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Inhibitory Regulation of Higher-Plant Myosin by Ca2+
Ions1 *Corresponding author; e-mail yokota/at/sci.himeji-tech.ac.jp; fax
81–7915–8–0175. Received July 20, 1998; Accepted October 3, 1998.  This article has been cited by other articles in PMC. | |||||||||||||||||
Abstract Myosin isolated from the pollen tubes
of lily (Lilium longiflorum) is composed of a 170-kD
heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177:
153–162). Both the motile activity in vitro and the F-actin-stimulated
ATPase activity of this myosin were inhibited by Ca2+ at
concentrations higher than 10−6 m. In the
Ca2+ range between 10−6 and 10−5
m, inhibition of the motile activity was reversible. In
contrast, inhibition by more than 10−5 m
Ca2+ was not reversible upon Ca2+
removal. An 18-kD polypeptide that showed the same mobility in
sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of
spinach calmodulin (CaM) was present in this myosin fraction. This
polypeptide showed a mobility shift in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis in a
Ca2+-dependent manner. Furthermore, this polypeptide was
recognized by antiserum against spinach CaM. By immunoprecipitation
using antiserum against the 170-kD heavy chain, the 18-kD polypeptide
was coprecipitated with the 170-kD heavy chain, provided that the
Ca2+ concentration was low, indicating that this 18-kD
polypeptide is bound to the 170-kD myosin heavy chain. However, the
18-kD polypeptide was dissociated from the 170-kD heavy chain at high
Ca2+ concentrations, which irreversibly inhibited the
motile activity of this myosin. From these results, it is
suggested that the 18-kD polypeptide, which is likely to be CaM, is
associated with the 170-kD heavy chain as a light chain. It is also
suggested that this polypeptide is involved in the regulation of this
myosin by Ca2+. This is the first biochemical basis, to our
knowledge, for Ca2+ regulation of cytoplasmic streaming in
higher plants. | |||||||||||||||||
In plant cells cytoplasmic streaming plays an essential role in
intracellular transport of organelles and molecules. The motive force
for cytoplasmic streaming is generated by the active sliding of myosin,
which is associated with organelles, along actin filaments (Williamson,
1976; Staiger and Schliwa, 1987; Shimmen and Yokota, 1994).
Physiological studies of Ca2+ regulation of
cytoplasmic streaming have been carried out extensively in the algae
family Characeae. In characean cells the cytoplasmic concentration of
Ca2+ increases to more than
10−6 m when an action
potential is generated at the plasma membrane (Williamson and Ashley,
1982). Concomitantly with this Ca2+ increase,
cytoplasmic streaming stops transiently (Hayama et al., 1979;
Williamson and Ashley, 1982). Microinjection of
Ca2+ into the cytoplasm of an internodal cell of
Nitella axillitormis causes a reversible inhibition of
cytoplasmic streaming (Kikuyama and Tazawa, 1982). The
membrane-permeabilized cell model prepared from characean cells
provides direct evidence that cytoplasmic streaming is reversibly
inhibited by an elevation of Ca2+ to
10−6 m (Tominaga et al.,
1983). For the molecular mechanism, involvement of myosin
phosphorylation has been suggested (Tominaga et al., 1987; McCurdy and
Harmon, 1992a, 1992b): Myosin is phosphorylated by a
Ca2+-dependent protein kinase, which is
inactivated by high Ca2+. The inhibitory effect of Ca2+ on cytoplasmic streaming has also been reported in some higher-plant cells such as lily (Lilium longiflorum) pollen tubes (Kohno and Shimmen, 1988b), stamen hair cells of Tradescantia (Doree and Picard, 1980), trichome cells of tomato (Woods et al., 1984), and leaf cells of Vallisneria gigantia (Takagi and Nagai, 1986). It has been suggested that pollen tube myosins have an inhibitory Ca2+ sensitivity. This is based on the facts that, along characean actin cables, movement of pollen tube organelles is inhibited by Ca2+ (Kohno and Shimmen, 1988a) and that characean actin cables are not equipped with a Ca2+-sensitizing mechanism (Shimmen and Yano, 1986). Biochemical studies of the molecular mechanism for Ca2+ regulation of the myosin activity responsible for cytoplasmic streaming are scarce in higher-plant cells because of difficulties in isolating myosin. Although Ca2+-sensitive