From the Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0127
Received for publication, October 13, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Myosin VIIA was cloned from rat kidney, and the
construct (M7IQ5) containing the motor domain, IQ domain, and the
coiled-coil domain as well as the full-length myosin VIIA (M7full) was
expressed. The M7IQ5 contained five calmodulins. Based upon native gel
electrophoresis and gel filtration, it was found that M7IQ5 was
single-headed, whereas M7full was two-headed, suggesting that the tail
domain contributes to form the two-headed structure. M7IQ5 had
Mg2+-ATPase activity that was markedly activated by
actin with Kactin of 33 µM and
Vmax of 0.53 s Myosins are mechanochemical proteins with a motor domain
containing an ATP and actin-binding region, a neck domain that
interacts with light chains or calmodulin, and a tail domain that
serves to anchor and position the motor domain so that it can properly interact with actin filaments at a specific intracellular location. Phylogenetic analysis revealed that myosin consists of at least 18 classes (1, 2). Myosin VIIs are found in human (3, 4), mouse (5),
porcine (6), zebrafish (7), bullfrog (8), Caenorhabditis
elegans (9), Dictyostelium discoidium (10), and
Drosophila (11). In vertebrates, two types of genes for type
VII myosin, myosin VIIA and VIIB, are identified. Among them, the
entire coding region has been determined for myosin VIIA. In mammals,
myosin VIIA is expressed in a variety of organs and tissues including
eye, inner ear, olfactory epithelium, brain, choroid plexus, intestine,
liver, kidney, adrenal gland, testis, and lymphocytes (12-14). In the
retina, myosin VIIA is found in the pigmented epithelial cells and is
postulated to play a role in phagocytosis of cell debris that
accumulates as a result of sloughing off of photoreceptor outer
segments. Of interest is that defects of myosin VIIA cause the mouse
shaker-1 phenotype and human Usher syndrome 1B,
which are characterized by deafness, lack of vestibular function, and
(in humans) progressive retinal degeneration (5, 15). In humans, two
forms of dominant and recessive nonsyndromic deafness, DFNB2 and
DFNA11, are also caused by myosin VIIA mutations (16-18).
Amino acid sequence analysis of myosin VIIA has indicated that this
myosin has a motor domain containing actin-binding and ATP-binding
sites, and five IQ motifs at the neck domain. In the tail domain, a
very short predicted coiled-coil region was found; therefore, it has
been assumed that myosin VIIA forms a two-headed structure. Two "band
4.1 protein, ezrin, radixin, moesin"
(FERM)1 domains that have
been implicated in cytoskeletal protein interactions have been found in
the tail region. There are two myosin tail homology 4 domains
found in the tail region. Although similar domains are found in myosin
IV and XV, the function of this domain is unclear. The tail domain of
unconventional myosins has been implicated to serve as the anchoring
site for the cellular target proteins. For myosin VIIa, several
tail binding proteins have been identified. The type I regulatory
subunit of protein kinase A binds to the FERM domain at the C terminus
of myosin VIIA (19), although the role of protein kinase A in the
function and regulation of myosin VIIA is unknown. It was shown that
the FERM domain is involved in anchoring with adherens junction via a
cadherin-catenins complex (20). Todorov et al. reported (21)
that the myosin VIIA tail has an affinity for microtubule-associated
protein 2B, suggesting the interaction of myosin VIIA with the
microtubule-based motility system. Quite recently, El-Amraoui et
al. (22) reported that myosin VIIa binds to myosin VIIa- and
Rab27-interacting protein, associates with melanosomes via Rab27A, and
plays a role in melanosome trafficking. On the other hand, little is
known about the motor function of myosin VIIa at a molecular level.
Unlike the case for the well characterized conventional myosins, it
becomes evident that the motor function among various members of the
unconventional myosin subfamily varies uniquely from one to another,
which is thought to be critical for specific physiological roles in
diverse cellular motile processes. For example, recent studies have
revealed that myosin V is a processive motor that can move in large
steps approximating the 36-nm pseudorepeat of the actin filament
(23-26). These characteristics are quite important to understand the
physiological function of myosin V, since the processive nature of
myosin V with large step size is suitable for the motors involved in
cargo movement in cells.
Recently, myosin VI was also demonstrated to be a processive motor with
a large step size (27, 28). A unique feature of myosin VI is that it
moves backward on the actin filament (29). It was originally proposed
that the myosin VI unique large insertion between the neck and
converter domains is responsible for the reverse directionality of
myosin VI (29). This view was based upon the hypothesis that the
orientation of the motor domain of myosin VI against actin filament is
the same direction as other myosins but the attachment of the position
of the "lever arm" (a long There is no doubt that myosin VIIA plays a role in various cellular
motile processes. How myosin VIIA functions in cellular motile events
is unknown. This is at least partly due to a lack of biochemical
information on myosin VIIA. Furthermore, little is known about the
motor characteristic of myosin VIIA at the molecule level. The present
study was initiated to clarify the motor function of myosin VIIA at a
molecular level.
Materials--
Escherichia coli strains Y1090,
XL1-Blue, and DH10BAC were purchased from Clontech
(Palo Alto, CA), Stratagene (San Diego, CA), and Invitrogen,
respectively. Vector plasmid pBluescript II SK( Preparation of Anti-myosin VIIA Antibodies--
A peptide,
GAETRKRSPTLSS, corresponding to residues 600-612 of rat myosin
VIIA was synthesized with an N-terminal Cys residue and conjugated to
keyhole limpet hemocyanin (Genemed Synthesis, Inc., South San
Francisco, CA). Antibodies (MVII600) were prepared by injecting two
rabbits with keyhole limpet hemocyanin-coupled peptide. The myosin VIIA
antibody affinity column was prepared from the purified MVII600
antibody and formyl-cellulofine (Seikagaku Kogyo, Tokyo, Japan)
according to the manufacturer's protocol.
cDNA Cloning and Sequencing--
Total RNA of rat adult male
was prepared from kidney by using an RNeasy minikit (Qiagen).
