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Functional expression of a chimeric myosin-containing motor domain of Chara myosin and neck and tail domains of Dictyostelium myosin II1
doi:10.1006/jmbi.2001.4883 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 311, 461±466
COMMUNICATION
Functional Expression of A Chimeric Myosin-containing Motor Domain of Chara Myosin and Neck and Tail Domains of Dictyostelium Myosin II Taku Kashiyama, Kohji Ito and Keiichi Yamamoto* Department of Biology, Chiba University, Inage-ku, Chiba 263-8522, Japan
We succeeded in expressing a chimeric myosin that comprises the motor domain of characean algal myosin, (the fastest known motor protein), and the neck and tail domains of Dictyostelium myosin II. Although the chimeric myosin showed an ATPase activity comparable to that of muscle myosin (15 times higher than that of the wild-type Dictyostelium myosin II), the motile activity of the chimera was only 1.3 times higher than that of the wild-type. However, this is the ®rst chimeric myosin that showed motile activity faster than at least one of the parent myosins. It was suggested, therefore, that the motor domain of Chara myosin has the potential for performing fast sliding movement. # 2001 Academic Press
*Corresponding author
Keywords: characean algae; chimeric myosin; cytoplasmic streaming; motor domain; plant myosin
Cytoplasmic streaming is quite important for plants because it enables the distribution of necessary nutrients and cellular components evenly throughout their large cells. The cytoplasmic streaming of characean alga is extremely fast (about 70 mm/second at 25 C).1 ± 3 It has been suggested that the sliding movement of unconventional myosin along the ®xed actin bundles in the cell generates this streaming.4 ± 6 The velocity of the streaming,therefore, should be comparable to the sliding velocity of the unconventional myosin along the actin bundles. Characean unconventional myosin was puri®ed from Chara corallina.7 The myosin can translocate ¯uorescently labeled actin ®laments at a velocity of about 50 mm/second in vitro. The velocity is about ten times higher than that of skeletal muscle myosin. That plant myosin moves much faster than animal myosin is very interesting, because the ability to move is one of the most important characteristics of animals, and animals must have evolved to move faster than Present address: T. Kashiyama, Department of Pharmacology, Juntendo University Medical School, Bunkyo-ku, Tokyo 113-8421, Japan. Abbreviations used: CCM, Chara corallina myosin; CMD, Chara motor domain; S-1, myosin subfragment 1. E-mail address of the corresponding author: [email protected] 0022-2836/01/030461±6 $35.00/0
other organisms. Recently, the cDNA sequence of characean myosin heavy chain was determined.8,9 The myosin has a motor domain that belongs to class XI, a neck domain comprising six tandem repeats of IQ motifs (calmodulin or light chain binding site), an a-helical coiled coil domain leading to dimer formation, and a globular tail domain. Overall, the structure predicted from the sequence agrees very well with that of biochemically puri®ed myosin observed by electron microscopy.10 Here, we report the functional expression of a chimeric myosin that comprises the motor domain of C. corallina myosin (CCM) and the neck and tail domains of Dictyostelium myosin II. We found that the motor domain of CCM has an ATPase activity comparable to that of skeletal muscle myosin and the potential to perform fast sliding movement. Expression in Dictyostelium The light chains of CCM are not characterized well, although calmodulin was copuri®ed with characean myosin.11 We, therefore, used Dictyostelium discoideum to express chimeric myosin heavy chain containing the motor domain of CCM, and the neck and tail domains of Dictyostelium myosin II. We thought that this chimeric heavy chain could use the endogenous light chains in Dictyostelium, so the uncertainty of characean myosin light # 2001 Academic Press
462 chains could be avoided. Chimeric Dictyostelium myosin in which the motor domain was replaced with that of Chara myosin (named Chara motor domain; CMD) was expressed in the Dictyostelium HS1 strain that lacks the myosin II heavy chain gene. Expression of functional myosin II usually allows the cells to grow in suspension. However, the cells expressing CMD could not grow in suspension culture, although the expression of CMD was detected by immunoblot analysis using antibody raised against the rod portion of Dictyostelium myosin II (data not shown) and the CMD showed an activity higher than that of wild-type Dictyostelium myosin II, as described below. One of the probable reasons is that the amount of functional (chimeric) myosin in the cells was not enough to form the contractile ring and the cells could not divide properly in the suspension culture. About 0.12 mg of CMD was obtained from 10 g of wet cells. This yield was about 0.05 that of wild-type Dictyostelium myosin II. A codon usage in Dictyostelium is quite unique (rich in A and T) and chimeric myosin containing the Chara myosin gene, which is not biased to A and T, may not be translated ef®ciently.