myosin was isolated from tomato fruits, its F-actin-activated ATPase activity is stimulated by Ca2+ (Vahey et al., 1982). Thus, it is unlikely that this myosin is involved in cytoplasmic streaming. Recently, we isolated a higher-plant myosin composed of a 170-kD heavy chain from the pollen tubes of lily (Yokota and Shimmen, 1994). In the present study this myosin is referred to as the 170-kD myosin. This 170-kD myosin was able to translocate F-actin in vitro at a velocity similar to that of cytoplasmic streaming in living pollen tubes of lily, and its ATPase was stimulated by F-actin (Yokota and Shimmen, 1994). Taken together, these biochemical results and immunocytochemical studies using antibodies against the heavy chain of 170-kD myosin (Yokota et al., 1995a, 1995b) suggest that this myosin is generally distributed in higher plants and that it is involved in generating the motive force for cytoplasmic streaming. In the present study we have shown that the activities of 170-kD myosin are inhibited by Ca2+ and suggest that CaM, a light chain of this myosin, is involved in Ca2+ regulation. To our knowledge, this is the first description of the biochemical basis for the Ca2+ regulation of cytoplasmic streaming in higher plants. | |||||||||||||||||
Isolation of 170-kD Myosin and CaM from Lily Pollen and of
Actin from Skeletal Muscle The isolation of 170-kD myosin from germinated pollen of lily
(Lilium longiflorum) was carried out according to the method
described previously (Yokota and Shimmen, 1994). The 170-kD myosin was
isolated from a crude extract of pollen tubes by cosedimentation with
F-actin and purified successively by a hydroxylapatite column
(Pharmacia LKB) and a Sephacryl S-300 gel-filtration column
(Pharmacia LKB). Finally, the 170-kD myosin was suspended in a solution
containing 0.1 m KCl, 1 mm
EGTA, 4 mm MgCl2, 50
μg/mL leupeptin, 0.5 mm PMSF, 1
mm DTT, and 30 mm Pipes-KOH
(pH 7.0).CaM was extracted from the TCA precipitate of germinating lily pollen according to the method of Yazawa et al. (1980) and further purified by a fluphenazine-affinity column (Kakiuchi et al., 1981). Unless otherwise noted, each procedure was carried out at 0°C to 4°C. In each column step described below, the fractions, including CaM, were monitored by SDS-PAGE. The germinating pollen grains were suspended in 4% (w/v) TCA and then homogenized by 15 strokes with a motor-driven glass-Teflon homogenizer. After the sample was centrifuged at 9,000g for 10 min, the pellet was suspended in a solution containing 5 mm EGTA, 100 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 20 mm Tris-HCl (pH 7.5). The pH of the suspension was adjusted to 7.5 with NaOH. After incubation on ice for 30 min, the suspension was centrifuged at 25,000g for 30 min. The supernatant was applied to an ion-exchange column (model DE-52, Whatman) preequilibrated with ET solution (0.2 mm EGTA, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 20 mm Tris-HCl [pH 7.5]) supplemented with 0.14 m ammonium sulfate. After the column was washed with the preequilibrated solution, the adsorbed materials were eluted with the ET solution supplemented with 0.3 m ammonium sulfate. Fractions containing CaM were pooled and diluted 1.5-fold with ET solution. After the addition of CaCl2 (final concentration at 0.4 mm), the dilutant was applied to a column of Sepharose 6B (Pharmacia) conjugated with fluphenazine (Sigma) according to the method of Kakiuchi et al. (1981). The column was first washed with CT solution (0.2 mm CaCl2, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 20 mm Tris-HCl [pH 7.5]) and subsequently with the CT solution supplemented with 0.5 m NaCl. CaM was then eluted with a solution containing 0.5 m NaCl, 2 mm EGTA, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 20 mm Tris-HCl (pH 7.5). After dialysis against a solution containing 60 mm KCl, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 30 mm Pipes-KOH (pH 7.0), CaM was stored at −80°C until use. F-actin used for the cosedimentation procedure, the in vitro motility assay, and the ATPase activity of 170-kD myosin was prepared from chicken breast muscle according to the method of Kohama (1981). Motility Assay in Vitro KEMP solution (30 mm KCl, 5 mm EGTA, 6
mm MgCl2 and 30 mm
Pipes-KOH [pH 7.0]) was used for the motility assay in vitro.
