an Alphascan flu-
orometer with a T-format for fluorescence measurements
(Photon Technology International). Samples were excited at
552 nm, and anisotropy was measured at 578 nm. The excita-
tion monochromometer bandwidth was 6 nm, and the emission
bandwidth was 4 nm. Fixed concentrations of Rho-VCA were
titrated with SpArp2/3 complex, and the fluorescence was
measured for both filter positions at 0.1 points/s for 40 s. Pho-
ton Technology International Felix software was used for data
collection and to calculate average anisotropy. For FRET meas-
urements fixed concentrations of OG-actin were titrated with
Rho-VCA with excitation at 480 nm, emission at 517 nm, and 3
nm slit widths.
Actin Polymerization Assays—We measured the nucleation
activity of wild-type and mutant Arp2/3 complex from the time
course of actin polymerization. Polymerization reactions of 100
l were assembled as follows: 2
lof10mMEGTA and 1 mM
MgCl
2
were added to 20
lof20
M15% pyrene actin mono-
mers in G-buffer (2 mMTris-HCl, pH 8.0, 0.2 mMATP, 0.1 mM
CaCl
2
, 0.5 mMDTT, and 0.01% NaN
3
) followed immediately by
adding 78
l of a solution containing Arp2/3 complex,
SpWsp1-VCA with buffers, and salts to bring the final reaction
conditions to 10 mMimidazole, pH 7.0, 50 mMKCl, 1 mM
EGTA, 1 mMMgCl
2
. Fluorescence measurements were made at
15-s intervals in a 96-well plate using a Gemini XPS spectroflu-
orometer (Molecular Devices) with an excitation wavelength of
365 nm and an emission wavelength of 407 nM. The rate of
polymerization was determined from the slope of the polymer-
ization curve at 50% polymer formation. The concentration of
barbed ends was calculated by setting the rate of polymer for-
mation equal to k
⫹
[ends][actin monomer], where k
⫹
⫽10
M
⫺1
s
⫺1
and solving for [ends].
Determination of Equilibrium Binding Constants—Binding
constants were determined by fitting the anisotropy curves to
Equation 1,
r⫽rf ⫹共rb
⫺rf 兲
冉
共Kd⫹关R兴⫹关L兴兲 ⫺
冑
共Kd⫹关R兴⫹关L兴兲2⫺4关R兴关L兴
2关L兴
冊
(Eq. 1)
where rf is the signal of the free receptor (R), rb is the signal of
the bound receptor, and [L] is the total concentration of the
ligand (species titrated). rb and K
d
were fit using KaleidaGraph
(Synergy Software). Determination of binding constants for
unlabeled SpWsp1-VCA measured using competition assay is
described in the supplemental materials.
Crystal Growth and Structure Determination—Crystals of
⌬Arp2 Arp2/3 complex grew in 750 mMammonium sulfate, 50
mMsodium citrate, and 7% glycerol at 4 °C to an average size of
150 ⫻80 ⫻80
m. ATP and calcium chloride were present in
the crystallization drop at 0.5 mMeach. The crystals diffracted
weakly and were indexed to the P4
2
22 space group with a large
unit cell (a ⫽219.1 Å, b ⫽219.1 Å, c ⫽315.2 Å) and two
molecules in the asymmetric unit (76% solvent). Attempts to
dehydrate crystals by increasing the precipitant concentration
did not improve diffraction. We used a homology model of the
S. pombe Arp2/3 complex based on the crystal structure of
bovine Arp2/3 complex (31) with the Arp2 subunit removed as
a molecular replacement search model. We also removed
inserts and regions with high B-factors in the bovine complex
structure from the search model. We found one solution using
the program Phaser (32) with a translational Z-score of 25.6.
The initial model was improved through three successive
rounds of rebuilding and restrained refinement carried out
using REFMAC with a weighting parameter of 0.005. B-factors
were refined by TLS refinement using one TLS group for each
subunit (33). The two molecules in the asymmetric unit were
symmetrically constrained throughout the refinement. To
improve density for model building, B-factors were sharpened
using a value of 70 Å
2
. The final model had an R
free
of 34.4% and
an R
work
of 32.4%. Coordinates were deposited in the Protein
Data Bank with accession code 3DWL.
Fluorescence Microscopy—Cells were grown to A
595
⫽0.2–
0.5 in YE5S at 25 °C and washed in Edinburgh minimal
medium (potassium phthalate (3 g/liter), Na
2
SO
4
(0.04 g/li-
ter), ZnSO
4
(0.4 mg/liter), Na
2
HPO
4
(2.2 g/liter), panto-
thenic acid (1 mg/liter), FeCl
2
(0.2 mg/liter), NH
4
Cl (5 g/li-
ter), nicotinic acid (10 mg/liter), molybdic acid (40
g/liter),
dextrose (20 g/liter), myo-inositol (10 mg/liter), potassium
iodide (0.1 mg/liter), MgCl
2
(1.05 g/liter), biotin (1 mg/liter),
CuSO
4
(40
g/liter), CaCl
2
(14.7 mg/liter), boric acid (0.5
mg/liter), citric acid (1 mg/liter), KCl (1 g/liter), MnSO
4
(0.4
mg/liter)) before mounting on 25% gelatin pads (34). Images
were acquired on an UltraView RS (PerkinElmer Life Sciences)
spinning disk confocal system installed on an Olympus IX-71
Arp2/3 Complex Crystal Structures
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microscope with a 100⫻, 1.4
numerical aperture PlanApo lens
(Olympus), and analyzed using
ImageJ software (rsb.info.nih.gov/
ij/). Z-series were collected in
0.6-
m steps with 200-ms sequen-
tial excitation of YFP and CFP.