Expression of Characean Myosin Motor Domain
ATPase activity Puri®ed CMD had essential and regulatory light chains of Dictyostelium in the same ratio as in wildtype Dictyostelium myosin II (Figure 1). The basal Mg-ATPase activity of CMD was 0.13 Pi/second per head. The Mg-ATPase activity was enhanced 188-fold in the presence of 2 mg/ml of F-actin (Table 1). Both enzymatic and motile activities of wild-type Dictyostelium myosin II were enhanced by phosphorylation of the regulatory light chain.12,13 Therefore, the actin-activated MgATPase activity was measured in both phosphorylated and non-phosphorylated conditions (Table 1). Phosphorylation of the regulatory light chain enhanced the actin-activated Mg-ATPase activity of CMD only 1.16-fold, although that of wild-type Dictyostelium myosin II was enhanced 6.5-fold, indicating that information of the phosphorylation of the regulatory light chain was not transmitted to the motor domain of CMD ef®ciently. Kapp and Vmax were determined from double-reciprocal plots (Figure 2). The Vmax of CMD was 25.8 Pi/second per head. The value is close to that of skeletal muscle myosin14 and is about 15 times higher than that of the wild-type Dictyostelium myosin II. The
Figure 1. SDS-PAGE of puri®ed CMD (lane 2) and wild-type myosin II (lane 3). The upper half is 8 % (w/v) polyacrylamide gel and the lower half is 15 %. Molecular mass marker was loaded onto lane 1. HC, myosin heavy chain; RLC, regulatory light chain; ELC, essential light chain.
463
Expression of Characean Myosin Motor Domain Table 1. Effect of phosphorylation on the ATPase activity of wild-type myosin II and CMD (Pi/second per head) Actin-activated Mg-ATPasea Wild-type CMD a
Basal ATPase
Untreated
Phosphorylated
0.05 0.13
0.25 20.9
1.62 24.3
Actin activated Mg2 ATPase was measured in the presence of 2 mg/ml of F-actin.
Kapp of CMD was 0.20 mg/ml and was almost the same as that of the wild-type. Motile activity The CMD supported smooth, continuous movement of actin ®laments without noticeable fragmentation, indicating that the preparation was essentially free of denatured heads that would interfere with the movement of active heads. The sliding velocities of wild-type Dictyostelium myosin II and CMD was 2.5 and 3.2 mm/second at 25 C, respectively (Table 2). Native CCM has six IQ motifs at the neck domain,8 which acts as a lever arm during the power stroke, and translocates an actin ®lament in vitro at about 50 mm/second at 25 C.7 The neck domain of CMD comprises only two IQ motifs and its length is one-third that of the native CCM. Thus, the sliding velocity of CMD is expected to be about 17 mm/second, one-third that of the native CCM.15 However, the sliding velocity observed was only 3.2 mm/second. We designed a chimeric heavy chain by connecting the motor domain of CCM to the neck and tail domains of Dictyostelium myosin II, expecting that endogenous Dictyostelium light chains would be used. Actually, they associated successfully with the chimeric heavy chain and copuri®ed (Figure 1). It is conceivable, however, that the Dictyostelium light chains were not fully compatible with the Chara myosin motor domain, and did not allow it to realize its potential activity. The fact that the
phosphorylation of regulatory light chain did not enhance the ATPase activity of CMD (Table 1) supports this idea. It is suggested that essential light chain interacts with the neck domain and with the carboxyl end region of the motor domain, sometimes called the ``converter'' domain, during the power stroke.16 The converter domain plays an important role in the regulation of the ATPase activity by means of light chain phosphorylation.17 In the case of a chimera of skeletal muscle myosin in which the neck domain was replaced by that of smooth muscle myosin, the sliding velocity was decreased to 0.025 that of the parent skeletal muscle myosin, and was even lower (0.25) than that of the smooth muscle myosin although the actin-activated Mg-ATPase activity was close to that of skeletal muscle myosin and was regulated by phosphorylation like smooth muscle myosin.18 A chimeric subfragment 1 (S-1) containing a smooth muscle myosin motor domain and a skeletal muscle myosin neck domain showed a motility slower than that of smooth muscle S-1, although the motility became slightly faster when smooth muscle light chains were attached to the neck domain in place of skeletal muscle light chains.17 Since the sliding velocity is determined by the length of the transient period during which the motor domain of myosin binds actin strongly and changes its lever arm orientation, the velocity is much more sensitive to the interaction between the converter region and the light chains on the lever arm than is the ATPase activity. Considering the
Figure 2. Double reciprocal plot of the actin-activated Mg-ATPase activity of CMD. Vmax was 25.8 Pi/ second per head and Kapp was 0.20 mg/ml.
464
Expression of Characean Myosin Motor Domain
Table 2. Motile activity of wild-type myosin II and CMD Sliding velocity of actin filaments (mm/s)a Wild-type CMD a
2.5 0.11 3.2 0.18
Values are the average (SD) of ten measurements.
