|
|
|
|||||||
|
|||||||||
(Received for publication, July 11, 1995; and in revised form, October
5, 1995) From the
During activation of the Limulus sperm acrosomal
process, actin filaments undergo a change in twist that is linked with
the conversion from a coiled to a straight scruin-actin bundle. Since
scruin had not been purified, its identity as an actin-binding protein
has not been demonstrated. Using HECAMEG
(methyl-6-O-(N-heptylcarbamoyl)-
In many examples of cell motility including: cytokinesis,
phagocytosis, exocytosis, chemotaxis, and extension of the lamella,
movement or force is generated by either actin-myosin interactions or
the reversible assembly of actin filaments(1) . Contrary to
these examples, extension of the acrosomal process in Limulus sperm may be a movement of an actin spring in which potential
energy, stored as a coiled bundle at the base of sperm body, is
unleashed at fertilization to uncoil and extrude the bundle through a
channel in the nucleus(2, 3) . During the uncoiling
process, the actin bundle untwists by an impressive 60° per 700 nm.
This action is accompanied by slippage and a modest (-0.23°
per subunit) untwisting of the actin filaments(4, 5) .
As a result of these events, the bundle forms a 60-µm-long membrane
extension, which bridges the egg jelly coat to fuse with the egg plasma
membrane. The factors that maintain the coiled state of the bundle
or signal its rotation and slippage are unknown, but the target of
their action must be scruin, an actin-associated protein in the
acrosomal process. Previous studies show that the acrosomal process
consists of a 1:1 complex of actin and scruin (M Based
on sequence analysis, limited proteolysis, and EM image
reconstructions, scruin is organized into two 40-kDa domains connected
by a highly helical protease-sensitive
neck(7, 8, 9) . Each domain consists of a
six-fold repeat of To identify the mechanism that causes
the dynamic conformational changes in the acrosomal process during the
acrosome reaction, we must first purify scruin and characterize its
actin binding properties. In this report, we describe the isolation of
scruin and report its association with calmodulin. Furthermore, we show
that the scruin-calmodulin complex cross-links F-actin into bundles
but, surprisingly, the cross-linking activity is independent of
calcium. Our results suggests that scruin is always bound to actin
filaments, and we hypothesize that during the acrosome reaction the
conformational changes in the actin filament and acrosomal process may
be caused by a subtle conformation change in scruin.
Treatment of true discharges, isolated in Triton X-100, with
1 M calcium disassociated scruin from actin. However, after a
few hours, the soluble scruin precipitated from solution (not shown).
Subsequent experiments determined that long term solubility depended on
the removal of the Triton X-100 by dialysis at high ionic strength
(0.45 M NaCl). Other treatments such as denaturing agents (8 M urea) and low pH (0.2 M glycine, pH 4.2) were found
to also disassociate the scruin actin bundles, but less than half of
the scruin remained active, as judged by high speed sedimentation with
rabbit skeletal actin (100,000
Figure 1:
Isolation and calcium treatment of
scruin-actin bundles and stoichiometry of scruin-actin-calmodulin
complex. A, SDS-PAGE of true discharges extracted with HECAMEG (lane T) show the presence of scruin, actin, and a 17-kDa
protein. The supernatant (lane S) of the calcium extract
contained mostly scruin and a 17-kDa protein, whereas the pellet (lane P) was enriched in actin. B, HPLC traces of
washed HECAMEG true discharges indicate that the ratio of
scruin:actin:calmodulin is essentially
1:1:1.
Figure 2:
Copurification of calmodulin and scruin by
gel filtration and quaternary amine ion exchange chromatography. A, the low speed supernatant was chromatographed through
Superdex 200 HR (top panel). SDS-PAGE (bottom panel)
of the fractions shows scruin and a 17-kDa polypeptide co-fractionate. B, a pool of scruin-containing fractions was loaded onto a
Q-HiTrap column. The chromatograph (top panel) indicates a
single peak elutes at at 40% buffer B. SDS-PAGE (bottom panel)
shows that scruin co-purifies with a 17-kDa band that was determined by
internal protein sequencing to be
calmodulin.
