No. 10] Proc. Japan Acad., 70, Ser. B (1994) 175

35. Purification of Based Motor from Chara corallina

By Keiichi YAMAMOTO , *) Munehiro KIKUYAMA,**) Noriko SUTOH-YAMAMOTO,*) and Eiji KAMITSUBO***)

(Communicated by Noburo KAMIYA, M.J. A., Dec. 12, 1994)

Abstract: Actin based was purified from Chara corallina using high performance liquid chromatography. Sodium dodecyl sulfate gel electrophoresis showed that the molecular weight of the main band was about 230 kDa. Although the molecular weight was quite similar to that of the heavy chain of muscle (myosin II), the motor protein was soluble at low ionic strength and antibody raised against the main band did not recognize smooth muscle myosin. The antibody also did not recognize the actin based motor protein from lily pollen tube. The motor protein translocated fluorescently labeled actin filaments at about 25 µm1s in the in vitro motility assay. The MgATPase activity of the motor protein was enhanced by F-actin about 150 fold. Calcium ion concentration had little effect on both the motor activity and the actin activated MgATPase activity. Key words: Cytoplasmic streaming; Chara corallina, motor protein.

Introduction. As is well known that the cytoplasmic streaming in characean cells is quite fast (about 70 um/s at 20°C). This streaming is caused by a certain actin based motor protein which runs, with bound vesicle (Nagai and Hayama,1979) or membranous network (Kachar and Reese, 1988), on the track of actin bundles attached to the chloroplasts lying under the plasma membrane (Kamitsubo, 1966; Nagai and Rebhun, 1966; Palevitz et al., 1974; Williamson,1974; Nothnagel and Webb,1982; Kamiya,1986). Speed of the streaming is much faster than that of the sliding of the thin actin filaments past the thick myosin filaments in free shortening muscle. Difference in the speed is due mainly to the motor protein itself because polystyrene beads coated with rabbit muscle heavy meromyosin moved only at about 5 ,um/s on Nitella actin bundles while crude extract of characean cells can support fast movement of fluorescently labeled rabbit actin (Sheetz and Spudich, 1983; Rivolta et al., 1990). To understand the reason of this high speed, it is essential to purify the motor protein from characean cells and compare it with muscle myosin. Kato and Tonomura reported the existence of muscle myosin like protein in the Nitella cells (1977). However, the protein has not been characterized well after that and its participation to the cytoplasmic streaming was not demonstrated. We, therefore, undertook the purification of this motor protein using in vitro motility assay to follow the activity. The in vitro motility assay uses fluorescently labeled actin and observes its sliding on coverslip coated with motor protein under the fluorescence microscope (Kron and Spudich, 1986). The assay requires very small amount of sample (about 20 ul) and is suitable to follow the motor activity of characean cells because it is not easy to obtain large