fact that CMD is the ®rst chimeric myosin that showed motility faster than at least one of its parent myosins, our results suggest that the motor domain of CCM has potential ability to perform fast sliding movement. A goal of our study is to elucidate the reason why the characean myosin can move at a velocity more than ten times faster than that of ordinary myosin II. It is clear that proper combination of the motor domain and the neck domain is required to achieve fast sliding movement of characean myosin. In the CMD that we produced, the mismatch with the neck domain seems to prevent the motor domain from realizing its potential activity. However, we do not know what the defective mismatch is, although improper interaction between the converter domain and the essential light chain is one possibility. Since the potential of the motor domain of the characean myosin is so high, this chimeric myosin might be a useful tool to investigate the proper combination with neck domain including light chains or to study the regulatory mechanism by means of light chain phosphorylation. Construction of a chimeric myosin expression vector Complementary DNA for the motor domain of Dictyostelium myosin II in an expression vector pTikl MyDap was replace with that of CCM. An actin 15 promoter was a generous gift from Dr Sutoh of Tokyo University in which a BamHI site was introduced at 3 bp from the initiation codon with one amino acid mutation, Gly3Pro. A BssHII site was introduced at 2239 bp from the initiation codon of Dictyostelium myosin II, with silent mutation by a mutual primed polymerase chain reaction (PCR). This region is located at the startpoint of long a helix, which serves as the light chain binding site, and is conserved between CCM and Dictyostelium myosin II over four amino acid residue lengths. Complementary DNA encoding CCM was used as a template for the PCR with two mutagenic oligonucleotide primers. The forward primer ¯anked the Glu4 of CCM and contained a BamHI site (50 -CGGGATCCAGAAAAGGCGAG GTCTTCGGCA-30 ) to join to the same site of the actin promoter. The reverse primer ¯anked the L727 of CCM and contained a BssHII site with the silent mutation. The PCR product was subcloned between the BamHI site of the actin 15 promoter
and the BssHII site of Dictyostelium myosin II. The resulting construct, referred to as CMD (Chara motor domain), codes for Met1-Asp2-Pro3- (Glu4Gln730 of CCM)-(Leu751-A2116 of Dictyostelium myosin II). Electroporation HS1 cells, myosin II null strain, were transfected with pTikl carrying the chimeric myosin gene by electroporation. Transformants were selected in HL5 medium in the presence of 18 mg/ml of G418 and were maintained in the same medium at 21-22 C. Purification of myosin Puri®cation of wild-type myosin and of CMD were performed using the method of Ruppel et al.}19 with slight modi®cation. Wild-type Ax2 cells were grown in 5 l ¯asks containing 2 l of HL-5 medium supplemented with 6 mg/ml each of penicillin and streptomycin on a rotary shaker at 21 C. HS1 cells transfected with the CMD gene did not grow well in suspension culture, and were cultured on 25 cm 25 cm square plastic plates containing 100 ml/plate of HL-5 medium supplemented with 6 mg/ml each of penicillin and streptomycin, and 18 mg/ml of G418 at 21 C. All procedures after that were carried out at 0-4 C. Cells at a density of 4 106-7 106 cells/ml were harvested by centrifugation at 1100 g for seven minutes. The pelleted cells were washed once with 10 mM Tris-HCl (pH 7.4), 0.04 % (w/v) NaN3, and resuspended in 4 vol./g cell of a lysis solution containing 25 mM Hepes (pH 7.4), 2.5 mM EDTA, 0.1 mM EGTA, 10 mM DTT, 50 mM NaCl, 0.04 % NaN3, and a mixture of protease inhibitors (100 mM p-toluenesulfonyl-L-lysine chloromethyl ketone, 200 mM phenylmethylsulfonyl ¯uoride (PMSF), 200 mM 1,10-phenanthroline, 20 mM leupeptine, 6 mM pepstatin, and 200 mM N-p-tosyl-L-arginine methylester). Then 4 vol./g cells of the lysis solution supplemented with 1 % (v/v) Triton X-100 were added and the resulting cell suspension was agitated gently. After 30 minutes on ice, the lysate was centrifuged at 61,000 g for one hour. The pellet was suspended in 8 vol./g cells of a washing solution (20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA, 5 mM DTT). The suspension was centrifuged again at 61,000 g for one hour. The pellet containing the actomyosin complex was suspended in 2 vol./g cells of an extraction solution (20 mM Hepes (pH 7.4), 125 mM NaCl, 3 mM MgCl2, 5 mM ATP, 5 mM DTT) and was centrifuged at 120,000 g for one hour. The supernatant was dialyzed against a solution containing 10 mM Pipes (pH 6.6), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT for more than eight hours in order to allow the myosin to form thick ®laments. The dialysate was centrifuged and the
Expression of Characean Myosin Motor Domain
resultant pellet was solubilized in 0.3 vol./g cells of 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 2 mM DTT, 4 mM ATP. This solution was centrifuged at 220,000 g for 30 minutes. The supernatant was diluted ®vefold with 10 mM Pipes (pH 6.8), 10 mM MgCl2, 2 mM DTT and left on ice for 40 minutes. The assembled myosin was collected by centrifugation at 110,000 g for 12 minutes and was dissolved by introducing 0.08 vol./g cells of 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 3 mM ATP, 2 mM DTT. The solution was clari®ed by centrifugation at 220,000 g for ten minutes to yield the myosin fraction. Phosphorylation of myosin Puri®ed wild-type and chimera myosins were incubated in 2 mM ATP, 3 mM MgCl2, 50 mM NaCl, 1 mM DTT, 10 mM Hepes (pH 7.4) for 30 minutes at 22 C in the presence of 0.1 mg/ml of bacterially expressed Dictyostelium myosin light chain kinase. The treated myosin was precipitated by centrifugation at 110,000 g for 20 minutes and dissolved in 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 3 mM ATP, 2 mM DTT. ATPase activity measurement The ATPase activity was measured at 30 C in 25 mM Hepes (pH 7.4), 25 mM KCl, 4 mM MgCl2, 1 mM DTT, 2 mM ATP together with 0 to 2 mg/ ml of rabbit skeletal muscle F-actin. The concentration of inorganic phosphate produced by ATP hydrolysis was determined by the method of Kodama et al.20 In vitro motility assay Motile activity of wild-type and chimera myosins was measured at 25 C using ¯uorescently labeled actin ®laments according to Kron & Spudich.21
Acknowledgements We thank Dr Uyeda of the National Institute for Advanced Interdisciplinary Research for helpful discussion, and Dr Sutoh of Tokyo University for the gift of actin 15 promoter. This work was supported by a Grantin-Aid for Scienti®c Research on Priority Areas from The Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Kamiya, N. (1962). Protoplasmic streaming. In Handbuch der P¯anzenphysiologie XVII/2 (Ruhland, F., ed.), p. 979-1035, Springer Verlag, Berlin. 2. Shimmen, T. & Yokota, E. (1994). Physiological and biochemical aspects of cytoplasmic streaming. Int. Rev. Cytol. 155, 97-139.