The presence of
calmodulin was also confirmed by immunofluorescence microscopy (Fig. 3). In unactivated sperm cells, calmodulin is localized to
the base of the nucleus as a ring of fluorescence, which colocalizes
with the rhodamine phalloidin staining pattern of F-actin or
immunostaining of scruin. Quantitation of the fluorescence showed that
71% (S.D. = ± 7.6, n = 7) of the
calmodulin was found to localize to the bundle at the base with a less
apparent staining in the nucleus and the perimeter of the acrosomal
vesicle. No calmodulin staining was observed within the flagellar
region of the sperm or the interior of the acrosomal vesicle.
Figure 3:
Distribution of scruin and calmodulin in Limulus sperm. a and b, DIC images of
unactivated sperm show the presence of long flagella and an apical
vesicle. c, phalloidin (green) and
To
determine the stoichiometry of the scruin, actin, and calmodulin in the
acrosomal process, we quantified the peak areas of samples separated by
gel exclusion chromatography in denaturing conditions. Based on the
integrated peak areas and the known molecular masses for the proteins,
the scruin:actin:calmodulin molar ratio was 1.00:1.15:0.97 (Fig. 1B). Although scruin and calmodulin were always
seen to co-purify, the molar ratio of the two proteins was sometimes
1:0.5, depending on the individual preparation. This variability in the
ratio of scruin to calmodulin after purification either is due to the
extraction conditions or is merely a consequence of an equilibrium for
binding and subsequent dissociation of calmodulin during purification.
Figure 4:
Scruin binds calmodulin avidly in calcium
based on blot overlays with
Although
scruin is predicted to be mainly
Figure 5:
The
putative calmodulin binding site. a, the protease-sensitive
neck region of scruin contains an amphipathic helix that is similar to
the calmodulin binding IQ motif of MYO 2. Bold text delineates
conserved residue matches. b, the helical wheel representation
of the peptide PSN1 identifies basic and hydrophobic faces, which are
characteristic of a calmodulin binding
motif.
Figure 6:
Cosedimentation of scruin with actin in
EGTA and calcium. A, various concentrations of scruin were
incubated with 2 µM actin in the presence of EGTA
(
Figure 7:
Reconstitution of scruin actin bundles.
Based on electron microscopy, scruin is capable of forming bundles with
rabbit skeletal F-actin in the presence of EGTA (a) and
calcium (b). These bundles have a similar packing to the Limulus bundles (c) but are not crystalline. In the
absence of scruin F-actin does not form bundles, and in the presence of
scruin alone no filamentous structures are observed (data not shown).
With the addition of exogenous sperm calmodulin at a 3.75-fold molar
excess over scruin, there is no apparent difference in the formation of
bundles (data not shown). Bar = 100
nm.
The long term goal of our studies is to understand how the
actin bundle uncoils during the acrosome reaction to form the
60-µm-long acrosomal process. We have made major strides toward
this goal through the development of methods to separate and purify
native scruin from actin. Although there are several three-dimensional
views of the filaments in the acrosomal
process(7, 8, 22) , this study provides the
first biochemical information about the protein components of the
acrosomal process. Two tricks enabled us to purify scruin as a soluble
and native protein and characterize it as an actin cross-linking
protein. First, because calcium disrupts the acrosomal
process(2) , we were able to extract scruin as a native protein
without using denaturants. Second, we modified the demembranation step
by replacing the detergent Triton X-100 with HECAMEG. This eliminated
the aggregation of the purified scruin and improved the overall yield
of protein. With the purified scruin, we first confirmed that scruin
is an actin cross-linking protein. Previously the only evidence for
actin binding was the observation that scruin is the only
actin-associated protein in the acrosomal process and that EM
reconstructions reveal the outside of the actin filament is decorated
in a 1:1 stoichiometry by a two-domain protein, approximately the mass
of scruin. Although these are powerful arguments that scruin is the
actin cross-linking protein in the acrosomal process, they do not
substitute for direct evidence. Thus, it was critical to show that
scruin binds actin filaments and reconstitutes actin filaments into
bundles. In the reconstituted bundles, the approximate equimolar ratio
of scruin and actin is maintained but the reconstituted actin bundles
do not display the characteristic banding pattern that is indicative of
the crystalline organization of F-actin and scruin in the acrosomal
process. This difference in apparent organization between the in
vitro and in vivo assembled bundles could easily be
attributed to the assembly conditions. For example, the slow assembly
of the actin bundle during spermatogenesis may be more favorable to the