Poly(A)+ RNA was isolated using an Oligotex mRNA
minikit (Qiagen). Random oligonucleotides were used to synthesize
cDNA from the poly(A)+ RNA. Sense
(5'-GAACTTCACTGTGAACAGCTTC-3') and antisense
(5'-GCATAGGCCTGCACGGTGATC-3') primers were synthesized according to the
nucleotide sequence of mouse myosin VIIa (5), and the myosin VIIA
cDNA of 1,193 bp, which corresponds to residues 443-846 of mouse
myosin VIIa, was amplified. The amplified cDNA was random labeled
with 32P by using the Megaprime labeling kit (Amersham
Biosciences) and used as a probe to screen rat kidney Production of M7IQ5 Construct--
Of the cloned rat myosin VIIA
cDNA, L1 clone (encoding residues 414-1,032) and L21 clone
(encoding residues 1-581) were used to construct M7IQ5 expression
vector. The ClaI site was introduced at nucleotide position
1,537 by site-directed mutagenesis without changing the amino acid. The
L1 clone in pBluescript II SK(
A sense primer, MVIIBam+, containing a BamHI site upstream
of the initiation codon and an antisense primer, MVII948 Preparation of Myosin VIIA Proteins--
To express M7IQ5, 200 ml of Sf9 cells (approximately 1 × 109) were
co-infected with two separate viruses expressing the M7IQ5 heavy chain
and calmodulin, respectively. The cells were cultured at 28 °C in
175-cm2 flasks and harvested after 72 h. Cells were
lysed with sonication in 100 ml of lysis buffer (20 mM
KPi, pH 8.0, 0.6 M KCl, 1 M
glutamic acid, 5 mM MgCl2, 5 mM
ATP, 5 mM 2-mercaptoethanol, 1 mM
phenylmethanesulfonyl fluoride, 0.01 mg/ml leupeptin, 0.002 mg/ml
pepstatin A, and 320 µg/ml calmodulin). After centrifugation at
100,000 × g for 30 min, the supernatant was incubated
with 50 mM glucose and 20 units/ml hexokinase at 4 °C
for 30 min to hydrolyze completely any residual ATP. After the addition
of F-actin (2.3 µM), the sample was centrifuged (120,000 × g for 20 min) to co-precipitate the
expressed myosin VIIA. The pellets were washed once with buffer A (20 mM KPi, pH 8.0, 0.6 M KCl, 0.1 mM EGTA, and 5 mM 2-mercaptoethanol) and
resuspended with buffer A containing 10 mM
MgCl2 and 10 mM ATP to release myosin VIIA from
F-actin. The supernatant was mixed with 0.2 ml of
Ni2+-NTA-agarose (Qiagen) in a 50-ml conical tube on a
rotating wheel for 30 min at 4 °C. The resin suspension was then
loaded on a column (1 × 10 cm) and washed with a 10-fold volume
of buffer containing 20 mM imidazole, pH 7.5, 0.6 M KCl, 0.1 mM EGTA, and 5 mM
2-mercaptoethanol. M7IQ5 was eluted with buffer containing 200 mM imidazole, pH 7.5, 0.6 M KCl, 0.1 mM EGTA, and 5 mM 2-mercaptoethanol. Fractions
containing M7IQ5 were pooled and dialyzed against 20 mM
imidazole, pH 7.5, 0.15 M KCl, and 5 mM
2-mercaptoethanol. The purified M7IQ5 was stored on ice and used within
2 days. Typically, 0.5 mg of isolated M7IQ5 was obtained.
The full-length myosin VIIA (M7full) was also expressed in Sf9
cells and purified as described above except for using immunoaffinity chromatography instead of Ni2+-NTA-agarose chromatography.
M7full was co-precipitated with F-actin and then dissociated from
F-actin in the presence of ATP as described above. Dissociated M7full
was applied to a MVII600-formyl cellulofine column (0.8 × 1.5 cm)
and was washed with 5 volumes of buffer B (20 mM imidazole,
pH 7.5, 0.6 M KCl, 1 mM EGTA, and 5 mM 2-mercaptoethanol). Bound full myosin VIIA was eluted by
buffer B containing 0.2 mg/ml antigen peptide. Fractions containing
M7full were pooled and dialyzed against 20 mM imidazole, pH
7.5, 0.15 M KCl, and 5 mM
2-mercaptoethanol.
Gel Electrophoresis and Immunoblot
Analysis--
SDS-polyacrylamide gel electrophoresis was carried out
on a 5-20% polyacrylamide gel using the discontinuous buffer system of Laemmli (42). Molecular mass markers used were smooth muscle myosin
heavy chain (200 kDa),
Native gel electrophoresis was performed as previously described (44).
The gel consisted of 4.75% acrylamide, 0.25% bisacrylamide, 40 mM sodium pyrophosphate, pH 8.8, 2 mM ATP, 2 mM MgCl2, 1 mM EGTA, 1 mM cysteine, and 10% glycerol. Electrophoresis was
performed for 16 h at 4 °C with a constant current of 20 mA.
For immunoblotting analysis, samples were electroblotted to
polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) after
separation with SDS-polyacrylamide gel or native gel electrophoresis.
Anti-myosin VIIA polyclonal, anti-calmodulin monoclonal (Upstate
Biotechnology, Inc., Lake Placid, NY), or anti-hexahistidine polyclonal
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies were used
as primary antibodies followed by horseradish peroxidase-conjugated
anti-rabbit or anti-mouse IgG secondary antibodies as described
previously (45). Signal detection was done with Supersignal West Femto
Maximum Sensitivity Substrate (Pierce).