465 3. Yamamoto, K., Hamada, S. & Kashiyama, T. (1999). Myosin from plants. CMLS Cell. Mol. Life Sci. 56, 227-232. 4. Sheets, M. P. & Spudich, J. A. (1983). Movement of myosin-coated ¯uorescent beads on actin cables in vitro. Nature, 303, 31-35. 5. Shimmen, T. & Yano, M. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma, 121, 132-137. 6. Kacher, B. & Reese, T. S. (1988). The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin ®laments. J. Cell Biol. 106, 1545-1553. 7. Yamamoto, K., Kikuyama, M., Sutoh-Yamamoto, N. & Kamitsubo, E. (1994). Puri®cation of actin-based motor protein from Chara corallina. Proc. Jpn. Acad. 70, 175-180. 8. Kashiyama, T., Kimura, N., Mimura, T. & Yamamoto, K. (2000). Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world. J. Biochem. (Tokyo), 127, 10651070. 9. Morimatsu, M., Nakamura, A., Sumiyoshi, H., Sakaba, N., Taniguchi, H., Kohama, K. & HigashiFujime, S. (2000). The molecular structure of the fastest myosin from green alga, chara. Biochem. Biophys. Res. Commun. 270, 147-152. 10. Yamamoto, K., Kikuyama, M., Sutoh-Yamamoto, N., Kamitsubo, E. & Katayama, E. (1995). Myosin from alga chara: unique structure revealed by electron microscopy. J. Mol. Biol. 254, 109-112. 11. Awata, J., Saitoh, K., Shimada, K., Kashiyama, T. & Yamamoto, K. (2001). Effects of Ca2 and calmodulin on the motile activity of characean myosin in vitro. Plant Cell Physiol. In the press. 12. Grif®th, L. M., Downs, S. M. & Spudich, J. A. (1987). Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. J. Cell Biol. 104, 1309-1323. 13. Uyeda, T. Q. P. & Spudich, J. A. (1993). A functional recombinant myosin II lacking a regulatory light chain binding site. Science, 262, 1867-1870. 14. Yano-Toyoshima, Y., Kron, S. J., McNally, E. M., Niebling, K. R., Toyoshima, C. & Spudich, J. A. (1987). Myosin subffragment-1 is suf®cient to move actin ®laments in vitro. Nature, 328, 536-539. 15. Uyeda, T. Q. P., Abramson, P. D. & Spudich, J. A. (1996). The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl Acad. Sci. USA, 93, 4459-4464. 16. Dominguez, R., Freyzon, Y., Trybus, K. M. & Cohen, C. (1998). Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell, 94, 559-571. 17. Trybus, K. M., Naroditskaya, V. & Sweeney, H. L. (1998). The light chain-binding domain of the smooth muscle myosin heavy chain is not the only determinant of regulation. J. Biol. Chem. 273, 1842318428. 18. Sata, M., Stafford, W. F., III, Mabuchi, K. & Ikebe, M. (1997). The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively. Proc. Natl Acad. Sci. USA, 94, 91-96. 19. Ruppel, K. M., Uyeda, T. Q. P. & Spudich, J. A. (1994). Role of highly conserved lysine 130 of myo-
466
Expression of Characean Myosin Motor Domain
sin motor domain. In vivo and in vitro characterization of site speci®cally mutated myosin. J. Biol. Chem. 269, 18773-18780. 20. Kodama, T., Fukui, K. & Kometani, K. (1986). The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modi®ed mala-
chite green method for determination of inorganic phosphate. J. Biochem. (Tokyo), 99, 1465-1472. 21. Kron, S. J. & Spudich, J. A. (1986). Fluorescent actin ®laments move on myosin ®xed to a glass surface. Proc. Natl Acad. Sci. USA, 83, 62726276.