formation of a crystalline bundle, while the relatively rapid assembly
of the bundle in vitro may cause the disorder. One novel
and important finding reported in this paper is that scruin is
complexed with a single calmodulin molecule. The identification of
calmodulin and its binding site in the neck region provides a more
detailed model of scruin structure. In EM reconstructions, a neck
region connects two large domains each of which binds a separate actin
subunit on the same filament. Based on secondary structure predictions
and similarity with galactose oxidase, the protein sequence of scruin
is organized as two large Our present evidence shows both calcium-dependent
binding of calmodulin to intact scruin and calcium-independent binding
to the PNS1 scruin neck peptide. We speculate that the flanking large
domains in scruin partially inhibit the binding of calmodulin in the
absence of calcium. In either case the immunofluorescence localization
of calmodulin within the membrane-limited acrosomal process suggests
the possibility that the local concentration of calmodulin could be
high. A high calmodulin concentration would ensure a calmodulin-scruin
complex is maintained in unactivated sperm. The presence of a
calmodulin subunit immediately suggests that actin binding by scruin is
calcium regulated. Normally, in many enzymatic complexes, calmodulin is
a regulatory subunit, which binds or dissociates from a target
catalytic subunit and thus acts as a calcium-dependent switch to
activate or inactivate the enzyme. However, our biochemical studies
suggest a different type of regulatory mechanism, because scruin binds
actin independently of calcium. We conclude that calmodulin does not
regulate actin binding activity in an on-off fashion. This finding
eliminates a simple on-off binding event as a mechanism for inducing
the conformation changes in the actin filament. Instead, our results
suggest that scruin is bound to actin before as well as after the
acrosome reaction. Although the coiled bundle of unactivated sperm has
not been studied, our speculation is supported by a related structure,
the supercoiled false discharge, which has been shown to contain
scruin(8) . Based on our studies, we propose a model that
calmodulin may instead control the twist of an actin filament or bundle
by regulating the conformation of scruin. We envision a mechanism in
which calmodulin acts as a wedge between the actin-binding domains (Fig. 8). In this position, changes in the conformation of
calmodulin could alter the relative positions of the actin binding
domains. The conformation change in scruin is then transmitted to the
underlying actin subunits which allows the actin filaments to untwist
by 0.23° between subunits (4, 5) . The local
change in twist is multiplied along the length of the filament causing
a large change in the filament. The change in filament twist breaks
scruin-scruin cross-links between neighboring filaments which allows
the filaments to slip as the bundle uncoils into the straight acrosomal
process. The caveat to this model is that we do not yet appreciate how
scruin-scruin interactions allow for the formation of bundled
filaments, since under the present conditions purified scruin appears
to be monomeric. It is possible that scruin interactions with itself
are much weaker than actin-scruin or scruin-calmodulin interactions.
Alternatively, the scruin-scruin binding site may not be exposed until
scruin binds actin.
Figure 8:
Model
for extension of the bundle during sperm activation. A cross-section
diagram (viewed from the tip) of a scruin-bound actin filament in the
acrosomal process before and after calcium binding. A pair of actin
subunits is associated with a scruin calmodulin complex that is not
drawn to scale, and the angular untwisting is exaggerated to show this
subtle change in structure. Upon sperm activation, calcium ions bind to
calmodulin, which initiates a series of conformation changes. First, in
calcium, calmodulin binds scruin more tightly, which then induces a
conformation change in scruin. Consequently, scruin binds more tightly
to actin, which causes a subtle rearrangement in the pair of actin
subunits. This rearrangement allows the unbending and extension of the
actin bundle.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2651-2657
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-glucopyranoside)
detergent extraction in concert with high calcium, we purified native
scruin and identified it as an equimolar complex with calmodulin. I-Calmodulin overlays and calmodulin-Sepharose indicate
that scruin binds calmodulin in calcium but not in EGTA. Overlay
experiments also map the calmodulin binding site between the putative
N- and C-terminal
-propeller domains within residues
425-446. Immunofluorescence microscopy reveals that calmodulin
colocalizes with scruin and actin in the coiled bundle. Although scruin
binds calmodulin, pelleting assays and electron microscopy show that
the scruin cross-links F-actin into bundles independently of calcium.
