Molecular Biology of the Cell
Vol. 14, 2617–2629, July 2003
The Saccharomyces cerevisiae Calponin/Transgelin
Homolog Scp1 Functions with Fimbrin to Regulate
Stability and Organization of the Actin Cytoskeleton□
Anya Goodman,* Bruce L. Goode,†Paul Matsudaira,* and Gerald R. Fink*‡
*Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge,
Massachusetts 02142; and†Biology Department and Rosenstiel Center, Brandeis University, Waltham,
Submitted January 21, 2003; Revised March 7, 2003; Accepted March 7, 2003
Monitoring Editor: Anthony Bretscher
Calponins and transgelins are members of a conserved family of actin-associated proteins widely
expressed from yeast to humans. Although a role for calponin in muscle cells has been described,
the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely
unknown. Herein, we have used genetic and biochemical analyses to characterize the budding
yeast member of this family, Scp1, which most closely resembles transgelin and contains one
calponin homology (CH) domain. We show that Scp1 is a novel component of yeast cortical actin
patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other
actin patch component that contains CH domains. Purified Scp1 binds directly to filamentous
actin, cross-links actin filaments, and stabilizes filaments against disassembly. Sequences in Scp1
sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH
domain. Overexpression of SCP1 suppresses sac6? defects, and deletion of SCP1 enhances sac6?
defects. Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize
the yeast actin cytoskeleton.
Actin filament assembly and organization are regulated by a
large number of actin-associated proteins that use a limited
set of structural modules to achieve great diversity of activ-
ities (Matsudaira, 1991). One such protein module, the cal-
ponin homology (CH) domain, is found in actin-associated
proteins that cross-link actin filaments (e.g., spectrin, fil-
amin, and fimbrin), link actin to other cytoskeletal systems
(e.g., fimbrin and plectin), and form signaling scaffolds (e.g.,
IQGAP, Vav; reviewed in Gimona et al., 2002). It is well
established that a pair of CH domains forms a classic actin
binding domain (e.g., ?-actinin and fimbrin; reviewed in
Matsudaira, 1991). In contrast, calponin family members
contain only a single CH domain, and it remains controver-
sial whether this domain can bind to actin filaments (Gi-
mona and Winder, 1998; Fu et al., 2000; Winder, 2003).
The calponin protein family, which includes calponins
and transgelins, is characterized by a single CH domain
located at the amino terminus and either one or more cal-
ponin-like repeats (CLR) located at the carboxy terminus
(Prinjha et al., 1994). Both mammalian transgelin and Saccha-
romyces cerevisiae calponin (Scp1) have a single CLR, whereas
mammalian calponin contains three CLRs (Figure 1). The
calponin family is highly conserved from yeast to humans.
Fungal genomes (S. cerevisiae, Schizosaccharomyces pombe, and
Neurospora crassa) contain a single transgelin-like gene,
whereas higher eukaryotic genomes have multiple transge-
lins and calponins. This evolutionary conservation of calpo-
nin family proteins suggests that they may have highly
conserved functions in vivo, yet our understanding of these
functions is limited. Calponin is a regulator of smooth mus-
cle contraction, but the functions of nonmuscle calponins are
not as well understood (reviewed in Morgan and Gango-
Transgelin (also called SM22 and WS3-10; Lees-Miller et
al., 1987a; Lawson et al., 1997) was named for its in vitro
gelation activity on actin filaments (Shapland et al., 1993),
but this activity has been questioned because it does not
occur at physiological salt concentrations (see DISCUS-
SION). In vivo, transgelin localizes to actin structures such
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E03–01–0028. Article and publication date are available at
‡Corresponding author. E-mail address: firstname.lastname@example.org.
VOnline version of this article contains video material for some
figures. Online version is available at www.molbiolcell.org.
Abbreviations used: CH calponin homology, CLR calponin-like
repeat. Supplementary video: Changes in GFP-Scp1 localization
over time (consecutive frames taken at 3-s intervals).
© 2003 by The American Society for Cell Biology2617
as stress fibers (Fu et al., 2000), yet the ability of transgelin to
bind directly to actin filaments in vitro also has been dis-
puted (Gimona and Mital, 1998; Morgan and Gango-
padhyay, 2001). Increased levels of transgelin expression
have been correlated with cell differentiation and senes-
cence, but the function, if any, of transgelins in these pro-
cesses has not been demonstrated (Thweatt et al., 1992; Liu et
al., 1994; Grigoriev et al., 1996). Thus, little is known about
the in vitro or in vivo functions of transgelins.
The S. cerevisiae genome contains a single open reading
frame with homology to the calponin protein family, and
this gene was annotated as S. cerevisiae calponin homolog,
Scp1 (Epp and Chant, 1997). However, the domain organi-
zation of Scp1 more closely resembles transgelin than calpo-
nin. As shown in Figure 1B, Scp1 contains a single CH
domain (residues 28–139; shaded) and one calponin-like
repeat (residues 174–200; underlined). The yeast genome
encodes two other proteins with readily apparent CH do-
mains, Sac6 (fimbrin) and Iqg1/Cyk1 (IQGAP), shown sche-
matically in Figure 1A. IQGAP has a single CH domain,
localizes to the bud neck, and functions in cytokinesis, but
little is known about its interactions with actin (Epp and
Chant, 1997; Lippincott and Li, 1998). Sac6/fimbrin binds to
and bundles actin filaments through a tandem pair of actin
binding domains, each comprised of two CH domains. Sac6
localizes to cortical actin patches and actin cables and is
important for actin organization, endocytosis, and cell po-
larity in vivo (Drubin et al., 1988; Adams et al., 1991; Kubler
and Riezman, 1993).
Herein, we show that the S. cerevisiae transgelin homolog
Scp1 is a novel component of the cortical actin cytoskeleton
and a bona fide actin filament binding and cross-linking
protein. The sequences in Scp1 critical for actin filament
binding and cross-linking reside outside of the CH domain.
Genetic interactions between SCP1 and SAC6/fimbrin and
similar biochemical activities suggest that these two CH
domain-containing proteins cooperate in vivo to regulate the
stability and organization of the cortical actin cytoskeleton.