RESULTS
Isolation of S. pombe Arp2/3
Complex Lacking Arp2—Arp2/3
complex from S. pombe purified
as a single component during
ammonium sulfate precipitation,
ion-exchangechromatography(Q-
Sepharose) and affinity chromatog-
raphy on GST-N-WASp-VCA on a
glutathione-Sepharose 4B column
but split into two peaks during
anion exchange chromatography on
Mono Q. The first Mono Q peak
eluted at a conductivity of 22
mS/cm, and the second peak eluted
at 29 mS/cm (Fig. 1A). We com-
pleted the purification of the two
fractions of Arp2/3 complex by gel
filtration on a column of Superdex
200. Both peaks contained Arp2/3
complex subunits, but the band on
SDS-PAGE previously shown to
contain both Arp2 and ARPC1 (7)
was less intense in pool A (Fig. 1B).
Two-dimensional PAGE (Fig. 1C)
showed that pool A lacked a cluster
of four spots corresponding to Arp2
(theoretical pI ⫽5.8). Immunoblots
with an S. pombe Arp2 antibody ver-
ified that Arp2 was missing (data
not shown). The fraction of com-
plex lacking Arp2 (⌬Arp2 Arp2/3
complex) ranged from 5 to 35%
(average ⫽13%) of total Arp2/3
complex in five preparations from
wild-type cells.
Arp2 Is Required for Arp2/3 Com-
plex Nucleation Activity—Polymer-
ization assays showed that 200 nM
⌬Arp2 Arp2/3 complex did not
nucleate actin filaments, whereas
200 nMof complete Arp2/3 complex
(pool B) increased the concentra-
tion of barbed ends to a maximum
of 3.5 nMin the presence of 1.6
M
SpWsp1-VCA and 4
Mchicken
skeletal muscle actin (Fig. 2A). Even
1
M⌬Arp2Arp2/3 complex had no
detectable nucleation activity (data
not shown).
FIGURE 1. Purification of S. pombe Arp2/3 complex lacking Arp2. A, elution of Arp2/3 complex from a Mono
Q column by a gradient of 100 –400 mMNaCl. Absorbance at 280 nm shows two peaks, both containing Arp2/3
complex subunits. B, SDS-PAGE (10–20% gradient of acrylamide) stained with Coomassie Blue of 6.3 pmol of
pools A and B from the Mono Q column further purified by gel filtration on a Superdex 200 column. ARPC1 and
Arp2 are not resolved under these conditions. C, two-dimensional gel electrophoresis of purified pool A and
pool B, stained with Coomassie Blue. Both pools contained a cluster of four spots at the apparent molecular
weight of Arp3, indicating multiple species with distinct isoelectric points (the theoretical pI of Arp3 is 5.8). Pool
B contained a cluster of four additional spots (arrow) just below Arp3, which we presumed to be Arp2 (theo-
retical pI ⫽5.8). This part of the gel shows the bands corresponding to Arp3, Arp2, ARPC1, and ARPC2. Arp2 is
missing from peak A. Theoretical pI values for each subunit are as follows: Arp3, 5.8; Arp2, 5.8; ARPC1, 8.2; and
ARPC2, 6.1.
FIGURE 2. Biochemical characterization of S. pombe Arp2/3 complex with and without Arp2. A, effect of
native and ⌬Arp2 Arp2/3 complex on the time course of polymerization of pyrene-labeled Mg-ATP actin.
Conditions: 4
M15% pyrene-labeled chicken skeletal muscle actin, 0.8
MSpWsp1-VCA, 200
Mcomplete
SpArp2/3 complex (“complete”) or ⌬Arp2 Arp2/3 complex (“⌬Arp2”) in 10 mMimidazole, pH 7.0, 50 mMKCl, 1
mMMgCl
2
,1mMEGTA, 0.13 mMATP, 63
MCaCl
2
, 0.3 mMDTT, 0.6 mMNaN
3
at 22 °C. Thick black line shows 4
M
actin and 0.8
MSpWsp1-VCA without Arp2/3 complex. Inset shows the concentration of barbed ends when
50% of the actin was polymerized plotted as a function of SpWsp1-VCA concentration for the complete
SpArp2/3 complex (pool B). High concentrations of VCA decrease the rate of polymer formation by inhibiting
nucleation and slowing pointed end elongation (23, 44). B, equilibrium binding of rhodamine-labeled and
unlabeled SpWsp1-VCA to ⌬Arp2 Arp2/3 complex and complete Arp2/3 complex measured by fluorescence
anisotropy. Conditions: 50 mMKCl, 10 mMimidazole, pH 7.0, 1 mMMgCl
2
,1mMEGTA, 0.1 mMATP, 1 mMDTT, and
0.2% thesit. Inset: titration of 100 nMSpWsp1-Rho-VCA with ⌬Arp2 (dashed line) and native Arp2/3 complex
(solid line). The K
d
values of SpWsp1-Rho-VCA were 120 ⫾13 nMfor the ⌬Arp2 and 49 ⫾5nMfor complete
Arp2/3 complex. Main plot: titration of 100 nMSpWsp1-Rho-VCA and 300 nM⌬Arp2 (dashed line) or native
Arp2/3 complex (solid line) with unlabeled SpWsp1-VCA. Curves were fit as described in the methods giving K
d
values of 0.4 ⫾0.1
Mand 0.9 ⫾0.1
Mfor unlabeled SpWsp1-VCA binding the ⌬Arp2 and complete com-
plexes, respectively. C, fluorescence resonance energy transfer to measure binding of SpWsp1-Rho-VCA to
OG-actin. Emission scans showing the dependence of the quenching of the fluorescence of 100 nMOG-actin on
the concentration of SpWsp1-Rho-VCA in the same buffer as in B.Numbers below the curves indicate Rho-VCA
concentrations, in nanomolar. Samples were excited at 480 nm. D, effect of ⌬Arp2 Arp2/3 complex and native
Arp2/3 complex on binding of OG-actin to Rho-VCA measured by FRET as in C. Plots of fraction of OG-actin
emission at 517 nm quenched verses Rho-VCA concentration. Fits of the data gave a K
d
of 15.5 ⫾1.7 nMfor
Rho-VCA binding to OG-actin (solid line,open squares). In the presence of 3
Mnative Arp2/3 complex (solid line,
filled circles), the K
d
increased to 23.9 ⫾1.3 nM. In the presence of 3
MArp2-less complex (dashed line,filled
triangles) the K
d
was 12.1 ⫾1.6 nM.