Based on our biochemical and structural studies, we suggest a model to
explain how scruin controls a change in twist of actin filaments during
the acrosome reaction. We predict that calcium subtly alters scruin
conformation through its calmodulin subunit and the conformation change
in scruin causes a shift in the relative positions of the scruin-bound
actin subunits.
102,000)(6) . In EM (
)reconstructions, scruin
decorates the outside of an actin filament, with each scruin molecule
bound to a pair of actin subunits along the actin one-start
helix(7) . Presumably, actin cross-links are maintained by
interactions between scruin proteins on neighboring filaments.50 amino acids that based on the work of Bork
and Doolittle(10) , is predicted to fold into a four stranded
-sheet motif(9) . This motif typifies a protein
superfamily, which includes galactose oxidase(10) , several
open reading frames in the genome of pox viruses(11) , a mouse
placental transcript, MIPP(12) , and kelch, the Drosophila gene that is important for nutrient transport
during oogenesis(13) . Although there is some understanding of
the structural organization of scruin, its regulation and biochemical
properties are not understood because the protein has not been purified
in a native, soluble state.
Materials
Pepstatin, leupeptin, aprotinin,
benzamidine, and ATP were purchased from Sigma. HECAMEG and calcium
ionophore A23187 were purchased from Calbiochem. I-Calmodulin was purchased from DuPont NEN. Artificial
sea water was purchased from Tropic Marin. Protein assays were
performed with the Bio-Rad assay reagent.
Purification of the True Discharge
The true
discharge was isolated using a protocol modified from Schmid et
al.,(6) . Twenty ml of sperm, collected from 30-40
adult male horseshoe crabs, was divided into six aliquots, layered on
33 ml of a 1:1 mixture of artificial sea water and 50 mM calcium chloride, and then gently mixed. Sperm were activated by
adding 100 µl of 19 mM A23187 (in dimethyl sulfoxide) to
each tube and incubated in the dark for 30 min. To prevent proteolysis,
a mixture of protease inhibitors were added to each step (final
concentrations: 0.1 mM phenylmethylsulfonyl fluoride, 0.1
mM benzamidine, 0.1 mM pepstatin A, 1.0 KIU/ml
aprotinin, and 0.2 µg/ml leupeptin). The activated sperm were
sheared three times through a 21-gauge needle and centrifuged twice at
2420 
g for 10 min to remove the sperm heads. The
supernatant was then centrifuged at 43,140 g for 15
min to pellet the acrosomal bundles. Each pellet was resuspended in 5
ml of scruin buffer A (10 mM Tris, pH 8.0, 1 mM dithiothreitol, 1 mM CaCl
, 100 mM NaCl, 0.01% NaN
) with 19 mM HECAMEG for 15
min on ice. The volume was diluted to 30 ml with scruin buffer A and
centrifuged at 43,140 g for 15 min. The pellets were
pooled into 1 ml of buffer A and pelleted at 16,000 g in a microcentrifuge for 15 min. To quantify the stoichiometry of
scruin and calmodulin by HPLC, this washing step was repeated 5 times
to reduce the protein contaminants. In a typical preparation, 5.2 mg of
true discharge were obtained as determined by the Bio-Rad assay. The
purity of the true discharge preparations was assessed by SDS-PAGE.Purification of Soluble Scruin
Scruin was
dissociated from actin by gently shaking the isolated bundles in 1 ml
of 1 M calcium for 1 h at room temperature, and then
centrifuging the extract for 15 min in a microcentrifuge (16,000
g) to remove most of the actin filaments. The low
speed supernatant containing primarily scruin, calmodulin, actin, and
some minor contaminants (3.7 mg of total protein) was successively
filtered through 0.8-, 0.45-, and 0.2-µm filters. In the later
stages of the project, nucleic acids in the extract were removed by
filtration through a 0.8-µm filter and then a Qiagen Tip 5. The
filtrate was loaded onto a Superdex 200 HR FPLC gel filtration column
pre-equilibrated and run in scruin buffer A supplemented with
inhibitors. The fractions that contained scruin were pooled (1.1 mg of
total protein), loaded onto a Q-HiTrap (Pharmacia Biotech Inc.) ion
exchange column with scruin buffer A, and eluted with a 0-1 M NaCl gradient in buffer A. Scruin protein eluted in 0.4 M NaCl. The scruin-containing fractions (300 µg of total
protein) were either shell-frozen in liquid nitrogen and stored as a
lyophilate or concentrated by Ultrafree-MC centrifugal membrane filters
(Millipore) and used immediately.Identification of Calmodulin
The stoichiometry of
the scruin-actin-calmodulin complex was determined by size exclusion
chromatography through a TSK 300SWLX column (Toyo Soda) under
denaturing conditions (6 M guanidine HCl). The absorbance was
measured at 230 nm. The molar ratio was calculated from the integrated
peak areas. The 17-kDa polypeptide was identified as calmodulin by
protein sequence obtained by mass spectrometry or chemical sequencing.