CaCl2 was added to the KEMP solution to modify
[Ca2+], and free [Ca2+]
was calculated from the dissociation constants by using the computer
analysis program (Kohno and Shimmen, 1988a). The pH of the KEMP
solution containing various [Ca2+] was finally
adjusted to 7.0 with KOH.A coverslip was treated with 0.2% (w/v) collodion dissolved in isopentyl acetate and then air dried. Sixty microliters of 170-kD myosin (45–65 μg/mL) was applied to a piece of Parafilm (American National Can, Neenah, WI), and the collodion-coated coverslip was laid on the drop of myosin. After 5 min on ice, the surface of the coverslip was rinsed with a washing solution prepared by adding 1 mm ATP, 2 mm DTT, and various concentrations of CaCl2 to the KEMP solution. To make a flow chamber with a volume of approximately 10 μL, a small amount of petroleum jelly was applied to the two opposite edges of the coverslip. The coverslip was then placed on a glass slide. The flow chamber was perfused two times with 60 μL of the washing solution and subsequently with 60 μL of an assay medium (1 mm ATP, 0.3 μg/mL RP-labeled F-actin, 0.216 mg/mL glucose oxidase, 36 μg/mL catalase, 4.5 mg/mL Glc, 0.6% methyl cellulose, 100 mm DTT, and various concentrations of CaCl2 in the KEMP solution). RP-labeled F-actin was prepared by incubating F-actin with RP (Molecular Probes, Eugene, OR), according to the method of Kohno et al. (1991). Movement of actin filaments over the surface of the coverslip was observed under a fluorescence microscope (model BH2, Olympus) equipped with epifluorescence optics (model BH2-RFC, Olympus).
Measurement of Myosin ATPase Activity The ATPase activity of 170-kD myosin in the presence or absence of
F-actin at 25°C was measured with the assay medium used for the
motility assay: KEMP solution supplemented with 0. 5 mm ATP
and various concentrations of CaCl2. The final
protein concentrations were 60 μg/mL for F-actin and 3 μg/mL for
170-kD myosin. The amount of Pi liberated was determined according to
the method of Anner and Moosmayer (1975). The ATPase activity of
F-actin alone was also measured under the same conditions. To determine
the F-actin-stimulated ATPase activity of myosin, the value of Pi
liberation in the presence of F-actin alone was subtracted from that in
the presence of both 170-kD myosin and F-actin.Immunoprecipitation using Antiserum against the 170-kD
Heavy Chain Two microliters of rabbit antiserum raised against the 170-kD
heavy chain (Yokota and Shimmen, 1994) was added to 200 μL of the
myosin (50 μg/mL) fraction. As a control, preimmune serum was used
instead of antiserum. The mixture was kept on ice for 1 h. Ten
microliters of protein A-conjugated Sepharose beads (Pharmacia) was
then added to the mixture. After further incubation for 1 h on
ice, the sample was centrifuged at 500g for 3 min. The
resultant pellet was washed three times with KEMP solution supplemented
with 0.05% (v/v) Tween 20. The final pellet containing proteins bound
to Sepharose beads was resuspended in SDS-PAGE sample buffer. An
aliquot of the sample was subjected to SDS-PAGE on a 6% polyacrylamide
gel to detect the presence of the 170-kD heavy chain. Another aliquot
was subjected to immunoblot using antiserum against CaM.The effect of Ca2+ on the immunoprecipitate was examined by the following two experiments. First, the myosin fraction was mixed with antiserum containing 1.5 mm CaCl2, followed by the addition of Sepharose beads. Then, the beads added to the mixture were treated as described above. Second, the myosin fraction was mixed with the antiserum without 1.5 mm CaCl2 and then with Sepharose beads. The beads added to the mixture were then washed with KEMP solution supplemented with 0.05% Tween 20 and various concentrations of Ca2+. The proteins bound to the beads were analyzed by SDS-PAGE and immunoblotting, as described above. Immunoblotting After SDS-PAGE, proteins in the gel were electrophoretically
transferred to a PVDF-nitrocellulose membrane (Millipore) according to
the method of Towbin et al. (1979). The nitrocellulose membrane was
blocked using PBS containing 2% BSA and 2% lamb serum for 1 h
and then incubated with the primary antiserum against the 170-kD heavy
chain (Yokota and Shimmen, 1994) or CaM (Muto and Miyachi, 1984), which
was diluted 2000- or 4000-fold, respectively, with PBS supplemented
with 1% BSA and 0.05% Tween 20. The detection of antibodies on the
nitrocellulose membrane by anti-rabbit IgG conjugated with alkaline
phosphatase (Sigma) was carried out according to the method described
previously (Yokota et al., 1995a). A spinach CaM (Sigma) was used as a
positive control for the anti-CaM antiserum.Other Methods SDS-PAGE was performed according to the method of Laemmli (1970).