Edited by J. Karn (Received 2 April 2001; received in revised form 25 June 2001; accepted 27 June 2001)
J. Mol. Biol. (2001) 311, 461±466
COMMUNICATION
Functional Expression of A Chimeric Myosin-containing Motor Domain of Chara Myosin and Neck and Tail Domains of Dictyostelium Myosin II Taku Kashiyama, Kohji Ito and Keiichi Yamamoto* Department of Biology, Chiba University, Inage-ku, Chiba 263-8522, Japan
We succeeded in expressing a chimeric myosin that comprises the motor domain of characean algal myosin, (the fastest known motor protein), and the neck and tail domains of Dictyostelium myosin II. Although the chimeric myosin showed an ATPase activity comparable to that of muscle myosin (15 times higher than that of the wild-type Dictyostelium myosin II), the motile activity of the chimera was only 1.3 times higher than that of the wild-type. However, this is the ®rst chimeric myosin that showed motile activity faster than at least one of the parent myosins. It was suggested, therefore, that the motor domain of Chara myosin has the potential for performing fast sliding movement. # 2001 Academic Press
*Corresponding author
Keywords: characean algae; chimeric myosin; cytoplasmic streaming; motor domain; plant myosin
Cytoplasmic streaming is quite important for plants because it enables the distribution of necessary nutrients and cellular components evenly throughout their large cells. The cytoplasmic streaming of characean alga is extremely fast (about 70 mm/second at 25 C).1 ± 3 It has been suggested that the sliding movement of unconventional myosin along the ®xed actin bundles in the cell generates this streaming.4 ± 6 The velocity of the streaming,therefore, should be comparable to the sliding velocity of the unconventional myosin along the actin bundles. Characean unconventional myosin was puri®ed from Chara corallina.7 The myosin can translocate ¯uorescently labeled actin ®laments at a velocity of about 50 mm/second in vitro. The velocity is about ten times higher than that of skeletal muscle myosin. That plant myosin moves much faster than animal myosin is very interesting, because the ability to move is one of the most important characteristics of animals, and animals must have evolved to move faster than Present address: T. Kashiyama, Department of Pharmacology, Juntendo University Medical School, Bunkyo-ku, Tokyo 113-8421, Japan. Abbreviations used: CCM, Chara corallina myosin; CMD, Chara motor domain; S-1, myosin subfragment 1. E-mail address of the corresponding author: [email protected] 0022-2836/01/030461±6 $35.00/0
other organisms. Recently, the cDNA sequence of characean myosin heavy chain was determined.8,9 The myosin has a motor domain that belongs to class XI, a neck domain comprising six tandem repeats of IQ motifs (calmodulin or light chain binding site), an a-helical coiled coil domain leading to dimer formation, and a globular tail domain. Overall, the structure predicted from the sequence agrees very well with that of biochemically puri®ed myosin observed by electron microscopy.10 Here, we report the functional expression of a chimeric myosin that comprises the motor domain of C. corallina myosin (CCM) and the neck and tail domains of Dictyostelium myosin II. We found that the motor domain of CCM has an ATPase activity comparable to that of skeletal muscle myosin and the potential to perform fast sliding movement. Expression in Dictyostelium The light chains of CCM are not characterized well, although calmodulin was copuri®ed with characean myosin.11 We, therefore, used Dictyostelium discoideum to express chimeric myosin heavy chain containing the motor domain of CCM, and the neck and tail domains of Dictyostelium myosin II. We thought that this chimeric heavy chain could use the endogenous light chains in Dictyostelium, so the uncertainty of characean myosin light # 2001 Academic Press
462 chains could be avoided. Chimeric Dictyostelium myosin in which the motor domain was replaced with that of Chara myosin (named Chara motor domain; CMD) was expressed in the Dictyostelium HS1 strain that lacks the myosin II heavy chain gene. Expression of functional myosin II usually allows the cells to grow in suspension. However, the cells expressing CMD could not grow in suspension culture, although the expression of CMD was detected by immunoblot analysis using antibody raised against the rod portion of Dictyostelium myosin II (data not shown) and the CMD showed an activity higher than that of wild-type Dictyostelium myosin II, as described below. One of the probable reasons is that the amount of functional (chimeric) myosin in the cells was not enough to form the contractile ring and the cells could not divide properly in the suspension culture. About 0.12 mg of CMD was obtained from 10 g of wet cells. This yield was about 0.05 that of wild-type Dictyostelium myosin II. A codon usage in Dictyostelium is quite unique (rich in A and T) and chimeric myosin containing the Chara myosin gene, which is not biased to A and T, may not be translated ef®ciently.