First, the scruin-calmodulin complex was dissociated in 6 M guanidine HCl and separated by size exclusion chromatography. The
purified calmodulin was cleaved with cyanogen bromide in 70% formic
acid for 12 h as described previously (Matsudaira, 1992). Following
incubation the sample was diluted in water and dried under vacuum in a
fume hood. The fragments were purified by reverse phase chromatography
on the HPLC. The fractions that were found to contain a single
polypeptide by mass spectrometry (LASERMAT, Finnigan MAT) were
N-terminally sequenced by gas phase sequencing on a model 2090E
sequencer (Beckman Instruments Inc.) equipped for the identification of
phenythiohydantoin amino acids. In later experiments, Limulus sperm calmodulin was purified from sperm with a phenyl-Sepharose
column on the FPLC using standard methods(14) . The purified
protein was confirmed to be calmodulin by calcium-dependent mobility
shift on SDS-PAGE and sequence by mass spectrometry using a PerSeptive
Biosystems Voyager Elite time of flight mass spectrometer.Identification of the Calmodulin Binding
Site
I-Calmodulin gel overlays were performed as
described previously (15) with minor
modifications(16) .
I-Calmodulin nitrocellulose
blot overlays were performed as described (17) using 0.05%
Tween 20 and 30 mg/ml bovine serum albumin as the blocking agents. For
scruin, we found that either method worked, but the nitrocellulose blot
overlays had a higher signal to noise ratio. In contrast, the myosin I
positive control only worked with the gel overlay method. The percent
binding was quantitated using NIH Image software and is represented as
the ratio of the signal intensity and the total signal in calcium and
EGTA
100. A value of zero indicates the signal was not above
the background levels. Competition assays with scruin and the peptide
PSN1 (described below) were performed in calcium only.Protein Expression of Scruin Fragments
The
calmodulin binding site in scruin was mapped using either expressed
scruin fragments or synthetic peptides to specific regions in scruin as
described below. The 454C and 590C domains of scruin were expressed in Escherichia coli and purified as described
previously(9) . Additionally, three constructs, GST1, GST2, and
GST3, consisting of residues 390-429, 430-469, and
470-509 of scruin fused to the GST domain, respectively, were
engineered by polymerase chain reaction using standard methods.
Briefly, an in-frame BamHI site was introduced 5` to the first
amino acid codon and a TAG stop/EcoRI site after the last
codon. The resulting polymerase chain reaction products were cloned
into the BamHI-EcoRI sites of pGEX-2T (Pharmacia) and
the fidelity of the final expression constructs confirmed by
sequencing. GST1 and GST3 were transformed into DH5 and expressed
upon addition of isopropyl-1-thio--D-galactopyranoside.