MATERIALS AND METHODS
Strains and Growth Conditions
The yeast strains used in this study are listed in Table 1. Standard
methods were used for growing and manipulating yeast (Guthrie
and Fink, 1991). To generate AGY189, the coding regions of SAC6
and SCP1 in AGY20 were replaced with LEU2 and HIS3, respec-
tively. AGY189 was sporulated and resulting haploid strains of
opposite mating type were crossed to generate AGY490, AGY491,
AGY492, and AGY493. For growth assays, homozygous diploid
strains carrying vectors (pRS313, pRS314, pRS315, and pRS316)
and/or plasmids (Table 2) were grown to log phase and then
serially diluted fivefold, spotted on plates, and grown for an addi-
tional 2–3 d. Latrunculin A sensitivity of cells was measured by halo
assays as described previously (Ayscough et al., 1997).
The coding region of the SCP1 gene (YOR367w), plus 397 bases of
sequence upstream of the translation start site, was amplified by
polymerase chain reaction (PCR) from wild-type yeast genomic
DNA. The PCR product was cloned into the ClaI and SmaI sites of
pRS316 (Sikorski and Hieter, 1989), generating pAG20. For addi-
tional SCP1 constructs, we introduced by site-directed mutagenesis
(QuikChange kit; Stratagene, La Jolla, CA), a BglII site at the start
codon of SCP1 in pAG20, generating pAG3. To construct an amino
terminal green fluorescent protein (GFP)-SCP1 fusion plasmid
(pAG9), we cloned GFP as a BamHI fragment from plasmid B3355
(Fink laboratory collection) into the BglII site of pAG3. To generate
an Escherichia coli expression amino terminal hexahistidine-SCP1
fusion construct (pAG22), SCP1 was excised from pAG3 as a BglII-
XhoI fragment and cloned into the BamHI and XhoI sites of
pTrcHisA (Invitrogen, Carlsbad, CA). To generate SCP1 Gal-over-
expression plasmids (pAG179), the BglII-XhoI SCP1 fragment was
cloned into the BamHI and XhoI sites of pRS426Gal1 (Christianson et
al., 1992). Point mutations in SCP1 (S185A and S185D) were gener-
ated by site-directed mutagenesis as described above. To generate
N136 and 136C constructs, sequences coding for the amino terminus
and carboxy terminus of Scp1 were amplified by PCR and cloned
into NcoI and HindIII sites of pProET™HTa (Invitrogen) and pBAT4
(Peranen et al., 1996). All mutant scp1 constructs were sequenced to
verify that no additional mutations had been introduced.
Domain organization of calponin and the three CH domain-contain-
ing proteins in budding yeast: Scp1, Sac6, and Iqg1/Cyk1. CH
domains are circled, CLRs are indicated as solid rectangles, the two
putative actin binding sequences in calponin are labeled (S1 and S2),
and the GTPase activating protein (GAP) domain in Iqg1/Cyk1 is
boxed. (B) Primary sequence of Scp1. The CH domain is shaded, the
CLR is underlined, and the sites of mutations in scp1 are indicated
by arrows. (C) Diagram of the mutant constructs generated in this
study and summary of their activities and in vivo functions.
Domain organization of Scp1 and related proteins. (A)
A. Goodman et al.
Molecular Biology of the Cell2618
Yeast actin was purified as described previously (Goode et al., 1999).
His6-tagged Scp1 proteins were expressed in BL21/DE3 E. coli and
purified on nickel resin as per manufacturer’s instructions (QIA-
GEN, Valencia, CA). Peak fractions eluted from the nickel column
were pooled and fractionated on a monoQ (5/5) column by using an
AKTA FPLC (Amersham Biosciences, Piscataway, NJ). Peak frac-
tions were pooled, concentrated in a Centricon 10 device (Millipore,
Bedford, MA), and exchanged into HEKG5 buffer (20 mM HEPES,
pH 7.5, 1 mM EDTA, 50 mM KCl, 5% glycerol). The proteins were
aliquoted, frozen in liquid nitrogen, and stored at ?80°C. Untagged
carboxyl-terminal fragment of Scp1 was expressed in E. coli. Cells
were lysed in HEKG5 buffer by using french press, and the lysate
was clarified by centrifugation at 313,000 ? g at 4°C for 30 min
(high-speed supernatant; HSS). HSS was fractionated on a 1-ml
HiTrap SP column (Amersham Biosciences). Peak fractions were
pooled, diluted in low salt buffer, and fractionated on a Mono S
column. Peak fractions were concentrated and fractionated on a
Superdex 75 (5/30) column (Amersham Biosciences) equilibrated in
HEKG5 buffer. Peak fractions were pooled concentrated, aliquoted,
frozen in liquid nitrogen, and stored at ?80°C. Untagged full-length
Scp1 was purified from yeast overexpressing SCP1 (BJ2168 carrying
pAG179). One liter of cells was grown to mid-log phase in SC-His
medium with 2% raffinose. Then, 2% galactose was added to the
medium, and cells were grown for an additional 12 h and harvested
by centrifugation. The cell pellet was resuspended in 0.3 volume of
water and frozen in droplets in liquid nitrogen. Next, the frozen
yeast cells were lysed in a coffee grinder by using liquid nitrogen,
described under “Lab Protocols” on the Goode Laboratory Web site
at www.bio.brandeis.edu/goodelab. An HSS was generated in
HEKG5 buffer supplemented with 0.5 mM dithiothreitol (DTT) and
protease inhibitors as described previously (Goode et al., 1999). The
HSS was fractionated on a 1-ml HiTrap SP column (Amersham
Biosciences), and proteins were eluted with a linear salt gradient
(50–500 mM KCl). Scp1 eluted at approximately 200 mM KCl. Peak
fractions were pooled and concentrated to 3 ml in a Centricon 10
device and then fractionated on a Superdex 75 (26/60) column
(Amersham Biosciences) equilibrated in HEKG5 buffer. Peak frac-
tions were pooled, concentrated as described above, aliquoted, fro-
zen in liquid nitrogen, and stored at ?80°C. Sac6 was purified as
described above for untagged Scp1 with the following exceptions:
AAY1918 strain was used for galactose induction; after the HSS was
fractionated on a HiTrap Q column, the Sac6-containing fractions
were pooled, desalted, and fractionated on a monoQ (5/5) column.