Arp2/3 Complex Crystal Structures
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The ⌬Arp2 Arp2/3 Complex Binds SpWsp1-VCA and Actin
Monomer—To determine the basis for the inactivity of ⌬Arp2
Arp2/3 complex, we used fluorescence anisotropy to measure
the affinity of rhodamine-labeled SpWsp1-VCA (Rho-VCA)
for complete and ⌬Arp2 Arp2/3 complex. Rho-VCA bound
complete Arp2/3 complex with a dissociation equilibrium con-
stant (K
d
)of49⫾5nMand the ⌬Arp2 complex with an affinity
of 120 ⫾13 nM(Fig. 2B,inset). Because the rhodamine label can
affect the affinity of VCA for Arp2/3 complex (14), we carried
out competition assays using unlabeled SpWsp1-VCA (VCA)
(Fig. 2B). VCA had a slightly higher affinity (K
d
⫽0.4 ⫾0.1
M)
for ⌬Arp2 Arp2/3 complex than native Arp2/3 complex (K
d
⫽
0.9 ⫾0.1
M). Therefore, the Arp2-less complex is not inactive
due to a loss of ability to bind this nucleation-promoting factor.
We next sought to determine if VCA bound to ⌬Arp2 Arp2/3
complex could recruit an actin monomer to form the ternary
complex of Arp2/3 complex, NPF, and an actin monomer. This
ternary complex is thought to assemble before binding to an
actin filament to initiate a branch (14). To detect interaction of
the VCA NPF with actin, we used fluorescence energy reso-
nance transfer (FRET) from an Oregon Green 488 label on Cys-
374 of actin (OG-actin) to Rho-VCA (Fig. 2C) (35). This assay
gave a K
d
of 16 ⫾2nMfor Rho-VCA binding OG-actin in the
absence of Arp2/3 complex (Fig. 2D). FRET was then measured
in the presence of 3
MArp2/3 complex, so that ⬎98% of the
Rho-VCA was bound to Arp2/3 complex. The presence of 3
M
complete Arp2/3 complex slightly increased the K
d
of Rho-
VCA and OG-actin to 24 ⫾1n
M, whereas the presence of 3
M
⌬Arp2Arp2/3 complex had no effect on the binding (K
d
⫽12 ⫾
2n
M). These results indicate that both complete and ⌬Arp2
Arp2/3 complexes can form a ternary complex with a VCA
nucleation-promoting factor and an actin monomer. Because
excess Arp2/3 complex does not increase the affinity of actin for
Rho-VCA, we conclude that actin does not make productive
contacts with Arp2/3 complex in the ternary complex.
Structure of ⌬Arp2 Arp2/3 Complex—To determine whether
loss of the Arp2 subunit perturbs the structure of Arp2/3 com-
plex, we solved the crystal structure of ⌬Arp2Arp2/3 complex
using molecular replacement with a homology model based on
the crystal structure of bovine Arp2/3 complex (31) to estimate
initial phases. We used this model to generate a solvent-flat-
tened electron density map averaged using the two molecules in
the asymmetric unit. The map showed density for a number of
features not included in the original model, including a section
of 33 residues of random coil inserted into ARPC1 and ATP in
the Arp3 cleft (see “Discussion”). Three successive rounds of
rebuilding and restrained refinement improved the model. Of
the 1615 total residues per complex, 1308 were modeled, but
287 of these were lacking density for side chains and were mod-
eled as alanine. Much of the backbone of ARPC3 was disor-
dered, so only 65% of this subunit could be modeled. The final
R
free
was 34.4%, and the final R
work
was 32.4% (Table 1), high
R-factors typical of this resolution range. Despite the low reso-
lution, omit electron density maps calculated at the start of the
refinement clearly showed not only the secondary structure,
but side-chain density for most residues (supplemental Fig. S1).
This is the first crystal structure of Arp2/3 complex other than
that from cow (10 –12). Given the limited resolution of the data,
we confine our discussion to comparisons of gross structural
features of S. pombe and bovine complexes, such as the overall
arrangement of the subunits and the conformation of long
backbone segments that adopt dramatically different confor-
mations in the structures.
The overall structure of the ⌬Arp2 Arp2/3 complex from S.
pombe closely resembles bovine Arp2/3 complex, except for the
absence of Arp2 (Fig. 3A). Arp2/3 complexes from these two
species can be overlaid with an overall root mean square devi-
ation of 1.85 Å for 1166 aligned C
␣
atoms. Therefore, dissoci-
ation of Arp2 does not cause major changes in the rest of the
complex (see supplemental Fig. S2 for detailed analysis).