However, because the GST2 fusion construct did not express in E.
coli under a number of expression conditions, a synthetic peptide,
PSN1 (CGAAKKVQRRWRRYIEQKSITKRM), was synthesized. The N-terminal
sequence CG was included to aid in generating a sequence-specific
polyclonal antibody. Two other synthesized peptides to a sequence near
the protease-sensitive neck region of scruin and a sequence in
-scruin (R34, CKAKPQPGSKPTSVK; R35, CTTRSGSRKTQKTLK, respectively)
were also used as controls(18) .Actin Cosedimentation Assays
Cosedimentation
assays with purified scruin (0-4 µM), sperm
calmodulin (15 µM), and rabbit skeletal actin (2
µM) were performed in the presence of 1 mM EGTA
or 1 mM calcium as described previously(16) . Briefly,
samples were incubated for either 4 °C overnight or for 1 h at room
temperature and were centrifuged for 30 min in a bench-top
ultracentrifuge (TL100, Beckman Instruments Inc., Fullerton, CA) at
75,000 rpm at 4 °C. Pellets and supernatants from the
centrifugation were adjusted to equal volumes and analyzed by SDS-PAGE,
and electron microscopy. To determine whether the ability of scruin to
bind and bundle F-actin was affected by calmodulin, exogenous sperm
calmodulin was added to scruin at 3.75-fold molar excess.Reconstitution of Scruin-Actin
Bundles
Scruin-actin bundles were formed by incubating scruin
and actin, with or without exogenous sperm calmodulin at a molar ratio
of at a 2:1:3.75, respectively, for 1 h at room temperature prior to
absorbing the samples onto carbon films. The samples were negatively
stained with 1.0% uranyl acetate and examined in a Phillips 410
electron microscope at an accelerating voltage of 80 kV. In some
instances calmodulin was added to the samples at 0-5-fold molar
excess of scruin.Immunofluorescence Microscopy
A rabbit polyclonal
antiserum (R213.5) was generated against scruin; the specificity to
crude sperm extracts has been described elsewhere(18) . The
mouse monoclonal anti-calmodulin antiserum was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). To reduce the nonspecific
background of the anti-scruin antibody for immunofluorescence, the
antibody was affinity-purified against scruin blotted to nitrocellulose
membrane (19) . Sperm were fixed and stained as described
elsewhere(18) . Coverslips were examined in a Zeiss Axioskop
microscope using differential interference contrast optics and a
100/NA 1.3 Plan Neofluar objective configured with a Bio-Rad
MRC600 Confocal ion laser. The ratio of the fluorescent intensity in
the bundle and the entire sperm was quantified using the standard
confocal image analysis software. For reproduction, the images were
transferred to Adobe Photoshop and Illustrator (Adobe Systems Inc.,
Mountain View, CA) for cropping, contrast/brightness adjustment,
placement of each image panel, and annotation.
g for 30 min: not
shown). Based on these findings, we replaced Triton X-100 with the non
ionic detergent HECAMEG. HECAMEG is easier to remove by gel filtration
or dialysis (critical micelle concentration 19.5 mM) and does
not absorb at 280 nm. After demembranation of the actin bundle with
HECAMEG and extraction of scruin with 1 M CaCl,
one half of the actin pellets at low g-forces leaving scruin
in the supernatant (Fig. 1A). The minor protein
contaminants, DNA, and HECAMEG in the calcium extract were removed by
gel filtration chromatography through Superdex HR 200 and ion exchange (Fig. 2). Based on relative molecular mass (M) standards, scruin fractionates as a monomer
with an apparent molecular weight of 107,000.
Identification of Calmodulin
At each stage of the
purification, a 17-kDa polypeptide consistently cofractionated with
scruin ( Fig. 1and Fig. 2). Although the N terminus was
blocked to chemical sequencing, we obtained sequence from two fragments
generated by cyanogen bromide cleavage (MKDTDSEEEI and
MIREADIDGDGQVNYEEFVTM). Data base searches with these two sequences
showed an exact match with calmodulin from Drosophila.
Additionally, this 17-kDa protein shows the characteristic
calcium-dependent mobility shift on SDS-PAGE gels and is recognized by
a monoclonal antibody to calmodulin (not shown).
-scruin (red) colocalize to the coiled actin bundle at the base of the
nucleus. d, calmodulin (green) primarily colocalizes
with phalloidin (red) in the bundle at the base of the sperm.