Protein bands were visualized by Coomassie brilliant blue staining.
Protein concentrations were determined by the method of Lowry et al.
(1951) using BSA as a standard. | |||||||||||||||||
Effect of Ca2+ on the Activities of 170-kD
Myosin In the presence of EGTA, RP-labeled F-actin was translocated
smoothly and continuously with an average velocity of 6.2 μm/s over
the glass surface coated with 170-kD myosin (Fig.
1). However, the motile activity was
greatly affected when the Ca2+ concentration was
increased to more than 10−6 m. Some
RP-labeled F-actin filaments that had been translocating stopped
temporarily for a few seconds and then began translocating again
(RP-labeled F-actin filaments numbered 1 and 3 in Fig.
2). Other RP-labeled F-actin showed
Brownian motion (RP-labeled F-actin numbered 2 and 4 in Fig. 2). In
some cases, RP-labeled F-actin detached suddenly from the glass surface
(data not shown). These results suggest that the interaction of
RP-labeled F-actin with 170-kD myosin is weak in the presence of
Ca2+. RP-labeled F-actin that moved more than
5 μm along its long axis was judged to be translocated by
myosin. The percentages of translocated RP-labeled F-actin among those
located at the surface of the coverslip are shown in Figure
3.
Figure 3A shows the motile activity of 170-kD myosin in vitro as a function of [Ca2+]. Both the percentage of translocated RP-labeled F-actin and the velocity of translocation were reduced by [Ca2+] higher than 10−6 m. The reversibility of the Ca2+ inhibition was examined (Fig. 3B). After the percentage of translocated RP-labeled F-actin was examined in the presence of Ca2+ at various concentrations, the flow cells were perfused with an assay medium without a supplement of Ca2+, and the percentage of translocated RP-labeled F-actin was again examined. Ca2+ at 2.5 μm (pCa [−log [Ca24]]-5.6) significantly decreased the percentage of translocated RP-labeled F-actin (Fig. 3B). The motile activity that had been inhibited by Ca2+ at 10−6 to 10−5 m was recovered by perfusion with an assay medium without CaCl2. However, only partial recovery was observed after inhibition by 10−4 m Ca2+. Next, we measured the ATPase activity of 170-kD myosin in the presence or absence of F-actin as a function of [Ca2+] (Fig. 4). The ATPase assay was carried out using the same buffer as that of the motility assay. The ATPase activity of 170-kD myosin alone showed similar values at all [Ca2+] examined (Fig. 4, •). It was stimulated by F-actin up to 20- to 30-fold at low Ca2+. However, it was only partially activated at [Ca2+] higher than 10−6 m, i.e. F-actin-stimulated myosin ATPase was inhibited at higher [Ca2+].