The complete S. pombe Arp2/3 complex did not crystallize
under the same conditions used to grow crystals of ⌬Arp2
Arp2/3 complex. Modeling Arp2 into the ⌬Arp2 Arp2/3 com-
plex crystal revealed that Arp2 sterically clashes with the Arp3
subunit from a symmetry-related complex, explaining why the
packing arrangement is not possible if Arp2 is present.
The presence of the ARPC1 insert was the most striking fea-
ture of the electron density map of S. pombe ⌬Arp2 Arp2/3
complex (Fig. 3, Aand B, and 4). Electron density for this region
was present in the first F
o
⫺F
c
map and subsequent rounds of
rebuilding/refinement allowed us to build 19 of 43 residues of
the insert. This region forms a random coil that inserts into the
groove between subdomains 2 and 4 of Arp3 from the other
molecule in the asymmetric unit (supplemental Fig. S3A). In
some crystals of bovine Arp2/3 complex, the most conserved
portion of the ARPC1 insert forms an
␣
-helix, which packs
against the barbed end of Arp3 between subdomains 1 and 3 in
a symmetry-related molecule (supplemental Fig. S3B) (10). The
conformations of the ARPC1 inserts differ markedly in crystals
of S. pombe and bovine Arp2/3 complexes (Fig. 3A). The
ordered region of the insert in the structure of ⌬Arp2 Arp2/3
complex consists of residues unique to S. pombe (Fig. 3C),
whereas the conserved region of the insert that forms an
␣
-helix
in some bovine Arp2/3 complex structures is disordered in the
TABLE 1
Data collection and refinement statistics
Data collection statistics
Resolution limits (Å) 29.0-3.80
Space group P4
2
22
Cell constants a ⫽b⫽218.98
c⫽315.05
␣
⫽

⫽
␥
⫽90.0
Mosaicity (°) 0.46
Measured reflections 1,179,649
Unique reflections 72,502
Mean I/
9.5 (2.6)
R
sym
(%) 19.8 (49.5)
Completeness (%) 99.4 (95.2)
Refinement statistics
Modeled atoms 18,717
R
free
reflections 3,847 (5%)
Average B-factor (Å
2
)74.7
Root mean square from ideal
Bond lengths (Å) 0.007
Bond angles (°) 1.042
Ramachandran statistics
Most favored 1,751 (76.2%)
Additionally allowed 492 (21.4%)
Generously allowed 54 (2.3%)
Disallowed 0
R
free
(%) 34.4
R
work
(%) 32.4
Arp2/3 Complex Crystal Structures
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structure of ⌬Arp2 Arp2/3 complex (Fig. 3C). The conserved
portion of the ARPC1 insert is proposed to interact with one or
more actin subunits in the mother filament (9, 10). The flexibil-
ity revealed by our new structure may be critical for this pro-
posed function of this part of Arp2/3 complex.
The N Terminus of ARPC5 Is Not Essential for Activity—A
model of Arp2/3 complex in an actin filament branch based on
a three-dimensional reconstruction from electron tomograms
has Arp2 positioned next to Arp3 as the second subunit in the
daughter filament (9). This requires a shift of Arp2 by 31 Å from
its position in the inactive conformations captured in crystals.
The “migration model” for activation (22) proposed that the N
terminus of ARPC5 serves as a tether for Arp2 as it dissociates
from Arp3, ARPC1, and ARPC4 and migrates into the active
conformation (Fig. 5, Aand B).
We tested this hypothesis by
mutating the conserved arginine
from the N terminus of ARPC5 to a
glutamic acid (ARPC5R3E) or delet-
ing the N terminus entirely
(ARPC5⌬N). Both mutant strains
grew normally at 30 °C on YES
plates but slower than wild type at
25 °C and 36 °C on YES ⫹1MNaCl
(data not shown). Additionally, both
mutants were defective in expelling
Phloxin-B at 25 °C and 36 °C, but
not at 30 °C (data not shown). The
ARPC5⌬N strain yielded ⬃50%
more purified complex lacking
Arp2 than wild-type cells (data not
shown). Purified ARPC5⌬N Arp2/3
complex (Fig. 5C) nucleated fila-
ments as efficiently as native
SpArp2/3 complex (Fig. 5D). Thus,
the N terminus of ARPC5 is not
necessary for branching nucle-
ation in vitro.
Arp2 Co-localizes with Other
Arp2/3 Complex Subunits through-
out the Cell Cycle—To determine if
Arp2/3 complex in some parts of
cells lacks Arp2, we imaged haploid
strains of S. pombe expressing both Arp2 C-terminally labeled
with cyan fluorescent protein (CFP) and ARPC3 C-terminally
labeled with yellow fluorescent protein (YFP). Both tagged
genes were expressed from native promoters and provided the
sole copy of the gene. Cells depending on both tagged proteins
were viable but often misshapen. Their actin patches turned
over slower than patches in cells with only one tagged Arp.
3
Arp2 and ARPC3 co-localized in actin patches during all stages
of the cell cycle (Fig. 6A). The relative fluorescence intensities of
Arp2-CFP and ARPC3-YFP in patches were well correlated
(Fig. 6B), showing that subpopulations of patches depleted of
Arp2 were not present. The ratios of Arp2-CFP and ARPC3-
YFP fluorescence were unequal in some patches moving away
from the plasma membrane, owing to the time lapse between
acquisition of CFP and YFP images. These results suggest that,
if Arp2 dissociates from the rest of Arp2/3 complex in S. pombe,
it does not leave the actin patches.