Peak fractions were pooled, concentrated in Centricon 10 devices,
and fractionated on a Superose12 (5/30) gel filtration column (Am-
ersham Biosciences) equilibrated in HEKG5 buffer. Sac6 peak frac-
tions were pooled, concentrated, aliquoted, frozen in liquid nitro-
gen, and stored at ?80°C. Tpm1 was purified as described
previously (Liu and Bretscher, 1989).
Actin Filament Binding and Cross-Linking Assays
Yeast actin was assembled as follows. Actin (50 ?M) in G-buffer (10
mM Tris, pH 7.5, 0.2 mM CaCl2, 0.2 mM DTT, 0.2 mM ATP) was
thawed on ice, precleared by centrifugation, and 20? initiation mix
(10 mM ATP, 40 mM MgCl2, 1 M KCl) was added to induce
polymerization. Reactions were incubated for 1 h at 25°C. Then,
actin filaments were diluted in F-buffer (10 mM Tris, pH 7.5, 0.2 mM
CaCl2, 0.2 mM DTT, 0.7 mM ATP, 2 mM MgCl2, and 50 mM KCl),
purified proteins (Scp1, Sac6, and Tpm1), and/or HEKG5 buffer was
added, and the reactions were incubated at room temperature for
1 h. For low-speed pelleting assays, reactions were centrifuged for
10 min at 10,000 ? g, 4°C. For high-speed pelleting assays, actin
filaments were pelleted by centrifugation for 30 min at 313,000 ? g
in a TLA100 rotor (Beckman Coulter, Fullerton, CA). In both assays,
supernatants and pellets were fractionated on SDS-PAGE gels,
stained with Coomassie, and bands were quantified by densitome-
try with NIH Image (version 1.61, available at sippy.nimh.nih.gov).
The binding constant (Kd) of Scp1 for actin filaments was defined as
the concentration of Scp1 at which half-maximal Scp1 binding oc-
curred. For light scattering assays, yeast actin was thawed on ice,
diluted in G-buffer, and mixed with 20? initiation mix plus Scp1
and/or HEKG5 buffer. Light scattering was monitored overtime at
360 nm in a fluorescence spectrophotometer (Photon Technology
International, Lawrenceville, NJ) held at a constant temperature of
25°C. Apparent viscometry of actin solutions was measured by the
falling ball assay, performed as described previously (Pollard and
Cooper, 1982). Rabbit muscle G-actin (Cytoskeleton, Denver, CO)
was clarified by centrifugation for 30 min at 313,000 ? g 4°C in a
TLA100 rotor. Capillary tubes were loaded with actin (4.2 ?M),
Table 1. Strains used in this study
MATa, ura3, trp1, leu2, his3, prb1, can1, sac6?LEU2, pep4?HIS3, pGal10-SAC6/CEN/URA
MATa/MAT?, his3 200/his3 200, leu2-3, 112/leu2-3,112, ura3-52/ura3-52, trp1?HisG/trp1?HisG
MATa/MAT?, his3 200/his3 200, leu2-3, 112/leu2-3,112, ura3-52/ura3-52, trp1?HisG/trp1?HisG,
MATa/MAT?, SAC6/SAC6, SCP1/SCP1
MATa/MAT?, sac6?LEU2/sac6?LEU2, SCP1/SCP1
MATa/MAT?, SAC6/SAC6, scp1?HIS3/scp1?HIS3
MATa/MAT?, sac6?LEU2/sac6?LEU2, scp1?HIS3/scp1?HIS3
MATa, pep4-3, prb1-1122, prc1-407, trp1, ura3-52, leu2, gal2
Sandrock et al., 1997
* Strains have the same genotype as AGY189, except at SAC6 and SCP1 loci.
Table 2. Plasmids generated in this study
Yeast Calponin Functions with Fimbrin In Vivo
Vol. 14, July 2003 2619
initiation salts, and varying concentrations of His6-Scp1 (0, 0.23,
0.46, 0.93, 1.4, 1.8, and 3.7 ?M) and incubated at room temperature
for 1 h before falling ball measurements. For electron microscopy, 2
?l of reactions were spotted onto freshly ionized carbon-coated
grids, stained with 1% uranyl acetate, and visualized using a Phil-
lips EM410 transmission electron microscope.
Actin Filament Disassembly Kinetics
Yeast actin (with 1% pyrene labeled rabbit skeletal muscle actin;
Cytoskeleton) was assembled at 35 ?M as described above. Actin
filaments (7 ?l) in F-buffer was mixed with 52.5 ?l of F-buffer and
7 ?l of Scp1 and/or HEKG5 buffer and incubated for 10 min at
room temperature. Actin filaments were agitated by vortexing for
10 s, and then mixed with 3.5 ?l of latrunculin A (400 ?M) in a
cuvette. The final reactions contained 3.5 ?M actin, 20 ?M latrun-
culin A, and variable concentrations of Scp1 (0–2 ?M). The
depolymerization kinetics of pyrene-labeled actin filaments was
monitored by excitation at 365 nm and emission at 407 nm in a
fluorescence spectrophotometer held at a constant temperature of
Fluorescence Light Microscopy
Images of cells were acquired using a Nikon TE300 inverted
fluorescence microscope equipped with a Hamamatsu Orca
charge-coupled device camera controlled by Openlab software
(Improvision, Lexington, MA). The localization pattern of GFP-
Scp1 fusion protein was examined in live yeast cells grown to log
phase. To disrupt the actin cytoskeleton, cultures were treated
with 200 ?M latrunculin A for 5 min before imaging (Figure 2A).