Additionally, the calmodulin also is also located in the nuclear region
and at the perimeter of the acrosomal vesicle. Bar =
5.0 µm.
Calmodulin Binding Site
To confirm that calmodulin
is a scruin subunit, we tested binding of exogenous bovine calmodulin
to scruin. By two criteria: I-calmodulin blot and gel
overlays (Fig. 4) and calmodulin-Sepharose chromatography (not
shown), calmodulin binds scruin in calcium but not in EGTA.
I-calmodulin. Purified scruin
was electrophoresed through SDS-PAGE gels and either stained with
Coomassie Blue (A) or electroblotted to nitrocellulose
membranes (B and C). The membranes were incubated
with
I-bovine calmodulin in the presence (B) or
absence (C) of calcium. The calmodulin bound only to scruin in
calcium; little or no binding was detected in EGTA. Similar results
were observed with gel overlays (not shown). Myosin I bound calmodulin
in calcium and EGTA as reported previously (data not shown; (24) ). D, competition experiments with scruin and
PSN1, the peptide containing the calmodulin binding site. Calmodulin
binding to scruin was inhibited by nanomolar concentrations of PSN1.
The estimated K
is <50 nM.
Each point is an average of two intensity
values.
-sheet, secondary structure
analysis predicted that the neck region (Fig. 5a)
between the N- and C-terminal domains of scruin is highly helical and
amphipathic. A comparison of this region with the calmodulin binding IQ
motif of MYO2 highlights the similar pattern of basic and hydrophobic
residues. A helical wheel representation of a portion of the neck
region clearly shows a basic and a hydrophobic face of the helix (Fig. 5b; Refs. 20 and 21). Because calmodulin binds to
the basic face of an amphipathic helix, we examined calmodulin binding
to various synthetic peptides, proteolytic fragments, and GST fusions
that spanned the neck region. Two GST fusion constructs, GST1 and GST3,
contain sequences that border outside of the predicted calmodulin
binding site. These fusion constructs did not bind the radiolabeled
calmodulin (Table 1). The third GST fusion (GST2) which contains
this region did not express in E. coli; however, a synthetic
peptide, PSN1, to the region 425-446 did bind calmodulin in EGTA
and calcium. The peptide at a concentration of 50 nM also
inhibited calmodulin binding to intact scruin by 61% (Fig. 4D). In other experiments, the C-terminal half of
scruin (454C and 590C, produced by expression in E. coli; (9) ), a tryptic digestion of scruin, or natural breakdown
products of scruin did not bind to the radiolabeled calmodulin in the
presence or absence of calcium (not shown). These proteolytic sites
have been previously mapped to the protease-sensitive neck region of
scruin(9) , suggesting that the calmodulin binding site is not
in the C- or N-terminal halves of scruin.
Characterization of Actin Cross-linking
Activity
Because scruin is the only actin-associated protein in
the acrosomal process, we tested it for actin binding activity using a
cosedimentation assay and electron microscopy. Scruin binds actin
independently of calcium. In calcium and EGTA, the majority of the
scruin is found associated with actin in the pellet (Fig. 6).
There is no difference between binding in EGTA and calcium.
Furthermore, the amount of actin found in the pellet is invariant
suggesting that scruin does not alter the F- to G-actin equilibrium. In
the absence of actin, scruin remains in the supernatant in high g-force sedimentation assays (not shown). Since exogenously
added calmodulin may affect the binding affinity of scruin for actin,
cosedimentation assays were also performed with a 3.75-fold molar ratio
of exogenously Limulus calmodulin (not shown). Examination of
the samples by electron microscope before centrifugation shows that
actin bundles form in the presence and absence of calcium (Fig. 7). The results from sedimentation assays and electron
microscopy show that the presence or absence of excess calmodulin does
not detectably affect actin binding activity of scruin.
) and calcium (
). SDS-PAGE samples of supernatants and
pellets show that in calcium and EGTA scruin binds avidly. B,
quantitation of the pelleting assays shows the slightly higher affinity
for actin in calcium than in EGTA. However, there is no apparent
difference in actin binding in the presence of exogenous sperm
calmodulin (data not shown). In the absence of actin, all scruin
remained in the supernatant (data not
shown).