Gel Filtration Chromatography--
M7IQ5 was dialyzed against a
solution containing 20 mM Tris-HCl, pH 7.5, 0.15 M KCl, 1 mM EGTA, 5 mM
MgCl2, 5 mM ATP, and 5 mM
2-mercaptoethanol and applied to a Sephacryl S-300HR column (1.0 × 46 cm). The protein was eluted with a dialyzed buffer at a flow rate
of 0.16 ml/min. Fractions were analyzed with immunoblotting with
anti-hexahistidine polyclonal antibody as described above. The
following molecular mass standards were also applied to the column:
tyroglobulin, immunogloblin, ovalbumin, myoglobin, and vitamin
B12.
ATPase Assay--
The actin-activated ATPase activity was
assayed in 20 mM imidazole, pH 7.0, 50 mM KCl,
2 mM MgCl2, 1 mM EGTA with or
without F-actin, at 25 °C. All assays were initiated by adding
[ Preparation of the Dual Labeled F-actin--
F-actin (30 µM) was first labeled with a 5 M excess of
tetramethylrhodamine-5-(or -6-) maleimide (Molecular Probes, Inc., Eugene, OR) in the presence of 8 µM phalloidin, 25 mM KCl, 5 mM MgCl2, 1 mM EGTA, and 25 mM imidazole, pH 7.5 (buffer C)
in the dark for 40 min at 4 °C. After the reaction was stopped by
adding 10 mM DTT, the fluorescently labeled F-actin was
obtained by centrifugation (250,000 × g for 10 min).
The pellet was homogenized with buffer C plus 5 mM DTT and
then precipitated again (250,000 × g for 10 min) to
remove the residual dye. The pellet was again homogenized with buffer C
with 5 mM DTT. The homogenate was diluted with the same
buffer (F-actin concentration should be ~0.8 µM) and
subjected to sonication to make minus end cap. The fragmentation of the fluorescently labeled F-actin was confirmed under fluorescence microscope. 0.1-1 µM G-actin was added to the fragmented
fluorescently labeled F-actin (2-15 µg/ml) in 25 mM KCl,
5 mM MgCl2, 1 mM EGTA, 25 mM imidazole, pH 7.5, and 10 units/ml fluorescein
phalloidin (Molecular Probes). The elongation of actin filament was
carried out overnight in the dark at 4 °C.
In Vitro Motility Assay--
The motile activities were measured
by in vitro motility assay. A coverslip was first coated
with nitrocellulose, and then M7IQ5 was then applied to the coverslip.
Actin filament motility was observed in 25 mM imidazole, pH
7.4, 25 mM KCl, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, 18 µg/ml catalase, 0.1 mg/ml glucose oxidase, 3.0 mg/ml glucose, 0.5% methylcellulose, and
various concentrations of Mg2+-ATP with or without an ATP
regeneration system (20 units/ml pyruvate kinase and 3 mM
phosphoenol pyruvate). Actin filament velocity was calculated from the
movement distance and the elapsed time in successive snapshots.
Student's t test was used for statistical comparison of
mean values. A value of p < 0.01 was considered to be significant.
Actin Co-sedimentation Assay--
The binding of calmodulin to
M7IQ5 heavy chain was determined by an actin co-sedimentation assay.
M7IQ5 was incubated in buffer containing 20 mM imidazole,
pH 7.5, 50 mM KCl, 20 µM F-actin, 1 mM EGTA, and various concentrations of ATP or ADP at
25 °C for 30 min. The sample was ultracentrifuged at 100,000 × g for 30 min, and the pellets were analyzed by
SDS-polyacrylamide gel electrophoresis. The amounts of the
co-sedimented M7IQ5 heavy chain and calmodulin were determined by
densitometry as described previously (43).
Cloning and Sequencing of cDNAs Encoding Rat Myosin
VIIA--
From ~5 × 107 recombinant phage plaques,
22 positive clones were obtained. Among them, four clones covered the
entire open reading frame of rat myosin VIIA as shown in Fig.
1. A nucleotide sequence of 6,807 bp with
an open reading frame of 6,531 bp at positions 272-6,805 was obtained.
No polyadenylation signal or poly(A)+ tail were found in
the 3'-untranslated region. From this nucleotide sequence, a
2,177-amino acid sequence was deduced with Mr = 251,100.
The deduced amino sequence was compared with other myosin VIIAs.
According to a computer search of the GenBankTM, EMBL,
NBRF-PIR, and SWISS-PROT databases, this sequence showed a high
homology (85-97%) with other vertebrate myosin VIIAs. It showed the
highest homology (97%) with mouse myosin VIIa (5). After residue
Arg1523, the 38 residues of the tail domain of mouse and
human (4) (GenBankTM accession number U39226) myosin VIIA
were missing in rat. The deletion of these residues was also found in
the human (12) (accession number U55208) and zebrafish isoforms (7). On the other hand, residues 2,079-2,080, Val-Lys, were found in rat, mouse, human (U39226), and zebrafish, but not human, isoforms (U55208).
Residues 94-114, 141-171, and 195-221, which correspond to
ATP-binding sites of rabbit skeletal myosin (GenBankTM
accession number U39226; residues 115-135, 162-192, and 229-255), represent high homology (57, 45, and 82%) with rabbit skeletal myosin.
The actin-binding site, residues 530-559 of rabbit skeletal myosin, is
also highly conserved in myosin VIIA (50%; residues 503-532). On the
other hand, lower homologies are shown in other actin-binding sites
(e.g. residues 404-417 and 568-579 of rabbit skeletal
myosin versus residues 372-285 and 537-548 of myosin VIIA
showed 14 and 17% homology, respectively). Five IQ motifs were
positioned at 745-857, suggesting the binding of five light chains.
The region of residues 862-933 was predicted by the program COILS to
form the coiled-coil structure (48). The tail region, residues
1,322-1,492 and 1,934-2,095, was found to contain two FERM domains.
At the upstream of each FERM domain, two myosin tail homology 4 domains
were positioned at 1,148-1,250 and 1,752-1,858, respectively. An Src
homology 3 domain was also found at residues 1,565-1,634.
Expression and Purification of Mammalian Myosin VIIA--
A rat
myosin VIIA construct was produced and expressed in Sf9 insect
cells. The construct (M7IQ5) contains the entire coiled-coil domain in
addition to the complete head and neck domains with a C-terminal
hexahistidine tag to aid purification (Fig.
2A). The histidine tagging at
the C-terminal end of the molecule has been performed with conventional
(49) as well as unconventional myosin (50), and no influence on motor
function has been observed. The cells were co-infected with an
appropriate ratio of myosin VIIA-expressing virus and
calmodulin-expressing virus, since it was reported that calmodulin
copurified with myosin VIIA heavy chain (21), suggesting that
calmodulin plays a role as the light chain subunits. It should be noted
that functional myosin VIIA was only obtained with co-infection of a
calmodulin-expressing virus, in contrast to myosin V, in which
functional protein can be obtained without calmodulin co-infection
(40). The purification of M7IQ5 involved basically two steps: F-actin
co-precipitation followed by ATP-induced dissociation from F-actin with
Ni2+-NTA-agarose affinity chromatography using the
hexahistidine tag (see "Experimental Procedures"). The former step
selects the functionally active molecules, and the second step
eliminates the endogenous Sf9 cell myosin and F-actin. Fig.
2B shows SDS-polyacrylamide gel electrophoresis of the
purified myosin VIIA. The purified M7IQ5 construct was composed of a
high molecular mass band and a low molecular mass band, free of the
200-kDa Sf9 conventional myosin and actin. The high molecular
mass band (110 kDa) was consistent with the calculated molecular mass
of M7IQ5 and was recognized by anti-rat myosin VIIA (MVII600)
antibodies (Fig. 2C) and anti-His6 antibodies
(Santa Cruz Biotechnology) (not shown), indicating that the high mass
band is the expressed myosin VIIA heavy chains. The small subunits
showed a mobility shift with a change in Ca2+ that is
characteristic of calmodulin, suggesting that the small subunits are
indeed calmodulin (not shown). The identification of the small subunit
was also confirmed using anti-calmodulin antibodies (Fig.
2C). The stoichiometries of calmodulin versus myosin VIIA heavy chain were determined by densitometry to be 4.9 versus 1.0, consistent with the five IQ motifs in the M7IQ5 construct. Fig. 2D shows the native gel electrophoresis of
M7IQ5. The mobility of M7IQ5 was similar to that of myosin II S1 but much larger than that of myosin II HMM. Based upon the calculated molecular mass of heavy chain plus five calmodulin light chains (194,717 Da), the results suggest that M7IQ5 is single-headed, although
it contains an entire short coiled-coil region of myosin VIIA. This was
also confirmed with gel filtration chromatography using Sephacryl
S-300HR. The estimated molecular mass of 206 kDa was obtained according
to the elution positions of the molecular mass standard proteins (Fig.
2F). On the other hand, the mobility of the full-length
myosin VIIA was much lower than M7IQ5 and similar to myosin II HMM
(Fig. 2E). To further confirm the two-headed structure of
the full-length myosin VIIA, we performed gel filtration chromatography. The elution position of M7full was similar to that of
myosin II HMM, suggesting the two-headed structure of M7full (Fig.
2F). These results indicate that the full-length myosin VIIA
is a dimer. The results obtained here suggest that the coiled-coil
domain is not sufficient to form the two-headed structure of myosin
VIIA and that its C-terminal domain contributes to form the stable
doubled-headed structure.
Actin-activated ATPase Activity--
Fig.
3 shows the myosin VIIA ATPase activity
as a function of ATP concentration in the presence of an ATP
regeneration system. In contrast to the conventional myosin, myosin
VIIA ATPase activity in the absence of actin required extremely high
ATP concentrations to saturate the activity. A
KATP of 154 µM was obtained (Table I). A high KATP
value (231 µM) was also obtained in the presence of actin
(Table I) (i.e. actin-activated ATPase). The results suggest
either extremely slow ATP binding or weak affinity of myosin VIIA for
ATP. While measuring the ATPase activity of M7IQ5, we found that the
rate of Pi liberation significantly decreased with time
(Fig. 4). This time-dependent
inhibition of the Pi liberation was abolished when the ATP
regeneration system was included in the reaction mixture (Fig. 4). In
contrast, the time-dependent inhibition of the ATPase
activity was not observed for skeletal HMM (Fig. 4, inset).
A similar observation has been reported for myosin V. Based upon the
equation, d[ADP]/dt = Vmax[ATP]/(KATP(1 + [ADP]/KADP) + [ATP]) and
KATP = 231 µM,
KADP was estimated from the initial phase of the
fitting curve to be 7.3 µM (Table I). To further confirm
this notion, the effect of ADP on the actin-activated ATPase activity
of M7IQ5 was examined (Fig. 5). ADP
markedly inhibited the actin-activated ATPase activity of M7IQ5 but not
skeletal HMM. Based upon the equation, V = Vmax[ATP]/KATP(1 + [ADP]/KADP) + [ATP],
KADP was calculated to be 9.1 µM
(Table I). The result was consistent with Fig. 4.
The actin activated-ATPase activity was measured as a function of actin
concentration (Fig. 6). The actin
concentration required for the saturation of the activation was
significantly higher than that of myosin V (50-52) but lower than that
of conventional myosin. A Kactin of 32.7 µM was obtained in 50 mM KCl (Fig. 6; Table
I). The addition of ADP (300 µM) markedly shifted the
actin dependence curve to the left, and a Kactin
of 17.5 µM was obtained (Fig. 6, inset; Table
I).
Binding of Myosin VIIA to Actin--
The dissociation of M7IQ5
from actin was examined as a function of ATP concentration. Myosin VIIA
was mixed with actin in the presence of Mg2+-ATP, and the
fraction of myosin VIIA dissociated from actin was determined by
sedimentation analysis (Fig.
7A). The dissociation of
myosin VIIA from actin required much higher ATP concentrations than
that required for conventional myosin II, and the complete dissociation
required 1 mM ATP. Assuming that ATP binding to myosin VIIA
dissociates myosin VIIA from actin, the dissociation constant (KATP) of 202 µM was obtained from
the ATP dependence of actomyosin VIIA dissociation (Table I). These
results suggest the weak ATP binding of myosin VIIA. On the other hand,
a significant fraction of myosin VIIA in the presence of 1 mM ATP becomes co-precipitated with actin in the presence
of ADP (Fig. 7B). In contrast, the addition of ADP did not
affect the binding of skeletal HMM to actin in the presence of ATP.
KADP values were estimated to be 6.9 µM based upon the equation, [bound heavy chain] = 1 The Motility Activity of Myosin VIIA--
The motility activity of
myosin VIIA was measured as a function of ATP concentration (Fig.
8). Consistent with the high ATP requirement of M7IQ5 for the actin-activated ATPase activity, a high
ATP concentration was also required for the actin-translocating activity of myosin VIIA. The velocity was saturated at 4 mM
ATP with a maximum velocity of 0.16 ± 0.02 µm. This value is
consistent with that obtained for the myosin VIIA prepared from tissue
(53). This extremely high ATP requirement for the actin-translocating activity of myosin VIIA was not observed for myosin II. On the other
hand, ADP significantly inhibited the motility activity of myosin VIIA
(Fig. 9A). The inhibition by
ADP was saturated at 2 mM ADP, and an approximate 50%
inhibition of the motility velocity was observed. The addition of
higher concentrations of ADP did not further inhibit the motility
activity. In contrast, ADP did not inhibit the actin-translocating
velocity of conventional myosin (skeletal muscle HMM) under the
conditions used.
Direction of the Movement--
Quite recently, it was shown that
the mammalian class VI myosin is a minus end-directed motor, in
contrast to other known myosins (25). It was originally hypothesized
that myosin VI's unique large insertion between the motor domain and
the neck domain was responsible for the reverse directionality of
motility (25). However, this view has been questioned recently (26),
opening up the possible presence of other minus end-directed motors
within the myosin superfamily.
To determine the direction of movement of the myosin VIIA,
we utilized F-actin filaments in the in vitro motility assay
that were labeled throughout with fluorescein and labeled with a
rhodamine cap at the filament's pointed end (see "Experimental
Procedures"). The dual fluorescence-labeled F-actin filaments were
visualized under the fluorescence microscope moving on coverslips
coated with M7IQ5. As shown in Fig.
10A, myosin VIIA moved the
dual fluorescence-labeled F-actin with the pointed end at the front of
the movement. This means that myosin VIIA moves toward the barbed end,
as is known for the conventional myosins. Fig. 10B shows a
histogram of the velocities of polarity-marked actin filaments on
myosin VIIA-coated cover slips. Whereas some variation of the sliding
velocity was observed, all actin filaments moved in the same direction.
The results show that myosin VIIA is a (+)-directed motor.
A number of unconventional myosins have been found during the last
decade, and it has been anticipated that these newly found myosins play
a key role in diverse cellular motile processes. Whereas their unique
C-terminal domain would be important for targeting each myosin to a
specific cellular component, it is anticipated that the uniqueness of
the motor properties of each myosin is also critical for its
physiological function, in particular motile processes in cells. For
myosin VIIA, where gene disruption is responsible for hereditary
deafness and blindness (5, 15, 16, 18), little is known regarding its
motor function at a molecular level. The present study has determined
the motor properties of myosin VIIA. In order to prepare mammalian
myosin VIIA, we decided to express recombinant myosin VIIA rather than
to purify it from tissues for several reasons. First, it is known that
various types of myosins are present in the same tissue and it would be difficult to completely eliminate the contamination of other myosins. This problem can be overcome by overexpressing myosin VIIA in Sf9 cells and purifying with a histidine tag. Also, large
quantities of the protein can be made and prepared in a short period of
time. This is critical for preventing the denaturation and degradation of the protein during preparation.
We produced a M7IQ5 construct that contains the entire head domain plus
coiled-coil domain. It has been thought that myosin VIIA is a
double-headed myosin because of the presence of the coiled-coil domain
(12). However, the length of the coiled-coil domain is relatively
short, and there is no conclusive evidence for the two-headed structure
of myosin VIIA. Our results clearly demonstrated that M7IQ5, having an
entire coiled-coil domain of myosin VIIA, is single-headed. However,
quite interestingly, the full-length myosin VIIA formed a two-headed
structure. The result suggests that the relatively short coiled-coil
domain of myosin VIIA is not sufficient to stabilize the two-headed
structure, and the tail domains of myosin VIIA in the two heavy chains
interact with each other, which contributes to form a stable
two-headed structure of myosin VIIA.
Myosin VIIA showed maximum actin-translocating velocity of 0.16 ± 0.02 µm/s at 25 °C. This value agrees with that of mouse myosin
VIIA (0.19 µm/s) as recently reported by Udovichenko et al. (53). However, to our surprise, myosin VIIA requires an extremely high ATP concentration for its motility activity.