Expression of Characean Myosin Motor Domain
ATPase activity Puri®ed CMD had essential and regulatory light chains of Dictyostelium in the same ratio as in wildtype Dictyostelium myosin II (Figure 1). The basal Mg-ATPase activity of CMD was 0.13 Pi/second per head. The Mg-ATPase activity was enhanced 188-fold in the presence of 2 mg/ml of F-actin (Table 1). Both enzymatic and motile activities of wild-type Dictyostelium myosin II were enhanced by phosphorylation of the regulatory light chain.12,13 Therefore, the actin-activated MgATPase activity was measured in both phosphorylated and non-phosphorylated conditions (Table 1). Phosphorylation of the regulatory light chain enhanced the actin-activated Mg-ATPase activity of CMD only 1.16-fold, although that of wild-type Dictyostelium myosin II was enhanced 6.5-fold, indicating that information of the phosphorylation of the regulatory light chain was not transmitted to the motor domain of CMD ef®ciently. Kapp and Vmax were determined from double-reciprocal plots (Figure 2). The Vmax of CMD was 25.8 Pi/second per head. The value is close to that of skeletal muscle myosin14 and is about 15 times higher than that of the wild-type Dictyostelium myosin II. The
Figure 1. SDS-PAGE of puri®ed CMD (lane 2) and wild-type myosin II (lane 3). The upper half is 8 % (w/v) polyacrylamide gel and the lower half is 15 %. Molecular mass marker was loaded onto lane 1. HC, myosin heavy chain; RLC, regulatory light chain; ELC, essential light chain.
463
Expression of Characean Myosin Motor Domain Table 1. Effect of phosphorylation on the ATPase activity of wild-type myosin II and CMD (Pi/second per head) Actin-activated Mg-ATPasea Wild-type CMD a
Basal ATPase
Untreated
Phosphorylated
0.05 0.13
0.25 20.9
1.62 24.3
Actin activated Mg2 ATPase was measured in the presence of 2 mg/ml of F-actin.
Kapp of CMD was 0.20 mg/ml and was almost the same as that of the wild-type. Motile activity The CMD supported smooth, continuous movement of actin ®laments without noticeable fragmentation, indicating that the preparation was essentially free of denatured heads that would interfere with the movement of active heads. The sliding velocities of wild-type Dictyostelium myosin II and CMD was 2.5 and 3.2 mm/second at 25 C, respectively (Table 2). Native CCM has six IQ motifs at the neck domain,8 which acts as a lever arm during the power stroke, and translocates an actin ®lament in vitro at about 50 mm/second at 25 C.7 The neck domain of CMD comprises only two IQ motifs and its length is one-third that of the native CCM. Thus, the sliding velocity of CMD is expected to be about 17 mm/second, one-third that of the native CCM.15 However, the sliding velocity observed was only 3.2 mm/second. We designed a chimeric heavy chain by connecting the motor domain of CCM to the neck and tail domains of Dictyostelium myosin II, expecting that endogenous Dictyostelium light chains would be used. Actually, they associated successfully with the chimeric heavy chain and copuri®ed (Figure 1). It is conceivable, however, that the Dictyostelium light chains were not fully compatible with the Chara myosin motor domain, and did not allow it to realize its potential activity. The fact that the
phosphorylation of regulatory light chain did not enhance the ATPase activity of CMD (Table 1) supports this idea. It is suggested that essential light chain interacts with the neck domain and with the carboxyl end region of the motor domain, sometimes called the ``converter'' domain, during the power stroke.16 The converter domain plays an important role in the regulation of the ATPase activity by means of light chain phosphorylation.17 In the case of a chimera of skeletal muscle myosin in which the neck domain was replaced by that of smooth muscle myosin, the sliding velocity was decreased to 0.025 that of the parent skeletal muscle myosin, and was even lower (0.25) than that of the smooth muscle myosin although the actin-activated Mg-ATPase activity was close to that of skeletal muscle myosin and was regulated by phosphorylation like smooth muscle myosin.18 A chimeric subfragment 1 (S-1) containing a smooth muscle myosin motor domain and a skeletal muscle myosin neck domain showed a motility slower than that of smooth muscle S-1, although the motility became slightly faster when smooth muscle light chains were attached to the neck domain in place of skeletal muscle light chains.17 Since the sliding velocity is determined by the length of the transient period during which the motor domain of myosin binds actin strongly and changes its lever arm orientation, the velocity is much more sensitive to the interaction between the converter region and the light chains on the lever arm than is the ATPase activity. Considering the
Figure 2. Double reciprocal plot of the actin-activated Mg-ATPase activity of CMD. Vmax was 25.8 Pi/ second per head and Kapp was 0.20 mg/ml.
464
Expression of Characean Myosin Motor Domain
Table 2. Motile activity of wild-type myosin II and CMD Sliding velocity of actin filaments (mm/s)a Wild-type CMD a
2.5 0.11 3.2 0.18
Values are the average (SD) of ten measurements.