DISCUSSION
Isolation and Activity of the ⌬Arp2 Arp2/3 Complex—We
considered multiple hypotheses to explain why some of puri-
fied fission yeast Arp2/3 complex lacks Arp2. Measurements
of fluorescently tagged Arp2/3 complex subunits in fission
yeast indicated that all subunits were present in the cyto-
plasm at near equal concentrations, with Arp2 the second
most abundantly expressed subunit (36). Therefore, low lev-
els of Arp2 expression cannot explain the existence of the
⌬Arp2 Arp2/3 complex.
3
V. Sirotkin, personal communication.
FIGURE 3. Crystal structure of SpArp2/3 complex lacking Arp2. A,C
␣
trace showing overlay of the ⌬Arp2
Arp2/3 complex (orange, Arp3; green, ARPC1; cyan, ARPC2; magenta, ARPC3; blue, ARPC4; yellow, ARPC5) onto
the bovine apo-protein complex (1K8K.pdb, gray C
␣
trace). The overall arrangement of the subunits is identical
in both Arp2/3 complexes with 1166 atoms overlaid with an overall root mean square deviation of 1.9 Å
2
. The
left panel shows the standard orientation of the complex, the right panel shows the complex rotated by 90°
about the vertical axis. All but the Arp2 and ARPC1 subunits of the bovine complex are omitted from the right
panel for clarity. B,ribbon diagrams of overlaid S. pombe (green) and bovine (blue) ARPC1. The seven blades of
the propeller are numbered 1–7 and the

-strands from one propeller are labeled A–D. The ARPC1 insert is
located between

D6 and

A7. C, sequence alignment of the ARPC1 insert from six diverse species. Abbrevi-
ations are as follows: Sp,S. pombe;Sc,S. cerevisiae;Bt,Bos taurus;Ce,Caenorhabditis elegans;Dd,Dictyostelium
discoideum; and Dm,Drosophila melanogaster. Secondary structure for S. pombe (green) and bovine complexes
(blue) is indicated above and below the alignment, respectively. Dashed lines indicate regions of disorder in the
structures.
FIGURE 4. Stereo figure of electron density of the ARPC1 insert. 2F
o
⫺F
c
electron density map contoured at 2.0
calculated with phase contributions
for the insert region from ARPC1 (subunits C and H, residues 291–311) omit-
ted. C
␣
trace of ARPC1 is shown in blue.
Arp2/3 Complex Crystal Structures
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Dissociation of Arp2 during purification is the most likely
explanation for the presence of ⌬Arp2 Arp2/3 complex. No
information is available on the affinity of individual subunits for
Arp2/3 complex in S. pombe, but no Arp2 dissociated from the
complete fission yeast complex during gel-filtration chroma-
tography, indicating that the complete complex is stable under
these conditions. A similar experiment with Acanthamoeba
Arp2/3 complex showed that the highest dissociation equilib-
rium constant for any subunit is 70 nM(37). However, ⬃7% of
Arp2 dissociated when the complete complex was subjected to
a second round of Mono Q purification. We conclude that Arp2
is the least tightly associated subunit in the S. pombe complex
and that some Arp2 dissociates during anion-exchange chro-
matography and elution with high salt. Electrospray mass spec-
trometry after high-pressure liquid chromatography showed
no mass differences for the individual subunits of the complete
and Arp2⌬complexes.
4
Therefore, neither proteolysis during
purification nor differences in post-translational modifications
to subunits common to both complexes is responsible for dis-
sociation of Arp2. We attempted to purify His-tagged Arp2
from the rest of the complex for
reconstitution experiments but
could not isolate adequate amounts
of purified Arp2 under either native
or denaturing conditions (data not
shown).
Previous work suggested that
Arp2 may not be essential in some
organisms, but no one had studied
the biochemical properties of
Arp2/3 complex lacking Arp2. Win-
ter et al. (38) found that deletion of
ARP2 from Saccharomyces cerevi-
siae is not lethal, suggesting that
actin might substitute for Arp2 dur-
ing filament branching. However,
the E316K mutation of S. pombe
Arp2 causes dissociation of Arp2
from the complex and septation
defects at 36 °C indicating the
importance of Arp2 (39). Gournier
et al. (21) expressed human Arp2/3
complex without the Arp2 subunit
in insect cells, but could not isolate
the Arp2⌬Arp2/3 complex.
Our preparation of fission yeast
Arp2/3 complex lacking Arp2 did
not nucleate actin filaments in an
assay with SpWsp1-VCA and
chicken skeletal muscle actin (Fig.
2A), so actin cannot substitute for
Arp2 in the branching reaction. S.
pombe Arp2 and chicken skeletal
muscle actin are 45% identical, but
many residues of Arp2 that contact
ARPC4, ARPC1, or ARPC5 are different in actin. For example,
the
␣
G helix in Arp2 (residues 226–236) and the loop immedi-
ately following it (residues 237–242) make extensive contacts to
ARPC4, and only four residues in this region are identical in
actin and Arp2. In addition, both Arp2 and Arp3 contain an
insert in the
␣
K/

15 (Arp2 residues 320 –334) loop not present
in actin. In Arp2, this insert forms a major part of the interac-
tion surface with the N terminus of ARPC5. In the model based
on the reconstruction of branch junctions (9), the DNase-bind-
ing loop of Arp2 (residues 39–51) interacts with an actin sub-
unit in the mother filament. The DNase-binding loop of Arp2
differs in sequence and is two residues longer than in actin.
These differences provide a convincing structural basis for the
failure of actin to substitute for Arp2 in the nucleation reaction.