Colocalization of GFP-Scp1 and actin (Figure 2B) was performed
essentially as described previously (Warren et al., 2002). Briefly, 1
ml of exponentially growing cells was fixed with 70% ethanol on
ice for 10 min, and cells were pelleted by centrifugation at 3000 ?
g and resuspended in 100 ?l of phosphate-buffered saline buffer
plus 1 mg/ml bovine serum albumin and 10 ?l of rhodamine-
phalloidin (300 U in 1.5 ml of methanol; Molecular Probes, Eu-
Scp1 to cortical actin patches. (A)
Localization of GFP-Scp1 in live
yeast cells untreated, treated with
dimethyl sulfoxide, or treated
with 200 ?M latrunculin A in di-
methyl sulfoxide. (B) Colocaliza-
tion of GFP-Scp1 and rhodamine
phalloidin actin staining in fixed
cells. Bar, 5 ?m.
Localization of GFP-
A. Goodman et al.
Molecular Biology of the Cell2620
gene, OR). After incubation on ice for 5 min, cells were washed
three times in phosphate-buffered saline buffer and mounted on
a slide for imaging.
Scp1 Localizes to Cortical Actin Patches In Vivo
To investigate the in vivo function of Scp1, we first exam-
ined localization of Scp1 in yeast cells. We were unable to
localize endogenous Scp1 by using anti-Scp1 antibodies or
hemagglutinin (HA)-tagged Scp1 by using anti-HA antibod-
ies, likely because Scp1 is expressed at low levels (see be-
low). Therefore, we examined the localization of GFP-Scp1
expressed under the control of the SCP1 promoter from a
low copy plasmid. As shown in Figure 2A, GFP-Scp1 local-
ized to motile cortical patches, largely polarized in the bud,
but also present in the mother cell. The GFP-Scp1 patches
disappeared rapidly after cells were treated briefly with 200
?M latrunculin A, an actin monomer-sequestering agent
(Figure 2A). Thus, filamentous actin is required for GFP-
Scp1 localization. In fixed cells, GFP-Scp1 patches colocal-
ized with rhodamine-phalloidin stained actin patches, dem-
onstrating that GFP-Scp1 localizes to actin patches (Figure
2B). Although all GFP-Scp1 patches overlapped with actin
patches, ?16% of actin patches (n ? 89) did not have a
corresponding GFP signal. This leaves open the possibility
that some actin patches do not contain Scp1.
Deletion of SCP1 Enhances sac6? Phenotypes
To further study SCP1 in vivo function, we generated a
complete deletion of the SCP1 gene. This mutation alone had
no salient phenotype in haploid or diploid cells, but did
show specific genetic interactions with sac6?. Among the
many phenotypes tested for the scp1? single mutants were
growth at a full range of temperatures, growth under vari-
ous stresses (e.g., NaCl, caffeine, and benomyl), cell mor-
phology, bipolar budding pattern, actin cytoskeleton orga-
nization, and endocytosis (assayed by lucifer yellow,
FM4-64 uptake and Ste6 internalization). The only detectable
phenotype of scp1? was a modest but reproducible sensitiv-
ity to latrunculin A (Table 3). Given the high degree of
functional redundancy among components of cortical actin
patches (reviewed in Pruyne and Bretscher, 2000; Goode and
Rodal, 2001), we tested for synthetic genetic interactions
between SCP1 and other genes that regulate actin function.
The scp1? mutants showed synthetic defects only with
sac6?, but not with abp1?, aip1?, arp2-1, cap2?, cof1-22,
crn1?, end3?, las17?, pan1-4, rvs167?, sla1?, sla2?, srv2?,
tpm1?, or tpm2?. Deletion of SCP1 enhanced many pheno-
types of sac6?, including temperature and caffeine sensitiv-
ity (Figure 3A), salt sensitivity (our unpublished observa-
tion), and latrunculin A sensitivity (Table 3). Deletion of
SCP1 did not further enhance the actin organization or en-
docytosis phenotypes of sac6? cells, which already have
depolarized actin cytoskeleton and fail to accumulate lucifer
yellow dye in the vacuole (our unpublished observations).
The scp1? sac6? double mutant cells provided a genetic
background that permits direct testing of the Scp1 function
in vivo. Mutation of a conserved serine residue in the CLR of
mammalian calponin (Ser175) and transgelin (Ser184) dis-
rupts actin binding in vitro (Tang et al., 1996; Fu et al., 2000).
To test whether the analogous residue in Scp1 (S185; Figure
1B) is important for in vivo function, we generated two
substitutions (S185A and S185D). scp1? sac6? cells trans-
formed with wild-type or mutant SCP1 constructs were
analyzed for growth phenotypes. Both wild-type SCP1 and
scp1S185D suppressed the growth defects of scp1? sac6?
cells, indicating that these constructs restore SCP1 function.
In contrast, cells expressing scp1S185A grew only slightly
better than control cells carrying an empty vector (Figure
3A). Stable expression of the mutant proteins was verified by
immunoblotting (our unpublished observations). These data
indicate that the conserved serine residue (located in the
CLR) is critical for Scp1 function in vivo.
Additional Copies of SCP1 Partially Suppress the
sac6? Growth Phenotype
SCP1 expressed from a low copy plasmid suppressed the
temperature sensitivity of sac6?scp1? double mutant cells
(Figure 3A). To investigate the basis of this effect, we quan-
tified the expression levels of actin, Sac6, and Scp1 in cells,
comparing cell extracts to standard curves of purified pro-
teins (actin, Sac6, and Scp1) by immunoblotting. The level of
Scp1 (?0.01 ng/?g total cellular protein) was considerably
lower than that of Sac6 (?0.15 ng/?g) and actin (?1 ng/?g).
From these values, we calculated that the molar ratio of
actin, Sac6, and Scp1 in cells is ?65:6:1. The expression level
of Scp1 in sac6?scp1? cells carrying a low copy SCP1 plas-
mid was two- to threefold higher than endogenous Scp1
levels in wild-type cells (our unpublished observations).
This suggested that extra copies of SCP1 can suppress sac6?
cell growth defects. To test this hypothesis directly, we
transformed sac6? cells with low copy plasmids expressing
wild-type and mutant Scp1 proteins. As shown in Figure 3B,
a wild-type SCP1 plasmid partially suppressed the temper-
ature sensitivity of the sac6? mutant. scp1S185D also par-
tially suppressed the sac6? phenotype, whereas scp1S185A
showed no suppression. Thus, low-level overexpression of
SCP1 partially suppresses the temperature-sensitive growth
phenotype of the sac6? mutant, and a specific mutation in
SCP1 abolishes this suppression.