fact that CMD is the ®rst chimeric myosin that showed motility faster than at least one of its parent myosins, our results suggest that the motor domain of CCM has potential ability to perform fast sliding movement. A goal of our study is to elucidate the reason why the characean myosin can move at a velocity more than ten times faster than that of ordinary myosin II. It is clear that proper combination of the motor domain and the neck domain is required to achieve fast sliding movement of characean myosin. In the CMD that we produced, the mismatch with the neck domain seems to prevent the motor domain from realizing its potential activity. However, we do not know what the defective mismatch is, although improper interaction between the converter domain and the essential light chain is one possibility. Since the potential of the motor domain of the characean myosin is so high, this chimeric myosin might be a useful tool to investigate the proper combination with neck domain including light chains or to study the regulatory mechanism by means of light chain phosphorylation. Construction of a chimeric myosin expression vector Complementary DNA for the motor domain of Dictyostelium myosin II in an expression vector pTikl MyDap was replace with that of CCM. An actin 15 promoter was a generous gift from Dr Sutoh of Tokyo University in which a BamHI site was introduced at 3 bp from the initiation codon with one amino acid mutation, Gly3Pro. A BssHII site was introduced at 2239 bp from the initiation codon of Dictyostelium myosin II, with silent mutation by a mutual primed polymerase chain reaction (PCR). This region is located at the startpoint of long a helix, which serves as the light chain binding site, and is conserved between CCM and Dictyostelium myosin II over four amino acid residue lengths. Complementary DNA encoding CCM was used as a template for the PCR with two mutagenic oligonucleotide primers. The forward primer ¯anked the Glu4 of CCM and contained a BamHI site (50 -CGGGATCCAGAAAAGGCGAG GTCTTCGGCA-30 ) to join to the same site of the actin promoter. The reverse primer ¯anked the L727 of CCM and contained a BssHII site with the silent mutation. The PCR product was subcloned between the BamHI site of the actin 15 promoter
and the BssHII site of Dictyostelium myosin II. The resulting construct, referred to as CMD (Chara motor domain), codes for Met1-Asp2-Pro3- (Glu4Gln730 of CCM)-(Leu751-A2116 of Dictyostelium myosin II). Electroporation HS1 cells, myosin II null strain, were transfected with pTikl carrying the chimeric myosin gene by electroporation. Transformants were selected in HL5 medium in the presence of 18 mg/ml of G418 and were maintained in the same medium at 21-22 C. Purification of myosin Puri®cation of wild-type myosin and of CMD were performed using the method of Ruppel et al.}19 with slight modi®cation. Wild-type Ax2 cells were grown in 5 l ¯asks containing 2 l of HL-5 medium supplemented with 6 mg/ml each of penicillin and streptomycin on a rotary shaker at 21 C. HS1 cells transfected with the CMD gene did not grow well in suspension culture, and were cultured on 25 cm 25 cm square plastic plates containing 100 ml/plate of HL-5 medium supplemented with 6 mg/ml each of penicillin and streptomycin, and 18 mg/ml of G418 at 21 C. All procedures after that were carried out at 0-4 C. Cells at a density of 4 106-7 106 cells/ml were harvested by centrifugation at 1100 g for seven minutes. The pelleted cells were washed once with 10 mM Tris-HCl (pH 7.4), 0.04 % (w/v) NaN3, and resuspended in 4 vol./g cell of a lysis solution containing 25 mM Hepes (pH 7.4), 2.5 mM EDTA, 0.1 mM EGTA, 10 mM DTT, 50 mM NaCl, 0.04 % NaN3, and a mixture of protease inhibitors (100 mM p-toluenesulfonyl-L-lysine chloromethyl ketone, 200 mM phenylmethylsulfonyl ¯uoride (PMSF), 200 mM 1,10-phenanthroline, 20 mM leupeptine, 6 mM pepstatin, and 200 mM N-p-tosyl-L-arginine methylester). Then 4 vol./g cells of the lysis solution supplemented with 1 % (v/v) Triton X-100 were added and the resulting cell suspension was agitated gently. After 30 minutes on ice, the lysate was centrifuged at 61,000 g for one hour. The pellet was suspended in 8 vol./g cells of a washing solution (20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA, 5 mM DTT). The suspension was centrifuged again at 61,000 g for one hour. The pellet containing the actomyosin complex was suspended in 2 vol./g cells of an extraction solution (20 mM Hepes (pH 7.4), 125 mM NaCl, 3 mM MgCl2, 5 mM ATP, 5 mM DTT) and was centrifuged at 120,000 g for one hour. The supernatant was dialyzed against a solution containing 10 mM Pipes (pH 6.6), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT for more than eight hours in order to allow the myosin to form thick ®laments. The dialysate was centrifuged and the
Expression of Characean Myosin Motor Domain
resultant pellet was solubilized in 0.3 vol./g cells of 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 2 mM DTT, 4 mM ATP. This solution was centrifuged at 220,000 g for 30 minutes. The supernatant was diluted ®vefold with 10 mM Pipes (pH 6.8), 10 mM MgCl2, 2 mM DTT and left on ice for 40 minutes. The assembled myosin was collected by centrifugation at 110,000 g for 12 minutes and was dissolved by introducing 0.08 vol./g cells of 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 3 mM ATP, 2 mM DTT. The solution was clari®ed by centrifugation at 220,000 g for ten minutes to yield the myosin fraction. Phosphorylation of myosin Puri®ed wild-type and chimera myosins were incubated in 2 mM ATP, 3 mM MgCl2, 50 mM NaCl, 1 mM DTT, 10 mM Hepes (pH 7.4) for 30 minutes at 22 C in the presence of 0.1 mg/ml of bacterially expressed Dictyostelium myosin light chain kinase. The treated myosin was precipitated by centrifugation at 110,000 g for 20 minutes and dissolved in 10 mM Hepes (pH 7.4), 250 mM NaCl, 3 mM MgCl2, 3 mM ATP, 2 mM DTT. ATPase activity measurement The ATPase activity was measured at 30 C in 25 mM Hepes (pH 7.4), 25 mM KCl, 4 mM MgCl2, 1 mM DTT, 2 mM ATP together with 0 to 2 mg/ ml of rabbit skeletal muscle F-actin. The concentration of inorganic phosphate produced by ATP hydrolysis was determined by the method of Kodama et al.20 In vitro motility assay Motile activity of wild-type and chimera myosins was measured at 25 C using ¯uorescently labeled actin ®laments according to Kron & Spudich.21
Acknowledgements We thank Dr Uyeda of the National Institute for Advanced Interdisciplinary Research for helpful discussion, and Dr Sutoh of Tokyo University for the gift of actin 15 promoter. This work was supported by a Grantin-Aid for Scienti®c Research on Priority Areas from The Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Kamiya, N. (1962). Protoplasmic streaming. In Handbuch der P¯anzenphysiologie XVII/2 (Ruhland, F., ed.), p. 979-1035, Springer Verlag, Berlin. 2. Shimmen, T. & Yokota, E. (1994). Physiological and biochemical aspects of cytoplasmic streaming. Int. Rev. Cytol. 155, 97-139.