*) Department of Bioengineering , Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192, Japan. **) Biological Laboratory , Faculty of Liberal Arts, The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba 260, Japan. ***) Biological Laboratory , Hitotsubashi University, Kunitachi, Tokyo 186, Japan. 176 K. YAMAMOTO et al. [Vol. 70(B), quantity of endoplasm from the cells. Characean cell has gigantic size but more than 90% of the cell was occupied by the central vacuole which contains strong proteolytic enzymes. Materials and methods. (a) Plant material and collection of endoplasm. Chara corallina was cultured in plastic buckets filled with well water and containing some soil on the bottom. The buckets were illuminated with diffused sun light under natural conditions. Internodal cells were isolated from neighboring cells and kept in artificial pond water containing 0.1 mM each of KCI, NaCI, and CaCl2 for one day before use. The cells were placed on Parafilm and exposed to the air until the turgor pressure was lost through transpiration. Then both ends of each cell were cut and the vacuolar sap was removed through internal perfusion (Tazawa,1964) with about 100 , t1of a solution containing 0.25 M sucrose, 30 mM 3-(N-morpholino)propane sulfonic acid (MOPS) buffer, pH 7.0, 3 mM MgCl2i 5 mM EGTA, 2 mM DTT, 3 mg/ml bovine serum albumin (BSA), 1 mM phenyl methyl sulfonyl fluoride (PMSF), 0.05 mg/ml leupeptin, and 0.01 mg/ml aprotinin. The endoplasm and chloroplasts which were left inside the cell were squeezed out together with the perfusion solution and collected in a test tube. In this process, care was taken not to contaminate the specimen with any organisms living on the outer surface of the cell. (b) and reagents. BSA, leupeptin, and pepstatin were purchased from Sigma Chemical Co. Aprotinin, MOPS, and PMSF were purchased from Wako Chemical Co., Dojin Chemical Co., and Nakarai Chemical Co., respectively. Actin was extracted from the acetone powder of rabbit skeletal muscle and purified according to Spudich and Watt (1971). Smooth muscle myosin was a generous gift of Dr. Shinsaku Maruta of Soka University. Prestained molecular weight marker protein mixture was purchased from BioRad Laboratories, Inc. (c) In vitro motility assay. F-actin was labeled with rhodamine-phalloidin as described by Yanagida et al. (1984). A flow cell was prepared as described by Kron et al. (1991) but coverslip was not coated with either nitrocellulose or silicone. Assay solution contained 25 mM imidazole buffer, pH 7.5, 4 mM MgC12, 3 mM ATP, 0.5 mM EGTA, and 5 mM DTT. An enzyme system to reduce oxygen in solution was also added to avoid photodynamic action of rhodamine-phalloidin on the motility (Harada et al., 1990). Movement of fluorescently labeled actin was observed under a microscope (Nikon Microphot-FX) equipped with epifluorescence optics. Images were taken by a microchan- nel plate-intensified CCD camera (CS2400-97, Hamamatsu Photonics), processed with Argus 10 (Hamamatsu Photonics), and recorded on videoptape with a video recorder (BR-5611, Victor). The average speed of actin filaments was measured as follows. A piece of plastic wrap was placed on the video screen and leading edges of moving actin filaments were marked at time zero. The displacement of these edges after several frames was measured and the speed calculated by dividing the displacement by the time (1/30 sec for 1 frame). (d) Antibodies. Main band of motor protein was cut out from polyacrylamide gel after electrophoresis and homogenized in Freund's complete adjuvant. The suspension was injected to a rabbit. Second injection of the antigen with Freund incomplete adjuvant was done 2 weeks after the first injection. The animal was bled 10 days after the second injection. Isolation of IgG from serum was done using ammonium sulfate fractionation and DEAE cellulose column chromatography. Anti-panmyosin monoclonal antibody which was raised against mouse 3T3 cell myosin was purchased from Amersham Co (RPN. 1169). (e) SDS polyacrylamide gel electrophoresis and immunoblotting. SDS polyacryla- mide gel electrophoresis was done according to Laemmli and Favre (1973) using slab gel of 1 mm in thickness. Protein bands in gels were visualized either with Coomassie Brilliant Blue or silver staining. Electroblotting of protein bands to nitrocellulose membrane was done using a semidry blotting apparatus at 1 mA/cm2 for 1.5 hr. Membrane was washed No. 10] Actin Based Motor Protein from Chara corallina 177

Fig. 1. SDS polyacrylamide gel electrophoresis of the motor protein from Chara corallina. Lanes A and B, smooth muscle myosin and the motor protein from Chara corallina, respectively. A gradient gel (4-20%) was used. The position and the molecular weight of prestained molecular weight marker proteins were indicated. The molecular weight marker kit contained phosphorylase b (130 K), bovine serum albumin (75 K), ovalbumin (50 K), carbonic anhydrase (39 K), soybean trypsin inhibitor (27 K), and lysozyme (17 K). Note that, due to the prestaining, these molecular weights are different from those of unstained proteins. Fig. 2. Results of immunoblotting. Lanes A and B, smooth muscle myosin and crude extract of Chara corallina, respectively, detected by anti-panmyosin. Lane C, the same amount of the crude extract of Chara corallina detected by the anti-motor protein. Prestained molecular weight marker proteins were also blotted, and their position and the molecular weight were indicated. A gradient gel (4-20%) was used. once with TBS-Tween (iris buffered saline, pH 7.5 plus 0.05% Tween-20) solution. Blocking was done in 1% gelatin in TBS-Tween solution for 1 hr. Anti-motor protein IgG was diluted with the blocking solution to 4 ,ug/ml. Anti-panmyosin monoclonal antibody was diluted 20 fold with the same solution. These antibodies were allowed to react for overnight. After thorough washing with TBS-Tween solution, bound anti-motor protein and anti-panmyosin were detected by goat anti-rabbit IgG and goat anti-mouse IgG conjugated with alkaline phosphatase (Promega Co.), respectively. Color development was done using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazorium dissolved in 0.1 M Tris-HCI, pH 9.5, 0.1 M NaCI, and 5 mM MgC12. Results. (a) Purification of the motor protein. Characean endoplasm and chloro- plasts were squeeze out together with the perfusion solution as mentioned above. To the solution, KCl and MgATP were added to make 0.3 M and 5 mM, respectively. The solution was centrifuged at 17000 x g for 5 min to remove chloroplasts. Resulting supernatant was passed through 0.45 um membrane filter and applied to a Superdex 200 column (Pharmacia) equilibrated with 0.1 M KCI, 20 mM imidazole buffer, pH 7.5, 3 mM MgCl2i1 mM ATP, 1 mM EGTA, and 1 mM DTT. Activity appeared near the void volume. The active fractions were pooled and applied to a DEAE cellulose column (DEAE- 178 K. YAMAMOTO et al. [Vol. 70(B),

TOYOPEARL 650, Tosoh) equilibrated with the same solution used for the Superdex 200 column. The column was washed with 2 column volume of the same solution. Proteins were eluted with a linear gradient of KCl from 0.1 M to 0.5 M. Activity was eluted at KC1 concentration of about 0.25 M. The active fractions were passed through a desalting column (BioRad 10 DG) equilibrated with 0.05 M potassium phosphate buffer, pH 7.0, 1 mM ATP, 3 mM MgC12, and 1 mM DTT. Protein fractions were then applied to a hydroxyapatite column (BioRad) equilibrated with the same solution used for the desalting column. Proteins were eluted with a linear gradient of potassium phosphate from 0.05 to 0.45 M. Activity appeared at potassium phosphate concentration of about 0.23 M. If the purity was not insufficient, the active fractions were concentrated with Ultrafree-PF (Millipore) and rechromatographed on a Superdex 200 column. Sodium dodecyl sulfate gel electrophoresis of the final product is shown in Fig. 1. The apparent molecular weight of the main band (lane B) was about 230 kDa because it was slightly higher than that of the smooth muscle myosin heavy chain (lane A). A band with apparent molecular weight of 190 kDa seems to be a degradation product of the main band because an antibody against the main band recognized this band. There is a faint band with apparent molecular weight of 27 kDa but we could not decide if this is a light chain component of the motor or just a contaminating protein. The antibody against the main band did not recognize this band. The main band and the 190 kDa band coprecipitated with F-actin in the absence of ATP and dissociated in the presence of ATP. Coprecipitation of the 27 kDa band was not clearly seen due to the paucity of the material. (b) Motor activity. The ability of active fractions to support the movement of fluorescently labeled rabbit skeletal muscle actin filaments changed during the purification process. The velocity of the actin movement was the highest when the active fraction of DEAE cellulose chromatography was used (about 50 , tm/s at 25°C) and it dropped to about 25 imJs when the active fractions of the following purification steps were used (Table I). After the purification, the velocity was still higher than that of rabbit skeletal muscle myosin (Harada et al. 1990).

Table I. Comparison of the sliding velocity

Table II. The MgATPase activity of the motor protein No. 10] Actin Based Motor Protein from Chara corallina 179