The presence of Arp2/3 complex lacking Arp2 in extracts of
S. pombe led us to wonder if dissociation of Arp2 plays a role in
regulating Arp2/3 complex activity in vivo. The inactivity of
⌬Arp2 Arp2/3 complex is consistent with the observation that
the E316K mutation in Arp2 causes defects in cell septation, a
process thought to require Arp2/3 complex (39, 40). Fluores-
cence microscopy of wild-type fission yeast strains expressing
Arp2-CFP and ARPC3-YFP showed that the ratio of Arp2 to
ARPC3 was uniform in actin patches throughout the cell cycle
4
S. Almo and W. Zenchek, Albert Einstein College of Medicine, personal
communication.
FIGURE 5. The N-terminal residues of ARPC5 are not required for nucleation activity of purified S. pombe
Arp2/3 complex. A, ribbon diagram of bovine Arp2/3 complex (1K8K) showing the “back side” of the complex
relative to the standard orientation in Fig. 3A. The N terminus of ARPC5 forms a random coil that wraps around
the back of the complex and binds to a groove between subdomains 3 and 4 of Arp2. Disordered residues of
ARPC5 (31–34) are indicated with a dashed yellow line.B, detail of the boxed region in Awith key side chains
shown as sticks. Arp2 is pink, and ARPC5 is yellow.C, SDS-PAGE of purified wild-type (left lane) and ARPC5⌬N
complexes stained with Coomassie Blue. Fourteen residues are deleted from the N terminus of ARPC5⌬N
Arp2/3 complex (see supplemental Fig. S4). The mutated ARPC5 subunit has a higher mobility than the native
subunit (arrow). D, comparison of the effects of native and ARPC5⌬N Arp2/3 complex on the time course of the
polymerization of pyrene-labeled actin. Conditions: same as those in Fig. 2Awith 80 nMwild-type (red)or
ARPC5⌬N(green) Arp2/3 complex, 1.0
MSpWsp1-VCA, 4
M15% pyrene-labeled Mg-ATP actin. The black line
shows actin polymerization in the absence of Arp2/3 complex and activator.
Arp2/3 Complex Crystal Structures
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(Fig. 6). Previous studies established that all actin patches con-
tain a 1:1:1:1:1 ratio of all five subunits measured (Arp3, Arp2,
ARPC1, ARPC3, and ARPC5) (36).
3
Although we cannot rule
out the possibility that Arp2 dissociates from the rest of the
complex but remains in the patches, this scenario seems
unlikely, because the E316K mutation, which favors dissocia-
tion of Arp2 from the complex in vivo, causes Arp2 to leave the
actin patches and adopt a diffuse localization (39). Therefore,
we have no evidence that cells use ⌬Arp2 Arp2/3 complex
selectively in patches or other structures.
Interaction of VCA and Actin Monomers with Arp2/3 Com-
plex Lacking Arp2—We found that the loss of Arp2 does not
decrease the affinity of SpWsp1-Rho-VCA for SpArp2/3 com-
plex. Similarly, GST-Bee1-VCA (a budding yeast WASp hom-
olog construct) pulled down similar amounts of Arp3 from
extracts of wild-type and ⌬arp2 strains of budding yeast (41).
This is consistent with experiments that showed that most of
the binding energy of GST-Bee1-VCA for ScArp2/3 complex
comes from its interaction with the ARPC1 (Arc40) subunit
(41). On the other hand, VCA constructs protect Arp2 of
bovine Arp2/3 complex from oxidation in synchrotron radia-
tion footprinting experiments (16) and have been chemically
cross-linked to the Arp2 subunit of Arp2/3 complex from mul-
tiple species (17–19). All of these observations may be recon-
ciled, if VCA contacts Arp2 without contributing strongly to
the binding affinity.
Our FRET assays confirmed that the affinity of OG-actin for
rhodamine-VCA is slightly weaker in the presence of the full
Arp2/3 complex (K
d
⫽16 ⫾2nMwithout Arp2/3 complex,
24 ⫾1n
Mwith Arp2/3 complex) (35). This indicates that VCA,
actin, and Arp2/3 complex can interact simultaneously, but
that the actin monomer in this ternary complex does not make
productive contacts with Arp2/3 complex. We suggest that
actin in the ternary complex must reorient relative to Arp2/3
complex subunits during the activation step (35) to establish a
nucleus for the polymerization of the daughter filament. The
lower affinity of actin for VCA in the presence of Arp2/3 com-
plex also suggests that actin and Arp2/3 complex compete for a
common binding site on VCA. The C region is the best candi-
date for this common site (14), because it binds weakly to both
actin (K
d
⫽12
M) and Arp2/3 complex (K
d
⬎200
M)ina
mutually exclusive manner (42).
The FRET assay showed that the affinity of VCA for OG-
actin is 2-fold stronger (K
d
⫽12 ⫾2nM) with ⌬Arp2 Arp2/3
than full Arp2/3 complex (K
d
⫽24 ⫾1nM). We speculate that
dissociation of Arp2 either relieves steric inhibition that pre-
vents the C region from interacting with actin and/or that Arp2
subunit completes with actin directly by weakly binding the C
region.
We note that our results differ from the interpretation of a
small angle x-ray scattering model of Arp2/3 with bound acti-
vator and monomeric actin (20), in which the C-region of the
activator makes extensive interactions with Arp2. Although we
do not rule out weak interactions between Arp2 and the activa-
tor, our data show that the loss of Arp2 increases the affinity of
VCA for Arp2/3 complex and does not affect recruitment of
actin to VCA bound to Arp2/3 complex.