Table 3. Synthetic latrunculin A sensitivities of scp1 and sac6 mu-
to latrunculin A
sac6? scp1?, pSCP1/CEN
The latrunculin A sensitivities of cells were measured by halo assays
as described previously (Ayscough et al., 1997). Relative sensitivity
was defined as the ratio of latrunculin A concentrations required to
produce halos of the same diameter for wild-type and mutant
Yeast Calponin Functions with Fimbrin In Vivo
Vol. 14, July 20032621
Scp1 Binds to and Cross-Links Actin Filaments
To test whether Scp1 interacts directly with actin filaments
in vitro, we overexpressed Scp1 in yeast by using a galac-
tose-inducible promoter, purified the protein, and measured
its ability to bind actin filaments in a high-speed cosedimen-
tation assay. As shown in Figure 4, A and B, Scp1 bound to
yeast actin filaments in a concentration-dependent manner
with micromolar binding affinity (Kd? 0.7 ?M) and a molar
saturation stoichiometry of 1:2 Scp1 to actin. Hexahistidine
(His6)-tagged Scp1 expressed and purified from E. coli
bound to actin filaments with a similar affinity (Figure 4C).
between SCP1 and SAC6. (A) Syn-
thetic genetic interactions of scp1
(AGY490, AGY491, AGY492, or
AGY 493) carrying vector alone
were serially diluted and grown
on YPD at different temperatures
in the presence or absence of 5
mM caffeine. (B) Suppression of
sac6? null mutant phenotypes by
additional copies of SCP1. Serial
dilutions of wild-type (AGY490)
and sac6?/sac6? (AGY491) ho-
mozygous diploid yeast cells car-
rying vector (pRS316) or low copy
SCP1 plasmids (pAG20, pAG16,
and pAG17) grown at different
A. Goodman et al.
Molecular Biology of the Cell2622
Mammalian calponin family members have been shown
to cross-link actin filaments (Shapland et al., 1993; Kola-
kowski et al., 1995; Tang et al., 1997). Using several comple-
mentary approaches, we found that Scp1 has a similar ac-
tivity. First, His6-Scp1 increased light scattering of yeast
actin filaments in a concentration-dependent manner (Fig-
ure 5A), suggesting that Scp1 organized filaments into larger
structures (e.g., bundles or networks). Second, we analyzed
the reactions from the light scattering experiment in a low-
speed pelleting assay. In the absence of Scp1, most actin
remained in the supernatant as expected, but with increas-
ing amounts of Scp1, actin shifted to the pellet (Figure 5B).
This concentration-dependent increase in actin pelleting cor-
related with the observed increase in light scattering (Figure
5A). To ensure that actin cross-linking by His6-Scp1 was not
due to the tag, we tested untagged Scp1 in the low-speed
actin-pelleting assay. Figure 5C shows that the effects of 1
?M untagged Scp1 are nearly identical to the effects of 1 ?M
His6-Scp1. These results demonstrate that Scp1 cross-links
actin filaments in vitro.
To characterize the actin cross-linking activity of Scp1
further, we used the falling ball assay (Pollard and Cooper,
1982) to measure the apparent viscosity of the actin filament
solution in the presence and in the absence of His6-Scp1. The
apparent viscosity of a 4 ?M actin filament solution in-
creased markedly when Scp1 concentration exceeded 1 ?M
(Figure 5D). We also examined by electron microscopy neg-
atively stained actin filaments in the presence and in the
absence of His6-Scp1. In the absence of Scp1, actin filaments
were distributed evenly throughout the grid (Figure 6A).
When actin was polymerized in the presence of Scp1, actin
filaments formed loose bundles tangled into networks (Fig-
ure 6, B and C). Scp1 cross-linked bundles seemed wavy,
and the spacing between filaments in a bundle was not
uniform. In contrast, Sac6/fimbrin bundles were straight
with uniform spacing between filaments (Figure 6D).
The Carboxy Terminus of Scp1 Alone Can Cross-
Link Actin Filaments
To better understand the molecular mechanism of actin
binding and cross-linking by Scp1, we expressed and puri-
fied amino-terminal N(1–136) and carboxyl-terminal C(136–
200) fragments of Scp1 (Figure 1C). Although we attempted
to generate both His6-tagged and untagged constructs in E.
coli, we were able to isolate only the His6-tagged N(1–136)
and untagged C(136–200). We tested the purified proteins
for actin binding in the high-speed pelleting assays. In con-
trast to the full-length Scp1, the N(1–136) fragment did not
copellet with actin filaments (Figure 7A). The C(136–200)
fragment, on the other hand, copelleted with actin filaments,
indicating that at least one actin-binding site resides in the
carboxy terminus of Scp1. We also tested the ability of the
truncated proteins to cross-link actin filaments. In a low-
speed pelleting assay, actin remained in the supernatant in
the absence of Scp1 and in the presence of N(1–136) (Figure
7B). However, actin was found mostly in the pellet in the
presence of the carboxyl-terminal fragment or the full-length
Scp1. Therefore, untagged carboxy terminus of Scp1 is suf-
ficient to cross-link actin filaments.
In addition, we addressed whether a specific residue in
the carboxy terminus (S185) critical for in vivo function of
Scp1 (see above), was also important for actin filament bind-
ing. We purified the His6-tagged S185A and S185D mutant
Scp1 proteins and compared their ability to bind and cross-
link actin filaments with the wild-type Scp1. Scp1S185D
bound to actin filaments in a high-speed actin pelleting
assay (Figure 7C), cross-linked actin filaments in the low-
speed actin pelleting assay (our unpublished observations),
and increased light scattering of the actin filaments similar
to wild-type Scp1 (Figure 7D). In contrast, Scp1S185A had
greatly diminished actin binding and cross-linking activities
(Figure 7, C and D; our unpublished observations). These
results suggest that Scp1S185D retains much of the wild-
assay using 2.5 ?M yeast actin filaments and varying concentrations
of Scp1. The samples are labeled below the pellet (P) and superna-
tant (S) lanes: A, 0.5 ?M; B, 1 ?M; C, 2 ?M; D, 3 ?M; and E, 4 ?M.