(c) The ATPase activity. The MgATPase activity of the final product is shown in Table II. The activity in the absence of actin was around 1 nmoles Pi/min mg. The activity was enhanced by rabbit skeletal muscle actin about 150-fold. Calcium ions did not suppress the actin activation. The activity in the presence of Ca2+ was rather higher than that in its absence. (d) Immunogenicity. The cross reactivity of the antibody raised against the motor protein was examined. The antibody did not recognize both skeletal and smooth muscle myosin and higher plant motor protein isolated from lily pollen tube (data not shown). Anti-panmyosin monoclonal antibody did not recognize the motor protein though the antibody recognized smooth muscle myosin under the same condition (Fig. 2). Discussion. Kato and Tonomura purified skeletal muscle myosin like protein from Nitella flexilis (1977). We first tried to obtain motor activity according to their method. However, the motor activity disappeared when characean cells were homogenized in their extraction solution. The activity seemed so labile (probably due to the proteolysis) that we adopted painstaking perfusion technique (Tazawa, 1964). We also used high performance liquid chromatography instead of ammonium sulfate fractionation and dialysis because it is fast and, therefore, seems to be able to reduce the risk of the proteolysis. It takes only 24 hr to obtain final product. If proteolytic enzymes mingled in the specimen were properly killed or removed, the activity is stable at least for 1 week. The protein we obtained has quite similar molecular weight to that reported by Kato and Tonomura (1977) but has different characteristics. First, our protein is soluble at low ionic strength. It does not form any aggregate at 30 mM KCl used by Kato and Tonomura to precipitate their protein. Second, the protein sticks strongly to the dialysis membrane. This is another reason why we do not use ammonium sulfate fractionation. Therefore, the protein we obtained seems to be different from that obtained by Kato and Tonomura. The Difference in the motor activity of active fractions obtained by various chromatographic column is quite puzzling (Table I). Contamination of some actin binding proteins will reduce the sliding velocity by acting as the dynamic resister. Removal of the actin binding proteins during the purification procedure should increase the sliding velocity but it was not the case. One simplest explanation is that we lost some factor(s) during the hydroxyapatite chromatography. Difference in the surface density of the motor protein on the coverslip may be another explanation. Concentration of the motor protein is probably the highest in the active fractions of the DEAE cellulose chromatography because active fractions of 4 to 5 runs of the gel filtration chromatography were applied to the DEAE cellulose column and proteins were eluted in one run. Theoretically, the velocity of the actin filament sliding is independent of the number of the motor protein molecules under an actin filament as long as at least one motor protein molecule interacts with it at any moment. However, the theory assumes that all the motor protein molecules stick to the glass surface with the same favorable configuration as to the force generation. The motor protein molecule can take several different configuration on the glass surface actually and some motor protein molecules in certain configuration may not be able to generate force but bind to actin filaments and thus reduce the sliding velocity. If the proportion of the motor protein molecules in such configuration depends on the concentration of the motor protein, the velocity of the actin sliding becomes a function of the motor protein concentration. Cytoplasmic streaming of characean cells stops instantaneously by various kinds of stimulation. Using the vacuolar perfusion technique, Tazawa and Kishimoto (1968) studied cytoplasmic streaming in Chara internodes and concluded that the sudden cessation of streaming upon stimulation was cuased by the temporary disappearance of its motive force. It is known that initial Ca2+ influx causes this cessation of streaming (Hayama et al., 180 K. YAMAMOTO et al. [Vol. 70(B),

1979; Williamsonand Ashley, 1982;Kikuyama and Tazawa,1982; Kikuyama et al.,1993). If the motor protein moving on actin bundle stops by Cat, both the motor activity and the actin activated MgATPase activity should change drastically depending on the concentra- tion of Cat+. However, such drastic change in these activities was not observed in our study. The actin activated MgATPase activity was even higher in the presence of Ca2+ than that in its absence (Table II). The motor activity of the purified protein was not affected also by Ca2+ up to 10-3 M. These results suggest that Ca2+ does not act on the motor protein directly. Calcium ion dependent kinase or phosphatase may phosphorylate or dephosphorylate the motor protein to exert the effect. It is interesting that the antibody against the motor protein from Chara corallina did not recognize higher plant motor protein from lily pollen tube nor smooth and skeletal muscle myosin. Grolig et al. reported that they detected 200 and 110 kDa bands in the crude extract of Chara using anti-panmyosin (1988) but our motor protein was not recognized by the antibody (Fig. 2). Recently, it was reported that the anti-panmyosin also did not recognize the motor protein from lily pollen tube (Yokota and Shimmen, 1994). In conclusion, this motor protein from Chara corallina is quite different from ordinary muscle myosin though it has similar molecular weight as that of muscle myosin. Acknowledgments. Authors thank Drs. Etsuo Yokota and Teruo Shimmen of Himeji Institute of Technology for their invaluable advice on the purification of the motor protein and their generous gift of lily pollen motor protein for our study on the cross-reactivity. We also thank Dr. Haruhiko Noda of Soka University for his encouragement and discussion.

References

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