Identification of CaM in Pollen Tube Myosin Results in Figure 5 suggest that the
myosin fraction contains CaM. In the myosin fraction two prominent
bands, 34 kD (Fig. 5A, arrowhead 1) and 18 kD (Fig. 5A, arrowhead 2),
were detected in addition to the 170-kD heavy chain (Fig. 5A, arrow,
lane a). Since a large amount of casein, a 34-kD polypeptide, was
supplemented in the homogenizing buffer as an antiproteolysis agent
(Yokota and Shimmen, 1994), the 34-kD polypeptide is likely to be
casein remaining in the 170-kD myosin fraction. The 18-kD polypeptide
showed the same mobility in SDS-PAGE as that of spinach CaM (Fig. 5A,
lane b). An antiserum against spinach CaM recognized the 18-kD
component (Fig. 5B, lane A). This antiserum did not cross-react with
the 170-kD heavy chain (Fig. 5B, lane a), whereas an antiserum against
the 170-kD heavy chain recognized neither the 18-kD polypeptide nor
spinach CaM (Fig. 5C).
In SDS-PAGE this 18-kD polypeptide exhibited a Ca2+-dependent mobility shift, which is one of the characteristics of CaM (Burgess et al., 1980). The mobility of this peptide in 170-kD myosin pretreated with SDS-PAGE sample buffer supplemented with 2 mm CaCl2 was faster than that in myosin pretreated with SDS-PAGE sample buffer supplemented with 1 mm EGTA (Fig. 6A). Furthermore, the mobility of this 18-kD peptide in the presence of CaCl2 or EGTA was the same as that of spinach CaM in the presence of CaCl2 or EGTA, respectively (Fig. 6). These results led us to the conclusion that the 18-kD polypeptide included in the 170-kD myosin fraction is CaM.
Interaction of CaM with the 170-kD Heavy Chain To examine the association of CaM with the 170-kD heavy chain, an
immunoprecipitation assay with an antiserum against the 170-kD heavy
chain was carried out. As described above, this antiserum recognized
only the 170-kD heavy chain, not CaM (Fig. 5C). Both the 170-kD heavy
chain (Fig. 7A, lane a) and CaM (Fig. 7A,
lane c) were immunoprecipitated with this antiserum. However, they were
only faintly detected in the immunoprecipitate when the preimmune serum
was used in the place of antiserum as a control (data not shown). The
large band above the 45-kD molecular marker (Fig. 7A, lane c)
corresponds to the rabbit IgG heavy chain in the serum.
It is interesting that CaM was not detected in the immunoprecipitate when the myosin fraction was pretreated with 1.5 mm CaCl2 (Fig. 7A, lane d). The intensity of the 170-kD heavy-chain band did not change when pretreated with CaCl2 (compare lanes a and b in Fig. 7A), indicating that Ca2+ does not inhibit the interaction of the 170-kD heavy chain with the antiserum. Therefore, it is suggested that CaM dissociates from the 170-kD heavy chain in the presence of high concentrations of Ca2+. Because 1 mm EGTA was included in the myosin fraction, as described in “Materials and Methods,” [Ca2+] in the myosin fraction should increase to above 10−4 m by the addition of 1.5 mm CaCl2. Next, the threshold concentration of Ca2+ required for the dissociation of CaM from the 170-kD heavy chain was determined by the second procedure described in Methods. The 170-kD myosin fraction was mixed with the antiserum against the 170-kD heavy chain in the presence of 1 mm EGTA, followed by the addition of protein A-conjugated beads. The 170-kD myosin bound to protein A beads through antibodies was treated with KEMP solution supplemented with Ca2+ of various concentrations (10−7 to 10−4 m). The bound material was then subjected to immunoblotting. CaM was recovered in the immunoprecipitate when the Ca2+ concentration in the KEMP solution was lower than 10−5 m (Fig. 7B, lanes a–d). In contrast, CaM was not detected when beads were treated with 10−4 m Ca2+ (Fig. 7B, lane e). The intensity of the 170-kD heavy-chain band in the immunoprecipitate was similar at all [Ca2+] examined (data not shown). Effect of Exogenous CaM on the Motile Inhibition by
Ca2+ The results presented above suggest that the motile activity of
170-kD myosin is inhibited irreversibly by treatment with
10−4 m Ca2+
because of the dissociation of CaM from the 170-kD heavy chain.