-sheet domains, which are separated by a
highly helical region. The domain organization suggested by the
sequence is in exact correspondence with the EM reconstructions; thus,
we propose that the helical region of sequence is the neck region.
Within this helical region is the calmodulin binding sequence (residues
425-446) of PNS1. Thus, calmodulin bound to the neck would be
flanked by a pair of actin-binding domains. We had not detected
calmodulin previously because of its low molecular weight and
relatively poor dye-binding
capacity(6, 8, 23) . Because the previous EM
reconstructions were of acrosomes that may have been partially depleted
of calmodulin (prepared in the absence of calcium), we are checking the
new preparations to see if the neck region is thicker from the presence
of calmodulin.
)-D-glucopyranoside;
HPLC, high performance liquid chromatography; PAGE, polymacrylamide gel
electrophoresis; GST, glutathione S-transferase.
We thank Dr. Dan Gibson for expertise with Limulus and Dr. Julie Theriot, Dr. David Fung, and Navin Pokala for
critical assessment of this manuscript. M. C. S. thanks Dr. Julie
Theriot for support during the later stages of the work. We thank
Ya-Huei Tu for assistance with the electron microscope and Matthew
Footer for the gift of scruin protein used in the competition
experiments. Finally, we also thank Associates of Cape Cod (Falmouth,
MA) for letting us collect sperm samples from their supply of horseshoe
crabs.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. H. Shin, L. Mahadevan, G. S. Waller, K. Langsetmo, and P. Matsudaira Stored elastic energy powers the 60-{micro}m extension of the Limulus polyphemus sperm actin bundle J. Cell Biol., September 29, 2003; 162(7): 1183 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Miyagawa, H. Tanaka, N. Iguchi, K. Kitamura, Y. Nakamura, T. Takahashi, K. Matsumiya, A. Okuyama, and Y. Nishimune Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein {alpha}3 (cp{alpha}3) Mol. Hum. Reprod., June 1, 2002; 8(6): 531 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Kelso, A. M. Hudson, and L. Cooley Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation J. Cell Biol., February 18, 2002; 156(4): 703 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Guild, P. S. Connelly, K. A. Vranich, M. K. Shaw, and L. G. Tilney Actin filament turnover removes bundles from Drosophila bristle cells J. Cell Sci., January 2, 2002; 115(3): 641 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lécuyer, J.-L. Dacheux, E. Hermand, E. Mazeman, J. Rousseaux, and R. Rousseaux-Prévost Actin-Binding Properties and Colocalization with Actin During Spermiogenesis of Mammalian Sperm Calicin Biol Reprod, December 1, 2000; 63(6): 1801 - 1810. [Abstract] [Full Text]
|
|
|
|
 |
|
 V. Aidinis, D. C. Dias, C. A. Gomez, D. Bhattacharyya, E. Spanopoulou, and S. Santagata Definition of Minimal Domains of Interaction Within the Recombination-Activating Genes 1 and 2 Recombinase Complex J. Immunol., June 1, 2000; 164(11): 5826 - 5832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Johnson and S. W. Craig Actin Activates a Cryptic Dimerization Potential of the Vinculin Tail Domain J. Biol. Chem., January 7, 2000; 275(1): 95 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S Grieshaber and N. Petersen The Drosophila forked protein induces the formation of actin fiber bundles in vertebrate cells J. Cell Sci., January 7, 1999; 112(13): 2203 - 2211. [Abstract] [PDF]
|
|
|
|
|
|
J. Philips and I. Herskowitz Identification of Kel1p, a Kelch Domain-containing Protein Involved in Cell Fusion and Morphology in Saccharomyces cerevisiae J. Cell Biol., October 19, 1998; 143(2): 375 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. Kranewitter, J. Ylanne, and M. Gimona UNC-87 Is an Actin-bundling Protein J. Biol. Chem., February 23, 2001; 276(9): 6306 - 6312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Kelso, A. M. Hudson, and L. Cooley Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation J. Cell Biol., February 18, 2002; 156(4): 703 - 713. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |