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Plant Cell Physiol. 43(6): 595–603 (2002) JSPP © 2002 TMBP200, a Microtubule Bundling Polypeptide Isolated from Telophase Tobacco BY-2 Cells is a MOR1 Homologue Hiroki Yasuhara 1, 4, Masaaki Muraoka 1, Hiroki Shogaki 1, Hitoshi Mori 2 and Seiji Sonobe 3 1 Department of Biotechnology, Faculty of Engineering, Kansai University, Yamate-cho, Suita, Osaka, 564-8680 Japan Department of Agriculture, Graduate School of Bioagricultural Science, Nagoya University, Nagoya, 464-8601 Japan 3 Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo, 678-1297 Japan 2 ; et al. 1995). As the formation of the cell plate progresses, microtubules in the central region of the phragmoplast depolymerize and the phragmoplast assumes a ring-like structure, expanding outwards with the centrifugal growth of the cell plate. The phragmoplast expands by polymerization of microtubules at its outer margins and tubulin dimers required for microtubule polymerization are supplied by depolymerized microtubules at its inner margins (Gunning 1982, Yasuhara et al. 1993, Nishihama and Machida 2001). Despite the critical importance of the well-ordered phragmoplast microtubule array during cell plate formation, mechanisms underlying its construction are not yet fully understood. The site of polymerization of phragmoplast microtubules is thought to be the equatorial region of the phragmoplast, since it has been demonstrated that exogenously applied tubulin is incorporated into phragmoplast microtubules at their plus ends, which are present at the equatorial regions, in membrane permeabilized Haemanthus endosperm cells (Vantard et al. 1990) and in glycerinated tobacco BY-2 cells (Asada et al. 1991). In glycerinated tobacco BY-2 cells, microtubules polymerized at the equatorial region were translocated away from the equatorial region in an ATP-dependent manner and the motor molecule responsible for such microtubule translocation has been identified as TKRP125 (Asada and Shibaoka 1994, Asada et al. 1997). To ensure the construction of the parallel array of phragmoplast microtubules, microtubules polymerized at, and translocated from, the equatorial region of the phragmoplast must be associated with each other. Cross-bridges between microtubules have been observed in phragmoplasts of Haemanthus endosperm cells (Hepler and Jackson 1968) and in phragmoplasts isolated from tobacco BY-2 cells (Kakimoto and Shibaoka 1988) but those components cross-linking phragmoplast microtubules have not been identified. The center to center distance of cross-bridged microtubules is 40 nm in Haemanthus endosperm cells and measures approximately 42 nm in isolated phragmoplasts. Cyr and Palevitz (1989) made the first report of reconstitution of microtubule cross-bridges by plant microtubule-associated proteins (MAPs) in vitro. They showed that a mixture of proteins between 39 and 114 kDa from carrot cells formed cross-bridges between neuronal microtubules of which center to center distances were 34 nm. Two types of cross-bridges have been observed between micro- Bundles of microtubules and cross-bridges between microtubules in the bundles have been observed in phragmoplasts, but proteins responsible for forming the crossbridges have not been identified. We isolated TMBP200, a novel microtubule bundling polypeptide with an estimated relative molecular mass of about 200,000 from telophase tobacco BY-2 cells. Ultrastructural observation of microtubules bundled by purified TMBP200 in vitro revealed that TMBP200 forms cross-bridges between microtubules. The structure of the bundles and lengths of the cross-bridges were quite similar to those observed in phragmoplasts, suggesting that TMBP200 participates in the formation of microtubule bundles in phragmoplasts. The cDNA encoding TMBP200 was cloned and the deduced amino acid sequence showed homology to a class of microtubule-associated proteins including Xenopus XMAP215, human TOGp and Arabidopsis MOR1. Keywords: Cross-bridge (microtubules) — Microtubuleassociated proteins (MAPs) — Phragmoplast — Cytokinesis — XMAP215 — Tobacco BY-2 cells. Abbreviations: DTT, dithiothreitol; EGTA, ethyleneglycol bis (2aminoethylether) tetraacetic acid; PIPES, piperazine-N,N¢-bis (2ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; RACE, rapid amplification of cDNA ends. The nucleotide sequence reported in this paper has been submitted to DDBJ under accession number AB080692. Introduction During telophase in plant cells, the phragmoplast develops centrifugally as it forms the cell plate until its outer margins reach the parental cell wall and the cytoplasm of the parental cell divides into two. The phragmoplast, a cytokinetic apparatus in plant cells, is composed mainly of two populations of microtubule arrays that are opposed to each other with their plus ends slightly overlapping on the division plane (Euteneuer and McIntosh 1980). The cell plate is formed through the coalescence of Golgi-derived vesicles that are transported along the phragmoplast microtubules to the division plane (Hepler 1982, Samuels 4 Corresponding author: E-mail, [email protected]; Fax, +81-06-6388-8609. 595 596 A MOR1 homologue from telophase tobacco cells Fig. 1 Loosening by KCl of microtubule bundles assembled in miniprotoplast extracts isolated from telophase tobacco BY-2 cells. A crude extract of miniprotoplasts isolated from telophase BY-2 cells was incubated in the presence of 1 mM GTP and 20 mM taxol and then centrifuged. Pelletted microtubule bundles (a) were suspended in PME buffer that contained 150 mM KCl (b), 300 mM KCl (c) or 300 mM KCl plus 5 mM MgAMPPNP (d) and centrifuged. Each pellet was negatively stained with uranyl acetate and observed by electron microscopy. The microtubule bundles were loosened in the presence of KCl. Bar: 200 nm. tubules assembled in a cytoplasmic extract obtained from miniprotoplasts of BY-2 cells. One is short (12–15 nm) and the other is long (20–25 nm) (Jiang et al. 1992). Assuming that the diameter of microtubules is 24 nm, the center to center distance of microtubules cross-linked by the shorter type of crossbridge is 36–39 nm and that of microtubules cross-linked by the longer type of cross-bridge is 44–49 nm. The 65-kDa MAP purified from BY-2 cells formed cross-bridges of similar length to the shorter type of cross-bridge (Jiang and Sonobe 1993). However, the 65-kDa MAP of carrot cells formed cross-bridges of similar length to the longer type of cross-bridge (Chan et al. 1999). This suggests that the 65-kDa MAP may be able to form both types of cross-bridges. Since the 65-kDa MAP forms cross-bridges comparable in length to those observed in phrag- moplasts and co-localizes with phragmoplast microtubules in BY-2 cells (Jiang and Sonobe 1993, Smertenko et al. 2000) and carrot cells (Chan et al. 1996), the 65-kDa MAP is a candidate component of the cross-bridges between the phragmoplast microtubules. However, the arrangement of microtubules bundled by the 65-kDa MAP is different from those observed in the phragmoplast. The 65-kDa MAP cross-linked microtubules laterally to form two dimensional ‘sheets’ (Jiang and Sonobe 1993, Chan et al. 1999), but such sheet-like bundles were not observed in transverse sections of developing cell plates (Hepler and Newcomb 1967, Cronshaw and Esau 1968, Hepler and Jackson 1968). Thus, microtubule cross-bridge proteins other than the 65-kDa MAP may participate in crosslinking phragmoplast microtubules. A MOR1 homologue from telophase tobacco cells 597 Fig. 2 SDS-PAGE analysis of MAPs in bundles of microtubules assembled in the extract of miniprotoplasts isolated from telophase tobacco BY-2 cells. Bundles of microtubules assembled in the extract from telophase BY-2 cells were suspended in PME buffer alone (lanes 1 and 2) or buffer that contained 150 mM KCl (lanes 3 and 4), 300 mM KCl (lanes 5 and 6) or 300 mM KCl plus 5 mM MgAMPPNP (lanes 7 and 8) and centrifuged. Each supernatant (S) and pellet (P) were analyzed by SDS-PAGE. The 200-kDa polypeptide was dissociated from microtubules in the presence of 300 mM KCl (lanes 5 and 6). Dissociation of polypeptides of about 120 kDa from microtubules by 300 mM KCl was inhibited by 5 mM MgAMPPNP (lanes 7 and 8). Molecular weights of standards are indicated on the left in kDa. The dark bands of about 50 kDa in the “P” lanes are tubulin derived from precipitated microtubules. In this paper, we describe the isolation of TMBP200, a novel microtubule bundling polypeptide of 200 kDa from telophase tobacco BY-2 cells. The arrangement of microtubules and the lengths of cross-bridges between microtubules in microtubule bundles formed by TMBP200 in vitro were comparable to those observed in phragmoplasts in Haemanthus endosperm cells (Hepler and Jackson 1968) and in phragmoplasts isolated from tobacco BY-2 cells (Kakimoto and Shibaoka 1988). Analysis of the cDNA encoding TMBP200 revealed that it shares homology with Arabidopsis thaliana MOR1 (Whittington et al. 2001), a plant member of the XMAP215 family of MAPs. Results Isolation of microtubule-bundling factors from telophase BY-2 cells Bundles of microtubules were formed in the extract from miniprotoplasts of telophase tobacco BY-2 cells following incubation in the presence of taxol (Fig. 1a). The microtubule bundles were suspended in PME buffer alone or in the same buffer that contained KCl or KCl plus MgAMPPNP and then centrifuged to sediment the microtubules. The sedimented microtubules were negatively stained and observed with an electron microscope (Fig. 1). The supernatants, as well as sedimented microtubules, were analyzed by SDS-PAGE (Fig. 2). The bundle structure was retained when the bundles were sus- Fig. 3 Microtubule bundling activity of S3 and S4 fractions of the extract from telophase tobacco BY-2 cells. Microtubules assembled in the extract from telophase tobacco BY-2 cells were incubated with 300 mM KCl and 5 mM MgAMPPNP and centrifuged to obtain a supernatant S3 and a pellet P3. P3 was further incubated with 300 mM KCl and 10 mM MgATP and again centrifuged to obtain a supernatant S4 and a pellet P4. The S3 and the S4 fractions were desalted and incubated with in vitro-assembled bovine brain microtubules and centrifuged. Pelletted microtubules were negatively stained with uranyl acetate and viewed by electron microscope. (a) Microtubules alone. (b) Microtubule bundles are observed after incubation with S3. (c) After incubation with S4, bundles of microtubules are not seen. Bar: 500 nm. 598 A MOR1 homologue from telophase tobacco cells Fig. 4 SDS-PAGE analysis of microtubule associated polypeptides in S3 and S4 fractions of the extract from telophase tobacco BY-2 cells. Both S3 and S4 (see legend to Fig. 3.) were incubated with (+MT) or without (–MT) in vitro-assembled bovine brain microtubules and centrifuged. Each supernatant (S) and pellet (P) were analyzed by SDSPAGE. Molecular weights of standards are indicated on the left in kDa. pended in the buffer alone (data not shown). Some bundles came loose by treatment with 150 mM KCl (Fig. 1b) and all were loosened by treatment with 300 mM KCl (Fig. 1c). Five mM MgAMPPNP inhibited the loosening of the bundles caused by 300 mM KCl (Fig. 1d). A number of polypeptides, including the major polypeptides of 200 kDa and 120 kDa, cosedimented with microtubules when the bundles were treated with PME buffer that contained no KCl (Fig. 2, lane 1). The 65-kDa polypeptides were also found in the microtubule pellet, but in lower amounts compared to the 120-kDa and 200kDa polypeptides (Fig. 2, lane 1). Some of the 200-kDa polypeptide was released from microtubules by treatment with 150 mM KCl (Fig. 2, lane 3). Both the 200-kDa polypeptide and the 120-kDa polypeptide were completely released from microtubules by treatment with 300 mM KCl (Fig. 2, lanes 5 and 6). Five mM MgAMPPNP inhibited the release of the 120kDa polypeptide from microtubules by 300 mM KCl, but not that of the 200-kDa polypeptide (Fig. 2, lane 8), suggesting that the 120-kDa polypeptide, but not the 200-kDa polypeptide, is associated with microtubules in an ATP-dependent manner. Since treatment with MgAMPPNP inhibited both the dissociation of the bundles (Fig. 1d) and the release of the 120-kDa polypeptide from microtubules (Fig. 2, lane 8), these results suggest that the 120-kDa polypeptide has the ability to retain the bundle structure even when microtubule-associated polypeptides other than the 120-kDa polypeptide are released. The microtubule proteins (MTPs) were separated into two fractions, S3 and P3, and the P3 was subsequently fractionated into S4 and P4 fractions according to the procedures described in the “Materials and Methods”. Both the S3 and the S4 fractions were desalted and incubated with microtubules, which Fig. 5 Binding of the purified 200-kDa polypeptide to microtubules. The 200-kDa polypeptide released from microtubules by 300 mM KCl in the presence of 5 mM MgAMPPNP (Fig. 2, lane 8) was purified by chromatography on FPLC Mono S column. The purified 200-kDa polypeptide was incubated with or without in vitro-assembled bovine brain microtubules and centrifuged at 100,000´g for 15 min. Each supernatant (S) and pellet (P) was analyzed by SDS-PAGE. The 200kDa polypeptide co-sedimented with the microtubules, indicating that the purified 200-kDa polypeptide has an ability to bind to microtubules. had been assembled from bovine brain tubulin in the presence of taxol, and centrifuged. The sedimented microtubules were observed with an electron microscope (Fig. 3) and the supernatants and the pellets were analyzed by SDS-PAGE (Fig. 4). As shown in Fig. 3b, the desalted S3, which contained more than 10 types of microtubule-associated polypeptide including the 200-kDa polypeptide (Fig. 4, lane 3), had microtubule bundling activity. The desalted S4 showed no microtubule bundling activity (Fig. 3c) despite the presence of the 120-kDa polypeptide in this fraction (Fig. 4, lane 7). Therefore, the 120kDa polypeptide does not appear to be able to form microtubule bundles alone as previously observed by Jiang and Sonobe (1993), although it is able to retain bundle structures formed by other microtubule bundling factors. Purification of the 200-kDa polypeptide We purified the 200-kDa polypeptide to determine if it could bundle microtubules. The S3 supernatant was fractionated by Mono S cation exchange chromatography and the 200-kDa polypeptide was eluted with about 240 mM NaCl. The fraction containing the 200-kDa polypeptide was desalted and incubated with or without in vitro-assembled microtubules, then centrifuged. The purified 200 kDa polypeptide cosedimented with microtubules (Fig. 5, lanes 3 and 4), indicating that the polypeptide did bind to microtubules. As shown in Fig. 6 the purified 200-kDa polypeptide was also able to bundle microtubules. Therefore, we named the 200-kDa polypeptide TMBP200 (tobacco microtubule bundling polypeptide of 200 kDa). A MOR1 homologue from telophase tobacco cells 599 Fig. 7 Electron micrographs of cross sections of microtubules and microtubule bundles formed by purified TMBP200. (a) In vitro-assembled bovine brain microtubules. (b) In vitro-assembled bovine brain microtubules bundled by TMBP200. Cross-bridges between microtubules are indicated by arrowheads. Hexagonal arrangements of microtubules are indicated by large arrows. Bars: 100 nm. Fig. 6 Bundles of microtubules formed in the presence of the purified 200-kDa polypeptide. In vitro-assembled bovine brain microtubules were incubated with (b) or without (a) the purified 200-kDa polypeptide and negatively stained for electron microscopy. Microtubule bundles were formed in the presence of the 200-kDa polypeptide (b). Bar: 500 nm. Formation of cross-bridge structures between adjacent microtubules by the 200-kDa polypeptide To observe detailed structure of the bundles of microtubules formed by TMBP200, ultra-thin transverse sections of the bundles were prepared and examined with an electron microscope (Fig. 7). Filamentous cross-bridges were frequently observed between adjacent microtubules (Fig. 7b, arrowheads). The center-to-center distance between the crosslinked microtubules measured 34.6±1.4 nm (n = 26). Hexagonal arrays, composed of seven microtubules, were frequently observed (Fig. 7b, large arrows). Analysis of the cDNA encoding TMBP200 The 200-kDa polypeptide purified by SDS-PAGE was digested with CNBr and two CNBr fragments were purified for amino acid sequencing. The sequence of the two fragments designated p200-2 and p200-3 were VTAVDDFGVSHLKLKDLIDFCKD and