Therefore, we examined the recovery effect of exogenously added CaM on
the Ca2+-inactivated 170-kD myosin. CaM was
isolated from germinating lily pollen (Fig.
8A). The motile activity of 170-kD myosin
remained suppressed after the following treatments: (a) A coverslip
coated with 170-kD myosin was rinsed in a solution containing
10−4 m Ca2+
and then used for a motility assay in an assay medium containing 2
μm CaM and low concentrations of
Ca2+ (10−6 and
10−7 m) or EGTA (data not shown).
(b) The coverslip coated with 170-kD myosin was rinsed in a solution
containing 10−4 m
Ca2+ and 2 μm CaM and then used for
a motility assay in an assay medium containing low concentrations of
Ca2+ or EGTA (Fig. 8B, white bars). In contrast,
when 2 μm CaM was added to both the rinsing solution and
the assay medium, the percentage of translocated RP-labeled F-actin was
recovered up to 45% to 60% (Fig. 8B, black bars). The sliding
velocity of RP-labeled F-actin was also restored to between one-half
and two-thirds of that induced by myosin that had not been rinsed with
10−4 m Ca2+
solution (data not shown).
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This is the first report, to our knowledge, demonstrating that Ca2+ inhibits both the motile activity in vitro (Fig. 3) and the F-actin-stimulated ATPase activity (Fig. 4) in higher-plant myosin (170-kD myosin). It has been reported that the organelles isolated from lily pollen tubes are translocated along actin filaments in characean cells and that this translocation is inhibited by Ca2+, indicating that myosin associated with organelles is equipped with a Ca2+-sensitive mechanism (Kohno and Shimmen, 1988a). The [Ca2+] required for this inhibition of organelle translocation along characean actin cables matches well with that required for the inhibition of 170-kD myosin (compare Fig. 3 in the present study with fig. 3 in Kohno and Shimmen [1988a]). These results, together with evidence that 170-kD myosin is associated with the membrane surface of organelles and that it is responsible for cytoplasmic streaming in lily pollen tubes (Yokota and Shimmen, 1994; Yokota et al., 1995a), suggest that Ca2+ sensitivity of 170-kD myosin may be a molecular basis for the Ca2+-sensitive translocation of organelles. In living lily pollen tubes, an intracellular Ca2+ gradient focused at the tip is present, and this is correlated with the tip growth (Nobiling and Reiss, 1987; Obermeyer and Weisenseel, 1991; Rathore et al., 1991; Miller et al., 1992). In the tip region, which is referred to as the clear zone, active cytoplasmic streaming is not observed (Pierson et al., 1990, 1994; Lancelle and Hepler, 1992). Recently, Pierson et al. (1994, 1996) demonstrated that at the tip the [Ca2+] is more than 3 μm (they considered 3–10 μm as a realistic range) and that it gradually decreases to a basal level of 0.2 μm within 20 μm from the pollen tube tip. When the [Ca2+] at the tip is reduced by microinjection of Ca2+ buffer 1,2-bis-(o-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid or by a treatment with caffeine (Miller et al., 1992; Pierson et al., 1994, 1996), the elongation of lily pollen tubes is blocked and cytoplasmic streaming begins to be observed close to the tip. The Ca2+inhibition of 170-kD myosin in vitro began at about 10−6 m and reached its plateau level at 2.5 μm (Fig. 3). This concentration range corresponds to that at the tip of elongating pollen tubes. In this [Ca2+] range, the inhibition of 170-kD myosin was reversible (Fig. 3B). The 170-kD myosin is associated with organelles but it also exists at the tip, where active streaming is not observed (Yokota et al., 1995a). It is suggested that the reversible Ca2+ inhibition of 170-kD myosin is responsible for the absence of active streaming at the tip region. It is well established that all myosins in nonplant cells whose primary structures have been determined contain at least one repeat of the IQ motif, which provides a binding site for CaM or a related protein of the EF-hand superfamily (Cheney and Mooseker, 1992; Mooseker and Cheney, 1995; Sellers et al., 1996). CaM has been shown to play a regulatory role in the activities of some of these myosins (Mooseker and Cheney, 1995; Wolenski, 1995). In plant myosin, heavy chains identified thus far by sequence analyses of their genes have several IQ motifs (Knight and Kendrick-Jones, 1993; Kinkema and Schiefelbein, 1994; Kinkema et al., 1994). However, no biochemical studies have been carried out to confirm whether CaM or a related protein is associated with the heavy chain as a light chain, since only a small number of plant myosins have been purified and characterized. In the present study we have shown the presence of an 18-kD polypeptide in the 170-kD myosin fraction (Fig. 5). Its molecular mass and mobility shift in SDS-PAGE, which are both dependent on Ca2+, were similar to those of spinach CaM (Figs. 5 and 6). This polypeptide was recognized by an antiserum against spinach CaM (Fig. 5). Immunoprecipitation using an antiserum against the 170-kD heavy chain showed the association of the 18-kD polypeptide with the 170-kD heavy chain (Fig. 7). These results indicate unequivocally that the 18-kD polypeptide is CaM and that it is associated with the 170-kD myosin heavy chain as a light chain. In characean cells it is hypothesized that the cessation of cytoplasmic streaming by Ca2+ is coupled to phosphorylation of myosin by a Ca2+-dependent protein kinase (Tominaga et al., 1987). The activity of this protein kinase from soybean is enhanced by a several-micromolar concentration of Ca2+ (Harmon et al., 1987; Putnam-Evans et al., 1990). In the case of Chara corallina, isolated myosin did not show Ca2+ sensitivity for its motile and F-actin-stimulated ATPase activities (Yamamoto et al., 1994). This is one of the characteristics of myosin regulated by its phosphorylation (indirect myosin-linked Ca2+ regulation). In contrast, isolated pollen tube myosin (170-kD myosin) showed significant Ca2+sensitivity (Figs. 3 and 4), suggesting that Ca2+ regulates myosin activity by binding to CaM, a light chain (direct myosin-linked Ca2+ regulation). It is likely that Ca2+ between 10−6 and 10−5 m causes reversible inhibition by an allosteric interaction of the 170-kD heavy chain and the CaM light chain. However, Ca2+ at concentrations higher than 10−5 m irreversibly inhibited the motility that was not reversible by Ca2+ removal (Fig. 3B). Concomitantly, CaM was dissociated from the 170-kD heavy chain (Fig. 7B). In myosin I from the brush border of the intestine (Collins et al., 1990; Wolenski et al., 1993) or liver (Williams and Coluccio, 1994) or in myosin V from the brain (Cheney et al., 1993), Ca2+ modulates the motile activity of these myosins in vitro by binding to their CaM light chain. Ca2+ inhibits the motile activity of such myosins by partial dissociation of CaM from the heavy chain. This impaired activity is restored by exogenously supplied CaM. Also, in the case of 170-kD myosin under low concentrations of Ca2+, the inactivated motility of 170-kD myosin was restored to some extent by exogenously supplied CaM that was isolated from lily pollen (Fig. 8B). However, it seems improbable that myosin encounters [Ca2+] higher than 10−5 m in living pollen tubes. Vahey et al. (1982) reported that the F-actin-stimulated ATPase activity of myosin isolated from tomato fruits is activated by increased Ca2+ concentrations. However, involvement of this myosin in cytoplasmic streaming seems unlikely, since several studies have shown that cytoplasmic streaming in somatic cells of higher plants is inhibited by Ca2+ (Shimmen and Yokota, 1994). We expect that our present results will provide a way to elucidate the molecular mechanism of the Ca2+ regulation of cytoplasmic streaming in higher plants. | |||||||||||||||||
ACKNOWLEDGMENT We thank the National Livestock Breeding Center, Hyogo Station (Tatsuno, Hyogo, Japan), for the gift of chicken breast muscle. | |||||||||||||||||
Abbreviations:
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Footnotes 1This work was supported by Grants-in-Aid for
Scientific Research (E.Y.) from the Ministry of Education, Science and
Culture of Japan (nos. 07740626 and 08740625). | |||||||||||||||||
LITERATURE  CITED
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![[filled square]](corehtml/pmc/pmcents/x25AA.gif)
). B, Reversibility of the Ca2+
inhibition