Consistently, a high ATP concentration was needed for the saturation of
the actin-activated ATPase activity of myosin VIIA and the ATP-induced dissociation of myosin VIIA from actin (Table I). These results suggest
either an extremely slow ATP binding or a weak affinity of myosin VIIA
for ATP. A very slow rate of ATP binding has been reported for
myr1 (54). On the other hand, ADP significantly inhibited
the actin-activated ATPase activity of myosin VIIA. Consistently, the
actin-translocating velocity of myosin VIIA was also inhibited by ADP.
These results suggest the relatively strong binding of myosin VIIA to
ADP. Of particular interest is the observation that ADP markedly
reduced Kactin, thus increasing the apparent
affinity for actin during the ATPase cycle (Table I). Consistently, the
addition of ADP markedly increased the binding of myosin VIIA to actin
filaments in the presence of ATP. Even in the presence of 1 mM ATP, the addition of ADP enhanced the binding of myosin
VIIA, with nearly 100% of myosin VIIA binding to actin filaments.
These results suggest that a significant fraction of myosin VIIA during
the ATPase cycle is in a strong binding state in the presence of ADP,
presumably in a myosin VIIA/ADP form.
If this is true, then it is plausible that myosin VIIA
plays a role in connecting actin cytoskeleton and cellular components to maintain stress. Myosin VIIA is associated with cross-links between
adjacent stereocilia (55), suggesting a role in maintaining their
structural integrity. The present results are consistent with this
earlier finding and provide a molecular basis for this notion.
The weak affinity for actin in the presence of ATP suggests
the low duty ratio of myosin VIIA. However, the tight binding of ADP
usually suggests slow ADP dissociation. If so, the myosin would tend to
give high duty ratio. In the presence of ADP, myosin movement is
inhibited, but the inhibition required a relatively high ADP
concentration. A likely scenario would be that the ADP affinity is not
very high, but because ATP binding is weak, relatively low ADP
concentration decreases the ATPase activity. In the presence of
sufficiently high ADP concentration, myosin movement is inhibited by
the presence of myosin-ADP complex. If this is the case, myosin VII may function to maintain tension in cells where a significant fraction binds ADP. Tension maintenance in cells has also been proposed
for myr1 (54).
Directionality of myosin movement is another important issue to
understand the physiological relevance of each myosin motor. The
present results clearly indicate that myosin VIIA is a (+)-ended motor,
unlike myosin VI. Quite recently, it was assumed that
Dictyostelium myosin VII would be a plus end-directed motor,
because it may function in the assembly and disassembly of adhesion
proteins at the plasma membrane, and the actin at areas of membrane
extension is arranged with the plus ends outermost (55, 56). The
present results agree with this assumption. Whereas myosin VI and VIIA are present in the sensory hair cells in the inner ear and the mutation
of these two classes of myosins causes auditory dysfunction, the
present results strongly suggest that the function of these two types
of myosins in the sensory hair cells must be distinct from each other.
1
head
1. Myosin VIIA required an extremely high ATP
concentration for ATPase activity, ATP-induced dissociation from actin,
and in vitro actin-translocating activity. ADP markedly
inhibited the actin-activated ATPase activity. ADP also significantly
inhibited the ATP-induced dissociation of myosin VIIA from actin.
Consistently, ADP decreased Kactin of the
actin-activated ATPase. ADP decreased the actin gliding velocity,
although ADP did not stop the actin gliding even at high concentration.
These results suggest that myosin VIIA has slow ATP binding or low
affinity for ATP and relatively high affinity for ADP. The
directionality of myosin VIIA was determined by using the
polarity-marked dual fluorescence-labeled actin filaments. It was found
that myosin VIIA is a plus-directed motor.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical region following the motor
domain) (30) is different so that the same movement of the catalytic
core would rotate the lever arm in the opposite direction on actin.
Since the insertion is located between the converter domain (a compact subdomain thought to amplify the conformational change of the motor
domain) (31, 32) and light chain binding helix, it was hypothesized
that the 53-amino acid unique insertion of myosin VI is critical in
determining the reverse directionality of myosin VI. However, a recent
study revealed that the myosin VI unique insertion is not important to
the reverse in directionality (33). Instead the motor core domain is
responsible for the change in the directionality of myosin movement
(33). This finding has raised the possibility that other classes of
myosin may be capable of moving to the minus-end of actin filaments.
Actually, it was found quite recently that myosin IX is another myosin
that shows reverse directionality (34).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), pCR-TOPO2.1, and
pFastBac1 were purchased from Stratagene, Invitrogen, and Invitrogen,
respectively. For screening of cDNA for rat myosin VIIA, oligo(dT)
and random primed rat, Sprague-Dawley, kidney 5'-stretch
plus
gt11 cDNA library was purchased from Clontech. Restriction enzymes and modifying enzymes
were purchased from New England Biolabs (Beverly, MA). Rabbit fast
skeletal muscle actin was purified according to Spudich and Watt (36).
Recombinant calmodulin from Xenopus oocyte (37)
was expressed in E. coli as described (38). Myosin II was
prepared from rabbit skeletal muscle according to Perry (39). Skeletal
muscle HMM was prepared from skeletal myosin by
-chymotryptic
digestion (40), and produced HMM was purified by Sephacryl S-300HR
(Amersham Biosciences) gel filtration. Hexahistidine tag-fused smooth
muscle myosin II HMM or S1 was co-expressed with two light chains in
Sf9 cells and purified with nickel-nitrilotriacetic acid-agarose
(Qiagen, Hilden, Germany).
gt11 cDNA
library. Plaque hybridization was carried out at 65 °C in Church
buffer (41). The cDNA inserts encoding rat myosin VIIA were
obtained by the EcoRI digestion of the cloned phage DNA and
then subcloned into pBluescript II SK(
). The nucleotide sequence was
analyzed with the PerkinElmer terminator ready reaction mix using a
model 377 DNA sequencer (Applied Biosystems, Foster City, CA).