Half of the supernatant was loaded in each lane compared with
pellet. (B) Resulting binding curve. The amount of Scp1 bound
(micromolar) in each reaction was calculated from densitometry
measurements of the gel in A and plotted versus the total concen-
tration of Scp1 in the reaction. (C) Cosedimentation assay using 5
?M yeast actin filaments and/or 1 ?M His6-Scp1 or untagged Scp1.
Equivalent amounts of pellet and supernatant were loaded in each
lane. The samples are labeled below the pellet and supernatant
lanes: 1, actin alone; 2, Scp1; 3, actin and Scp1; 4, His6-Scp1; and 5,
actin and His6-Scp1. Note that in A, a contaminant that does not
pellet with actin is marked with an asterisk; and in C, a proteolytic
fragment of His6-Scp1 is visible below the full-length protein.
Binding of Scp1 to actin filaments. (A) Cosedimentation
Yeast Calponin Functions with Fimbrin In Vivo
Vol. 14, July 2003 2623
type Scp1 interaction with actin. However, Scp1S185D
showed reduced actin binding affinity compared with wild-
type Scp1 at higher salt concentrations (150 mM KCl; our
unpublished observations). Therefore, the Scp1S185D inter-
action with actin may be weakened, but not nearly to the
extent of Scp1S185A.
Scp1, Like Fimbrin, Decreases the Rate of Actin
Both Sac6/fimbrin (Bretscher, 1981; Adams et al., 1991) and
Scp1 (Figures 4 and 5) bind to and cross-link actin filaments;
in addition, Sac6 stabilizes actin filaments against disassem-
bly (see Figure 5 in Goode et al., 1999). To address whether
Scp1 similarly can stabilize actin filaments, we compared
pyrene-actin filament disassembly kinetics in the presence
and absence of His6-Scp1. In the absence of Scp1, actin
filaments disassembled rapidly, and pyrene fluorescence
reached steady state by 600 s (Figure 8A). His6-Scp1 reduced
the rate of actin filament disassembly in a concentration-
dependent manner. Similar effects were observed using un-
tagged Scp1 (our unpublished observations). Thus, Scp1,
like Sac6/fimbrin, stabilizes actin filaments.
Scp1 and Sac6 Compete for Actin Filament Binding
Given that Scp1 and Sac6/fimbrin show genetic interactions
and have similar biochemical activities on actin, we investi-
gated whether they also have overlapping binding sites on
actin filaments. We tested the ability of Scp1 to compete with
Sac6 for binding to actin filaments in a high-speed cosedi-
mentation assay, by using constant concentrations of Sac6
(0.5 ?M) and actin filaments (2 ?M) and variable concentra-
tions of His6-Scp1 (0–7 ?M). In the absence of Scp1, nearly
90% of the Sac6 bound to actin filaments (Figure 8, B and C).
The percentage of Sac6 bound to actin filaments decreased
proportionally with increasing concentrations of Scp1. To
test the specificity of the competition, we assayed Sac6 bind-
ing to actin in the presence of another actin binding protein,
tropomyosin (Tpm1). A range of Tpm1 concentrations (1–10
?M) had no effect on Sac6 binding to actin filaments (our
unpublished observations). Thus, Scp1 specifically competes
with Sac6 for binding to actin.
Calponins and transgelins comprise one of the most widely
conserved actin-associated protein families, yet their in vivo
assembly and organization monitored by light scattering. Mono-
meric yeast actin (4 ?M) was polymerized in the absence or pres-
ence of His6-Scp1 (1 or 2 ?M) and monitored for change in light
scattering (360 nm) overtime. (B) Low-speed pelleting assay for
actin filament cross-linking. The reactions shown in A were centri-
fuged at low speed (10,000 ? g) to precipitate cross-linked actin
Actin filament cross-linking by Scp1. (A) Actin filament
filament structures. Pellets and supernatants were analyzed by SDS-
PAGE and Coomassie staining. (C) Low-speed pelleting assay of 2.5
?M actin filaments incubated with and without untagged Scp1
(middle two lanes) or His6-Scp1 (last two lanes). Note that a pro-
teolytic fragment of actin is marked by a single asterisk. (D) Effect of
His6-Scp1 on viscosity of actin filaments in solution. Actin (4 ?M)
was polymerized in capillary tubes in the presence of varying
concentrations of His6-Scp1 (0, 0.23, 0.46, 0.93, 1.4, 1.8, and 3.7 ?M).
Viscosity was measured after 1-h incubation. Viscosity is inversely
proportional to the velocity of the ball moving through the sample
(Pollard and Cooper, 1982). Gelation (as indicated by the ball-
bearing remaining stationary beneath the meniscus) was observed
at Scp1 concentrations above 1.4 ?M.
A. Goodman et al.
Molecular Biology of the Cell 2624
function in nonmuscle cells has remained elusive. Further-
more, controversy has surrounded the issue of whether
transgelins even bind to actin filaments and/or localize to
actin structures in vivo (reviewed in Small and Gimona,
1998; Morgan and Gangopadhyay, 2001). Herein, we have
shown unambiguously that the yeast transgelin homolog
Scp1 binds directly to actin filaments, cross-links and stabi-
lizes actin filaments in vitro, and localizes to actin filament
structures in vivo. Furthermore, we established that the sites
on Scp1 necessary and sufficient for actin cross-linking re-
side in the carboxy terminus, outside the CH domain. We
also provide the first in vivo evidence for transgelin cellular
function, showing that Scp1 cooperates with Sac6 (fimbrin)
to organize and stabilize the actin cytoskeleton. Finally, our
mutant analysis revealed a correlation between in vivo phe-
notypes and in vitro activities on actin, demonstrating that
actin binding is required for Scp1 in vivo functions.