465 3. Yamamoto, K., Hamada, S. & Kashiyama, T. (1999). Myosin from plants. CMLS Cell. Mol. Life Sci. 56, 227-232. 4. Sheets, M. P. & Spudich, J. A. (1983). Movement of myosin-coated ¯uorescent beads on actin cables in vitro. Nature, 303, 31-35. 5. Shimmen, T. & Yano, M. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma, 121, 132-137. 6. Kacher, B. & Reese, T. S. (1988). The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin ®laments. J. Cell Biol. 106, 1545-1553. 7. Yamamoto, K., Kikuyama, M., Sutoh-Yamamoto, N. & Kamitsubo, E. (1994). Puri®cation of actin-based motor protein from Chara corallina. Proc. Jpn. Acad. 70, 175-180. 8. Kashiyama, T., Kimura, N., Mimura, T. & Yamamoto, K. (2000). Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world. J. Biochem. (Tokyo), 127, 10651070. 9. Morimatsu, M., Nakamura, A., Sumiyoshi, H., Sakaba, N., Taniguchi, H., Kohama, K. & HigashiFujime, S. (2000). The molecular structure of the fastest myosin from green alga, chara. Biochem. Biophys. Res. Commun. 270, 147-152. 10. Yamamoto, K., Kikuyama, M., Sutoh-Yamamoto, N., Kamitsubo, E. & Katayama, E. (1995). Myosin from alga chara: unique structure revealed by electron microscopy. J. Mol. Biol. 254, 109-112. 11. Awata, J., Saitoh, K., Shimada, K., Kashiyama, T. & Yamamoto, K. (2001). Effects of Ca2 and calmodulin on the motile activity of characean myosin in vitro. Plant Cell Physiol. In the press. 12. Grif®th, L. M., Downs, S. M. & Spudich, J. A. (1987). Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. J. Cell Biol. 104, 1309-1323. 13. Uyeda, T. Q. P. & Spudich, J. A. (1993). A functional recombinant myosin II lacking a regulatory light chain binding site. Science, 262, 1867-1870. 14. Yano-Toyoshima, Y., Kron, S. J., McNally, E. M., Niebling, K. R., Toyoshima, C. & Spudich, J. A. (1987). Myosin subffragment-1 is suf®cient to move actin ®laments in vitro. Nature, 328, 536-539. 15. Uyeda, T. Q. P., Abramson, P. D. & Spudich, J. A. (1996). The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl Acad. Sci. USA, 93, 4459-4464. 16. Dominguez, R., Freyzon, Y., Trybus, K. M. & Cohen, C. (1998). Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell, 94, 559-571. 17. Trybus, K. M., Naroditskaya, V. & Sweeney, H. L. (1998). The light chain-binding domain of the smooth muscle myosin heavy chain is not the only determinant of regulation. J. Biol. Chem. 273, 1842318428. 18. Sata, M., Stafford, W. F., III, Mabuchi, K. & Ikebe, M. (1997). The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively. Proc. Natl Acad. Sci. USA, 94, 91-96. 19. Ruppel, K. M., Uyeda, T. Q. P. & Spudich, J. A. (1994). Role of highly conserved lysine 130 of myo-
466
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sin motor domain. In vivo and in vitro characterization of site speci®cally mutated myosin. J. Biol. Chem. 269, 18773-18780. 20. Kodama, T., Fukui, K. & Kometani, K. (1986). The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modi®ed mala-
chite green method for determination of inorganic phosphate. J. Biochem. (Tokyo), 99, 1465-1472. 21. Kron, S. J. & Spudich, J. A. (1986). Fluorescent actin ®laments move on myosin ®xed to a glass surface. Proc. Natl Acad. Sci. USA, 83, 62726276.
Edited by J. Karn (Received 2 April 2001; received in revised form 25 June 2001; accepted 27 June 2001)