) was digested with ClaI and
SacI, and the excised fragment was ligated into the L21
clone in pBluescript II SK(
) digested with the same enzymes.
and
MYCHIS
, containing c-myc and hexahistidine, and
EcoRI sequences downstream of residue 948 were made and used
for PCR amplification of myosin VIIA cDNA (2,895 bp) using L1/L22
as a template. This fragment was subcloned into pCR-TOPO2.1. The myosin
VIIA cDNA fragment was obtained with
BamHI/EcoRI digestion and subcloned into
pFastBac1 baculovirus transfer vector (Invitrogen). The full myosin
VIIA expression vector containing 2,177 amino acid residues was also constructed using the cDNA clones of myosin VIIA (L21, L1, L7, and
L9 clones encoding residues 1-582, 414-1,040, 985-1,856, and 1,754-2,177, respectively). Hexahistidine tag was fused at the C
terminus of myosin VIIA. After the sequence was confirmed, bacmid DNA
was prepared by manufacturer's protocol to produce the recombinant virus expressing M7IQ5.
-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), myosin regulatory light chain (20 kDa), and
lactalbumin (14.2 kDa). Gels were stained with Coomassie Brilliant Blue R-250. To estimate bound calmodulin with M7IQ5, the gel
was stained with GelCode Blue Stain Reagent (Pierce, Rockford, IL) by
the manufacturer's protocol, and densitometric analysis was performed
using the NIH Image version 1.62 software as described (43).
-32P]ATP (Amersham Biosciences) to the reaction
mixture. The liberated 32P was measured as described
previously (46) to determine ATPase activity. The actin-activated
ATPase activity was also assayed in the presence of 20 units/ml
pyruvate kinase and 3 mM phosphoenol pyruvate for
determining ATP concentration dependence of the activity. The liberated
pyruvate was determined as described previously (47).
RESULTS
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DISCUSSION
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Fig. 1.
Schematic drawing of rat kidney myosin VIIA
cDNA. The stippled and open
flanking boxes represent the open reading frame
and untranslated regions, respectively. The locations of several
restriction sites are shown. Solid bars and
arrows show the positions of nucleotide sequences of each of
the cloned cDNAs and probes used for screening, respectively. The
nucleotide sequence is available from GenBankTM with
accession number AB091825.
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Fig. 2.
Expression and purification of rat myosin
VIIA. A, schematic diagram of expressed truncated rat
myosin VIIA (M7IQ5). c-myc tag and His6 tag are
-EQKLISEEDL and -HHHHHH, respectively. B, purification of
M7IQ5 from Sf9 cell extracts. total, total cell
lysate homogenized with buffer containing 20 mM
KPi, pH 8.0, 0.6 M KCl, 1 M
glutamic acid, 5 mM MgCl2, 5 mM
ATP, 5 mM 2-mercaptoethanol, 1 mM
phenylmethanesulfonyl fluoride, 0.01 mg/ml leupeptin, 0.002 mg/ml
pepstatin A, and 320 µg/ml calmodulin. sup.1 and
p.p.t.1, supernatant and precipitation of cell homogenate
after centrifugation, respectively. sup.1+actin, supernatant
added to F-actin. sup.2 and p.p.t.2, supernatant
and precipitation of sup.1+actin after centrifugation;
sup. 3 and p.p.t.3, supernatant and precipitation
of p.p.t.2 homogenized with buffer containing ATP after
centrifugation. elution, eluate from
Ni2+-NTA-agarose column. C, immunoblot of
lane elution in Fig. 2B with anti-rat myosin VIIA
and anti-calmodulin antibodies. D, native gel
electrophoresis of M7IQ5. The proteins were expressed and purified as
described under "Experimental Procedures." The purified proteins
were analyzed by native gel electrophoresis followed by Western blot
using anti-hexahistidine antibodies as probes. HMM, smooth
muscle myosin II HMM; S1, smooth muscle myosin II S1;
M7IQ5, truncated myosin VIIA. E, native gel
electrophoresis of M7full. M7full, full-length myosin VIIA.
F, estimation of the molecular mass by gel filtration.
Sephacryl S-300HR gel filtration chromatography was performed as
described under "Experimental Procedures." Inset,
immunoblotting of the fractions probed by the anti-hexahistidine
antibodies.
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Fig. 3.
ATP dependence of the actin-activated
Mg2+-ATPase activity of M7IQ5. The ATPase activity was
measured in 20 mM imidazole, pH 7.0, 50 mM KCl,
2 mM MgCl2, 1 mM EGTA, 100 µM actin, various Mg2+-ATP concentrations,
and ATP regeneration system (20 units/ml pyruvate kinase and 3 mM phosphoenol pyruvate). Inset, in the absence
of F-actin. Solid lines, calculated based upon
the equation V = Vmax[ATP]/(KATP + [ATP]). According to the analysis, Vmax and
KATP were obtained for 0.46 s 1
head
1 and 231 µM in the presence of actin
and 0.04 s
1 head
1 and 154 µM
in the absence of actin, respectively.
The kinetic parameters of M7IQ5
[ATP]/([ATP] + KATP + [ADP]KATP/KADP).
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Fig. 4.
Time course of the actin-activated ATPase
activity of M7IQ5 and skeletal HMM with and without the
ATP-regenerating system. ATPase activity was measured in the
presence (triangle) and absence (circle) of 20 units/ml pyruvate kinase and 3 mM phosphoenol
pyruvate. Inset, the actin-activated ATPase activity of
skeletal HMM in the presence (triangle) and absence
(circle) of the ATP regeneration system. Other assay
conditions are as described in Fig. 3, except 0.2 mM ATP
was used to assay the ATPase activity of M7IQ5.