Activities of Scp1 on Actin Filaments In Vitro
Scp1 binds directly to actin filaments with an affinity (Kdof
?0.7 ?M) similar to that reported for other members of the
calponin family: 1 ?M for calponin (Lu et al., 1995; Tang et
al., 1996) and 1.3 and 1.4 ?M for transgelin (Shapland et al.,
1993; Kobayashi et al., 1994). Scp1 also cross-links actin fila-
ments. Previous studies have reported actin filament bun-
dling for calponin (Kolakowski et al., 1995) and actin fila-
ment gelation for transgelin (Shapland et al., 1993).
However, the ability of transgelin to cross-link actin fila-
ments has been questioned (Morgan and Gangopadhyay,
2001), in part because the gelation activity occurs specifically
in low ionic strength buffer and is blocked by the addition of
10 mM KCl (Shapland et al., 1993). Using four independent
assays, we showed that His6-Scp1 and untagged Scp1 each
cross-link actin filaments in buffer containing 50 mM KCl.
One possible explanation for the discrepancy between pre-
vious results and ours is that previous experiments tested
transgelin and actin from different species, whereas our
experiments used transgelin (Scp1) and actin from the same
organism. This raises the possibility that other transgelins
besides Scp1 also cross-link actin filaments.
By what mechanism does Scp1 cross-link actin filaments?
Cross-linking requires either the presence of two actin bind-
ing sites within a single polypeptide chain or dimerization of
an actin binding protein. All of the data available for calpo-
nin family members suggest that they do not dimerize,
because they behave as monomers in sedimentation veloc-
of negatively stained actin fila-
ments cross-linked by Scp1 or
Sac6. Actin filaments (15 ?M)
were polymerized in the presence
of control buffer (A), 7.5 ?M His6-
Scp1 (B and C), or 7.5 ?M Sac6
(D), negatively stained with ura-
nyl acetate, and photographed at
21,000? magnification. Bar, 100 nm.
Yeast Calponin Functions with Fimbrin In Vivo
Vol. 14, July 20032625
ity, sedimentation equilibrium, and gel filtration experi-
ments (Lees-Miller et al., 1987b; Stafford et al., 1995). Simi-
larly, we found no evidence for Scp1 dimerization by using
several methods: gel filtration, yeast two-hybrid assay, and
coimmunoprecipitation of HA-tagged Scp1 with untagged
Scp1 (our unpublished observations). We cannot rule out the
possibility that Scp1 dimerizes (e.g., it may dimerize specif-
ically when bound to actin). However, based on the avail-
able data, we speculate that Scp1 (and possibly other cal-
ponins) cross-link actin filaments via two distinct actin
The location of the two sites required for actin filament
cross-linking was revealed by the analysis of the mutant
proteins. One actin binding site probably resides in the CLR
(Figure 1B), because specific mutation of a single residue in
the CLR (S185A) abolished actin filament cross-linking and
greatly reduced actin binding affinity of Scp1. These results
are in agreement with the previous studies that identified
the analogous serine residue to be critical for actin binding
of other calponin family members (Winder et al., 1993; Tang
et al., 1996; Fu et al., 2000). These data also suggest that the
mechanism of actin binding by calponins is highly con-
Our analysis of the truncated Scp1 constructs revealed the
location of a second site required for actin filament cross-
linking. The amino-terminal Scp1 fragment His6-N(1–136)
containing CH domain did not bind to actin filaments,
whereas the carboxy-terminal fragment C(136–200) not only
bound to actin filaments, but also cross-linked them. It is
formally possible that the hexa-histidine tag interfered with
the actin binding of the amino-terminal fragment, yet it
seems unlikely, given that the full-length his-tagged Scp1
bound to actin filaments. In addition, single CH domains of
mammalian calponin and transgelin are also not sufficient
for in vitro binding to actin filaments (Gimona and Mital,
1998; Fu et al., 2000). The second actin binding site or dimer-
ization site of Scp1 must reside in the sequences between the
CH domain and the CLR. This site may be analogous to
the actin binding site S1 of calponin (Mezgueldi et al., 1995;
Mino et al., 1998; Figure 1A). Although the sequence of S1 is
not conserved among calponin isoforms or in transgelins
(Gimona and Mital, 1998), this region is enriched in posi-
tively charged amino acids in all calponin family members.
Mutations of the positively charged amino acids in this
region decrease actin binding affinity of calponin and trans-
gelin (Gong et al., 1993; Fu et al., 2000). Further mutational
analysis of Scp1 will be required for precise identification of
the residues required for cross-linking of the actin filaments.
Does the CH domain of Scp1 contribute to actin binding?
Our data show clearly that the CH domain is neither suffi-
tions on actin filament binding and
cross-linking. (A) High-speed actin
filament cosedimentation assay.
Reactions contained 0 or 5 ?M
yeast actin filaments and 5 ?M
Scp1: full-length His6-Scp1, amino
terminus His6-Scp1N(1–136), or
carboxy terminus Scp1C(136–200)
(designated WT, N, and C, respec-
tively). Actin filaments were pel-
leted by high-speed centrifugation
and equivalent amounts of the pel-
let and supernatant were analyzed
staining. (B) Low-speed pelleting
assay for actin filament cross-link-
ing. Actin filaments (5 ?M) were
mixed with 5 ?M Scp1 [His6-Scp1,
200)] or control buffer and centri-
fuged for 10 min at 10,000 ? g. The
pellets and supernatants were an-
alyzed as described above. (C)
High-speed actin filament cosedi-
mentation assay comparing wild-
type and mutant Scp1 proteins.
Yeast actin filaments (10 ?M) were
mixed with 6 ?M His6-Scp1 (wild-
type, S185A, or S185D mutant) or
buffer alone. Actin filaments were
pelleted by high-speed centrifuga-
tion and pellets and supernatants
were analyzed by SDS-PAGE and
Coomassie staining. Lanes: 1, actin
and Scp1; 2, actin and Scp1 S185A;
3, actin and Scp1 S185D; and 4:
Effects of Scp1 muta-
actin alone. Note that proteolytic fragments of Scp1 are marked by asterisks. (D) Actin filament assembly and organization monitored by light
scattering. Monomeric yeast actin (10 ?M) was polymerized in the absence or presence of 5 ?M wild-type or mutant His6-Scp1 and monitored for
change in light scattering (360 nm) over time.