Insights into the Mechanism of Arp2/3 Complex Activation—
Our structure of the ⌬Arp2 Arp2/3 complex shows that disso-
ciation of the Arp2 subunit did not perturb the remaining sub-
units in the complex. This establishes the feasibility that Arp2
partially dissociates during the conformational change that
activates the complex (9). However, the N terminus of ARPC5,
which has been proposed to tether Arp2 to the complex during
the proposed conformational rearrangement (22), is not neces-
sary for Arp2/3 complex activity in vitro, suggesting that Arp2
maintains contacts with other subunits during activation. This
observation supports a model where a twisting motion rotates a
rigid body composed of Arp2, ARPC1, ARPC4, and ARPC5 into
the active conformation (10). The interpretation of small angle
x-ray scattering from Arp2/3 complex with bound activator
and actin monomer also supports the rotation model (20).
Although the N-terminal tether of ARPC5 does not play a
fundamental role in the activation of Arp2/3 complex, it may be
important in stabilizing interactions of Arp2 with the rest of the
complex. Consistent with this hypothesis, we recovered more
⌬Arp2 Arp2/3 complex from the ARPC5⌬N strain than wild-
type cells. The residues involved in the interface between
ARPC5 and Arp2 are conserved in most species (supplemental
Fig. S4), but in most plant species the N terminus of ARPC5 is
FIGURE 6. Arp2-CFP and ARPC3-YFP co-localize to actin patches. A, spin-
ning disk confocal fluorescence micrographs of a haploid strain of S. pombe
with Arp2 tagged on its C terminus with CFP and ARPC3 tagged on its C
terminus with YFP, both expressed from their native promoters. Maximum
projection images created from fourteen 0.6
m Z-sections. Left panel, Arp2-
CFP intensity; center panel, ARPC3-YFP intensity; and right panel, merged
images with Arp2-CFP intensity shown in red and ARPC3-YFP intensity shown
in green.B, correlation of the intensity of CFP and YFP fluorescence in individ-
ual actin patches. Plot of mean CFP and YFP intensity for 163 patches in 3 cells.
The linear correlation coefficient ⫽0.81.
Arp2/3 Complex Crystal Structures
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too short to reach Arp2 in models based on structures of inac-
tive bovine Arp2/3 complex (10 –12) or the model of the branch
junction based on electron tomography (9). Therefore, the sta-
bilizing function of the N terminus of ARPC5 is unlikely to
occur in plants.
We were surprised that Arp2 does not contribute to the affin-
ity of VCA for Arp2/3 complex and that, in fact, the affinity is
slightly higher without Arp2. This observation suggests that
VCA facilitates the movement of Arp2 next to Arp3 without
strongly interacting with Arp2 Perhaps Arp2 is a passive partic-
ipant in activation, with interactions of other subunits of
Arp2/3 complex with NPFs and a mother filament providing
most of the free energy for the conformational change that cre-
ates a favorable binding site for the first actin subunit of the
daughter filament with Arp2 and Arp3. Alternatively, VCA may
interact strongly with Arp2 only after the complex is bound to
mother filament. Consistent with this hypothesis, kinetic and
thermodynamic data demonstrated multiple modes of NPF
binding to Arp2/3 complex (42), and sequence similarities
between C and V regions suggest that the C region may interact
with Arp2 just as V interacts with actin during activation (43).
Elucidation of this complex activation mechanism will require
much more structural, biophysical, and biochemical informa-
tion than is currently available.
Acknowledgments—We thank Kathleen Gould for the S. pombe Arp2
antibody, Chris Beltzner for yeast strains with Arp2 and Arp3 tagged
with CFP and YFP, Vladimir Sirotkin for help with microscopy, Aaron
Downs for help with 2D gels, Yong Xiong for advice on analyzing low
resolution data, Hongli Chen for assistance with protein preparations,
Aditya Paul for comments on the manuscript, and Shih-Chieh Ti and
Julien Berro for deriving the formula for fitting the competition bind-
ing curve.
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Arp2/3 Complex Crystal Structures
26498 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 39•SEPTEMBER 26, 2008
by guest on November 7, 2015http://www.jbc.org/Downloaded from
Supplemental Experimental Procedures
Determination of binding affinity of unlabelled VCA in competition assay. The following
equations were used to determine the binding affinity of VCA:
TB
AB
rfrbrfr ][
][
)( −+=
)][][][(2
][][][2][][2][2][][][2
][
][
1
2
11212
22
1
2
1
22
1
2
12
2
12
2
1
2
211212
TTT
TTTTTTTTT
TAKKXKAKKK
AKAXKXKAKKXKKKKAKXKAKKK
B
AB
++−−−
+−+++++−−−
=
where r is the anisotropy of Rho-VCA, rf is the anisotropy of free Rho-VCA, rb is the
anisotropy of Rho-VCA bound to Arp2/3 complex, [AB] is the concentration of bound
Rho-VCA, [B]T is the total concentration of Rho-VCA, K1 is the binding affinity of Rho-
VCA for Arp2/3 complex, [X]T is the total concentration of VCA, [A]T is the total
concentration of Arp2/3 complex and K2 is the affinity of VCA for Arp2/3 complex.
SUPPLEMENTAL FIGURE S1. 2Fo-Fc omit electron density of ARPC4. An omit electron
density map was generated by removing subunit F (ARPC4) from the molecular replacement
solution and improving the structure with one round of rigid body refinement and simulated
annealing with NCS restraints. The resulting pdb file was used to generate an omit map in which
none of the atoms in subunit F contribute to the calculated phases. The map was improved
through NCS averaging and is shown here contoured at 2 σ.
SUPPLEMENTAL FIGURE S2. Rfree values are sensitive to small changes in the orientation
of individual subunits in the ΔArp2 Arp2/3 complex structure. One of the twelve subunits
(Arp3, subunit A) in the asymmetric unit (this subunit includes 2557 of the 18726 atoms in the
asymmetric unit) was rotated by small angle increments and the Rfree value of the resulting
structure was calculated. The axis of rotation goes through the center of mass of the subunit, so
displacements of the atoms in the subunit are small even at relatively large rotation angles. Figure
B shows that rotation by as little as 0.5 degrees causes a detectable increase in the Rfree. Rotation
of 2.5 degrees causes an increase in Rfree of over 2%.
SUPPLEMENTAL FIGURE S3. Interaction of the ARPC1 insert with Arp3 from
symmetryrelated molecules in bovine and fission yeast crystal structures. The insert in
ARPC1 (green) interacts with symmetry-related Arp3 (orange) molecules in both the bovine and
fission yeast structures.
A, the ARPC1 insert (276-311,337-343) forms a random coil that interacts with subdomains 1 and
2 of Arp3 from the second molecule in the asymmetric unit in fission yeast structure.
B, residues (297-309) of the bovine ARPC1 insert form an α-helix which binds to the
groove between subdomains 1 and 3 in a symmetry-related Arp3 molecule (1K8K). In the right
panel the structures are rotated ~90° about the y-axis relative to the left panel. Arrows point to the
ARPC1 insert.
SUPPLEMENTAL FIGURE S4. Alignment of N-terminal sequences of ARPC5 from diverse
species. Species abbreviations are as follows: Bt: B. taurus (gi17943205), Hs: H. sapiens
(NP_005708), Ec: E. caballus (XP_00149039), Ce: C. elegans (NP_491099), Dr: D. rerio
(AAL55526), Xl: X. laevis (NP_001086165), Dm: D. melanogaster (NP_608693), Sp: S. pombe
(NP_593727), Sc: S. cerevisiae (NP_012202), Af: A. funestus (ABI83786), An: A. oryzae
(BAE57777), Vv: V. vinifera (CAN73657), At: A. thaliana (CAB77741), Os: Oryza sativa
(NP_001068095). Alignment was created using the Dialign server (45). Secondary structure from
1K8K.pdb is blue. Lines represent random coil, dashes are disordered regions, and boxes indicate
α-helices. Conserved residues that interact with Arp2 in 1K8K are boxed (hydrophobic residues)
or colored red (charged residues).
Figure S1
Arp3 r otat ion: effect on Rfree
30.0
34.0
38.0
42.0
46.0
-30.0-20.0-10.00.010.020.030.0
angle
Rfree (%
)
Figure S2
~90°
AB
1
1
2
2
3
3
4
4
2
1
4
Figure S3
Bt MSKNTVSSARFRKVDVDEY-------------------DENKFVDEDDGGDGQ-A--------GPDEGEVDSCLRQ-------GNMTAALQAALKNPPIN
Hs MSKNTVSSARFRKVDVDEY-------------------DENKFVDEEDGGDGQ-A--------GPDEGEVDSCLRQ-------GNMTAALQAALKNPPIN
Ec MSKNTVSSARFRKVDVDEY-------------------DENKFVDEEDGGDGQ-A--------GPDEGEVDSRLRQ-------GNMMAALQAALKNPPIN
Ce M-SKNMQNTSYRKLDVDSF-------------------DPEQYDENDETVDTPGL--------GPDERAVQGFLSS-------NRLEDALHAALLSPPLK
Dr M-SKNTVSDRFRKVDVDEY-------------------DENKFVDEEDGGENQ-L--------GPDEAEVDSLIRS-------GNLMGALQAVLKNPPIH
Xl M-AKNTLSSRFRKVDIDEY-------------------DENKFVDDQLQEEPVEP-------QGPDEAEVDSLIRQ-------GDLLRAFQSALINSPVN
Dm M-AKNTSSNAFRKIDVDQY-------------------NEDNFREDDG-VESAAA--------GPDESEITTLLTQ-------GKSVEALLSALQNAPLR
Sp --------MTFRTLDVDSI-------------------TEPVLTEQDIFPIRNET-------AEQVQAAVSQLIPQARSAIQTGNALQGLKTLLSYVPYG
Sc ------MEADWRRIDIDAF-------------------DPESGRLTAADLVPPYETTVTLQELQPRMNQLRSLATS-------GDSLGAVQLLTTDPPYS
Af -MAKNTSSSAFRKIDVDQY-------------------NEDNFKEDDADQASSGM-------IVPDEAEINSLLNQ-------GRNIDALKTVLQNAPLM
An -----MAQINYRTINIDVLDPESSVNFPMETLLPPTLPAPTT--------SSE-A--------ANVAAQVRQLLRS-------GDPEGALRAVLDTAPLG
Vv ------MAGTKEFVEADN--------------------AEAI--------ITR-I--------EHKSRKIESLLKQ-------HKPIEALKTALEGSPPN
At ---------MAEFVEADN--------------------AEAI--------IAR-I--------ETKSRKIESLLKQ-------YKHVEALKTALEGSPPK
Os -----MASSAAAYLDADEN-------------------LEAI--------ISR-I--------EQKSRKIETLLKQ-------SKPVEALKTALEGTPLK
Figure S4
Brad J. Nolen and Thomas D. Pollard
Arp2 Subunit
Fission Yeast Arp2/3 Complex Lacking the
Structure and Biochemical Properties of
Developmental Biology:
Molecular Basis of Cell and
doi: 10.1074/jbc.M802607200 originally published online July 18, 2008
2008, 283:26490-26498.J. Biol. Chem.
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