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Fig. 5.
Inhibition of the actin-activated ATPase
activity of M7IQ5 by ADP. The ATPase activity was measured as
described in the legend to Fig. 4, except 1 mM ATP and
various concentrations of ADP were added in the absence of the ATP
regeneration system. ADP concentration dependence of the
actin-activated ATPase activity of M7IQ5 was plotted. Solid
line, calculated based upon the equation V = Vmax[ATP]/(KATP(1 + [ADP]/KADP) + [ATP]). According to the
analysis, KADP value was obtained for 9.1 µM. Inset, ADP concentration dependence of the
actin-activated ATPase activity of skeletal HMM.
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Fig. 6.
Actin concentration dependence of the
Mg2+-ATPase activity of M7IQ5. The actin-activated
ATPase activity of M7IQ5 was measured as a function of actin
concentration in 1 mM ATP in the presence of the ATP
regeneration system and in the presence of 1 mM ATP and 0.3 mM ADP without the ATP regeneration system
(inset). Solid lines, calculated based
upon the equation V = Vmax[actin]/(Kactin + [actin]). According to the analysis, Vmax and
Kactin were obtained for 0.53 s 1
head
1 and 32.7 µM in the absence of ADP. In
the presence of 0.3 mM ADP, Vmax and
Kactin were reduced to 0.081 s
1
head
1 and 17.5 µM, respectively.
[ATP]/([ATP] + KATP + [ADP]KATP/KADP) (Table
I). This result is consistent with those of Figs. 5 and 6, suggesting
that a significant fraction of M7IQ5 forms myosin plus ADP complex even
in the presence of ATP. These results imply that, in the presence of
ATP and ADP, a significant fraction of myosin VII may be present in a
myosin plus ADP form, a strong actin binding form.
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Fig. 7.
Effect of nucleotides on the dissociation of
M7IQ5 from actin. A, dissociation of acto-M7IQ5 by ATP.
Inset, dissociation of skeletal myosin II HMM from actin.
The co-sedimentation assay was performed as follows. M7IQ5 or skeletal
HMM (0.1 mg/ml) was incubated in buffer containing 20 mM
imidazole, pH 7.5, 50 mM KCl, 20 mM F-actin, 2 mM MgCl2, and various concentrations of
Mg2+-ATP in the presence of the ATP regeneration system (20 units/ml pyruvate kinase and 3 mM phosphoenol pyruvate) at
25 °C for 30 min. The samples were ultracentrifuged at 100,000 × g for 30 min, and the pellets were analyzed by
SDS-polyacrylamide gel electrophoresis. The amount of the co-sedimented
heavy chain was determined by densitometry as described previously
(43). Solid lines, calculated based upon the
equation [bound M7IQ5] = KATP/([ATP] + KATP). According to the analysis,
KATP value was obtained for 202 µM. B, effect of ADP on the ATP-induced
dissociation of acto-M7IQ5. The conditions are as described for
A, except 1 mM ATP and various concentrations of
ADP were used. Solid lines are the calculated
ones based upon the equation (bound M7IQ5) = 1 [ATP]/([ATP] + KATP + [ADP]KATP/KADP).
According to the analysis, KADP value was
obtained for 6.9 µM in the presence of EGTA.
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Fig. 8.
Actin-translocating activity of M7IQ5 as a
function of ATP concentration. The actin-translocating activity
was measured in 25 mM imidazole, pH 7.4, 25 mM
KCl, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, 18 µg/ml catalase, 0.1 mg/ml glucose oxidase, 3.0 mg/ml glucose, 0.5% methylcellulose, and various ATP concentrations at
25 °C. The experiments were done in the presence of an ATP
regeneration system (20 units/ml pyruvate kinase and 3 mM
phosphoenol pyruvate). The bars represent the S.D. with
14-26 actin filaments observed for the motility assay. A
and B, plots of M7IQ5 and rabbit skeletal HMM,
respectively.
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Fig. 9.
The inhibition of actin-translocating
activity of M7IQ5 by ADP. The actin-translocating activity was
measured as described in the legend to Fig. 8, except various
concentrations of ADP were added in the presence of 5 mM
ATP and the absence of the ATP regeneration system. The bars
represent the S.D. with 12-29 actin filaments observed for the
motility assay. A and B, M7IQ5 and rabbit
skeletal HMM, respectively.
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Fig. 10.
Direction of the movement of M7IQ5.
A, movement of the dual labeled F-actin on an M7IQ5-coated
coverslip. Times are indicated on the left. Data were
obtained with a conventional in vitro motility assay with
dual fluorescence labeled F-actin. The bright tip
on the actin filament represents the minus end of the filament. The
white arrows in A indicate the leading
part of the dual labeled actin filaments at the front. The pointed
end of actin filament leads the movement, indicating that M7IQ5
moves toward the barbed end of F-actin. B, histogram of the
velocities of actin filaments having polarity markers. Movement toward
the barbed end of F-actin is defined as positive value.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR 41653, HL 60381, and GM 55834. The preliminary form of this study has appeared in abstract form (35).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB091825.
To whom correspondence should be addressed: Dept. of Physiology,
University of Massachusetts Medical School, 55 Lake Ave. N., Worcester,
MA 01655-0127. Tel.: 508-856-1954; Fax: 508-856-4600.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210489200
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ABBREVIATIONS |
---|
The abbreviations used are: FERM, band 4.1 protein, ezrin, radixin, moesin; M7full, full-length myosin VIIA; DTT, dithiothreitol; NTA, nitrilotriacetic acid.
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