A. Goodman et al.
Molecular Biology of the Cell2626
cient nor necessary for actin filament binding by Scp1, yet
Scp1 competes for actin binding with Sac6/fimbrin, which
binds actin via two tandem pairs of CH domains. This
apparent discrepancy may be explained by a recently pro-
posed model. Based on comparing cryo-electron microscopy
reconstructions of calponin and fimbrin decorated actin fil-
aments, it was suggested that the CH domain of calponin
may serve as a “locator” domain, helping to position the true
actin binding motifs in calponin (reviewed in Winder, 2003).
The CH domain of Scp1 may act similarly.
Scp1 Functions with Sac6 to Regulate the Actin
Cytoskeleton In Vivo
Our genetic and biochemical data, as well as subcellular
localization, reveal a functional relationship between Scp1
and Sac6/fimbrin. Both GFP-Scp1 (this work) and Sac6
(Drubin et al., 1988) localize to cortical actin patches. Sac6
was also reported to colocalize faintly with actin cables by
immunofluorescence; however, this was not observed with
GFP-Sac6 (Doyle and Botstein, 1996). Therefore, it is possible
that Scp1 localizes in vivo to both actin patches and cables,
but that we have only been able to detect patch localization
with GFP-Scp1. Biochemical analyses show that Scp1 and
Sac6 have similar activities on actin. Like Sac6/fimbrin, Scp1
cross-links actin filaments in vitro. Sac6 cross-links actin
filaments into tight bundles, and Scp1 cross-links actin into
loose bundles and networks. Scp1, like Sac6/fimbrin, not
only organizes actin filaments but also decreases the rate of
actin filament disassembly (filament stabilization). The
shared role of SAC6 and SCP1 in stabilizing the actin cy-
toskeleton is supported further by the latrunculin A sensi-
tivities of sac6? and scp1? mutant cells. Together, these in
vitro and in vivo observations suggest that Scp1 and Sac6
cooperate in organizing and stabilizing the actin cytoskele-
The overlapping genetic functions of SCP1 and SAC6 may
be related to their relative abundance in cells and their
ability to compete for actin binding. Using quantitative im-
munoblotting, we defined the in vivo molar ratios of actin,
Sac6, and Scp1 to be ?65:6:1 (actin to Sac6 to Scp1). The
higher levels of Sac6 compared with Scp1 suggest that Sac6
may provide the more “dominant” activity on actin. This
idea is supported by the relative strengths of their respective
null phenotypes. Furthermore, this could explain why as
little as two- to threefold higher expression of Scp1 partially
suppresses defects in sac6? cells.
To demonstrate the importance of the Scp1–actin interac-
tion for in vivo functions, we have used a mutant of Scp1
that has a weak affinity for actin filaments in vitro.
scp1S185A failed to suppress loss of SAC6 or loss of SCP1
function in a sac6? background. On the other hand,
scp1S185D mutant, which retained actin filament binding in
vitro, suppressed the phenotypes associated with the loss of
SCP1 and SAC6 in vivo. These results provide strong evi-
dence that Scp1–actin interactions are required for in vivo
functions of Scp1 shared with Sac6.
A functional relationship between calponins and fimbrin
may be conserved in other organisms. Both protein families
are widely expressed in different vertebrate tissues and have
overlapping subcellular locations. In fibroblasts, fimbrin and
calponin are both found on stress fibers (Shapland et al.,
1988; Messier et al., 1993; Babb et al., 1997; Jiang et al., 1997;),
where they might function together to regulate actin cross-
linking and stabilization. In addition, fimbrin and calponin
may play a role in adhesive actin structures, linking the actin
cytoskeleton to the cell membrane. Fimbrin is found at focal
adhesions and podosomes (Messier et al., 1993; Babb et al.,
fimbrin. (A) Effects of His6-Scp1 on the rate of actin filament depo-
lymerization. Actin filament disassembly was initiated by the addi-
tion of 20 ?M latrunculin A to 2 ?M preformed actin filaments (1%
pyrene labeled) in the presence of 0, 67, 200, 670, and 2000 nM
His6-Scp1, and change in pyrene-actin fluorescence was monitored
overtime. (B) Competition of Scp1 with Sac6 for binding to actin
filaments. Cosedimentation assay using 2 ?M yeast actin filaments
0.5 ?M Sac6, and variable concentrations of His6-Scp1 (0, 0.35, 0.7,
1.75, and 7 ?M). The pellets and supernatants were analyzed by
SDS-PAGE and Coomassie staining. (C) The concentrations of Sac6
(Œ) and Scp1 (?) bound to actin filaments were calculated from gel
densitometry and plotted versus the total concentration of His6-
Scp1 in the reactions.
Activities of Scp1 on actin filaments similar to Sac6/
Yeast Calponin Functions with Fimbrin In Vivo
Vol. 14, July 2003 2627
1997), and calponin is found in dense plaques, a type of
adherence junction similar to podosomes and focal adhe-
sions (North et al., 1994). Finally, it has been proposed that
fimbrin may link the actin cytoskeleton to the vimentin
intermediate filament cytoskeleton, and a vimentin-binding
site has been mapped to the first CH domain of fimbrin
(Correia et al., 1999). A similar function for calponin has been
suggested by in vitro binding studies and overlapping in
vivo localization of desmin and calponin (North et al., 1994;
Mabuchi et al., 1996; Wang and Gusev, 1996). Thus, calponin
and fimbrin may have shared in vivo functions that are
conserved across a wide range of organisms.
We thank Brian Cali for generous help during the initiation of this
project, Nicki Watson for assistance with microscopy, and Heath
Balcer and Avital Rodal for helpful advice and critical reading of the
manuscript. The microscopy was conducted in the W.M. Keck
Foundation Biological Imaging Facility (Whitehead Institute, Cam-
bridge, MA). A.G. was supported by a predoctoral fellowship from
the Howard Hughes Medical Institute. B.G. was supported by a
Pew Scholars award, a Basil O’Conner award, and a grant from the
National Institutes of Health (GM-63691). P.M. was supported by a
grant from the National Institutes of Health (GM-/AR57418). G.F.
was supported by a grant from the National Institutes of Health
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