VSKNLKDIQGPALAIVVERLRPY, respectively. Both sequences showed close homology to a putative MAP found in genome sequences of A. thaliana. During the progression of our work, this putative protein was reported to be a product of the MOR1 gene in which mutations cause temperature-dependent disruption of cortical microtubules (Whittington et al. 2001). The full-length cDNA was obtained using RT-PCR and RACE-PCR methods. The cDNA for TMBP200 encodes a polypeptide of 2,029 amino acids with an estimated relative molecular mass of 223 kDa (Fig. 8). Searches of current databases revealed that TMBP200 belongs to a family of weakly conserved MAPs including, Xenopus XMAP215 (Tournebize et al. 2000), human TOGp (Charrasse et al. 1998), Drosophila MSPS (Cullen et al. 1999), C. elegans ZYG-9 (Matthews et al. 1998), Dictyostelium DdCP224 (Gräf et al. 2000), yeast Stu2p (Wang and Huffaker 1997) and Dis1 (Nabeshima et al. 1998), 600 A MOR1 homologue from telophase tobacco cells Fig. 8 The predicted amino acid sequence of TMBP200. The deduced amino acid sequence contained the sequences of p200-2 (underline) and p200-3 (dashed underline). The complete nucleotide sequence of the TMBP200 cDNA is available from DDBJ/EMBEL/GenBank under accession number AB080692. A MOR1 homologue from telophase tobacco cells Fig. 9 A phylogenetic tree of TMBP200 and other members of the XMAP215 family. The N-terminal conserved region of each member of the XMAP215 family was aligned and represented as a phylogenetic tree using the programmes CLUSTALW (Thompson et al. 1994) and TreeView (Page 1996). The horizontal bar indicates 0.1 nucleotide substitutions per site. Accession numbers are shown in brackets. and Arabidopsis MOR1 (Whittington et al. 2001), the only member identified in plants. A phylogenetic analysis showed that TMBP200 is most similar to MOR1 protein (Fig. 9). Discussion The lengths of cross-bridges formed by TMBP200 were comparable to those observed in phragmoplasts in Haemanthus endosperm cells (Hepler and Jackson 1968) and in phragmoplasts isolated from BY-2 cells (Kakimoto and Shibaoka 1988). Arrangements of microtubules bundled by TMBP200 were significantly different from those bundled by the 65-kDa MAP. The 65-kDa MAP cross-linked microtubules laterally to form two-dimensional ‘sheets’ (Jiang and Sonobe 1993, Chan et al. 1999), whereas TMBP200 formed three-dimensional bundles. In an earlier electron microscopical study of phragmoplasts, microtubule clusters with a hexagonal arrangement were observed in a transverse section of a developing cell plate (Hepler and Jackson 1968). The hexagonal arrangement is very similar to that observed in microtubule bundles formed by TMBP200, suggesting that TMBP200 participates in forming the cross-bridges between phragmoplast microtubules. However, before we reach any conclusions, immunolocalization of TMBP200 in the phragmoplast should be determined. We are now examining whether TMBP200 is present on cross-bridges between phragmoplast microtubules using peptide antibodies directed against TMBP200. The deduced amino acid sequence of TMBP200 revealed that it shares strong homology to Arabidopsis MOR1, a plant member of the XMAP215 MAP family. MOR1 is thought to play an important role in controlling cell elongation through 601 organization of the cortical microtubule array, since mutations in the MOR1 gene cause temperature-sensitive cortical microtubule disorganization and abnormal cell elongation at restrictive temperatures (Whittington et al. 2001). Our result that TMBP200 was isolated from telophase tobacco cells provides new evidence that these plant MAPs may also be very important for microtubule organization during the cell cycle, as well as during cell elongation. All known homologues of TMBP200 in yeasts and animals are present in the mitotic apparatus, suggesting that they play an important role in cell division. The human TOGp homologue was reported to be present in the midbody (Charrasse et al. 1998), an apparatus in which microtubules are arranged similarly to those in the phragmoplast (Euteneuer and McIntosh 1980). Mutations in Stu2p (Wang and Huffaker 1997), Dis1 (Nabeshima et al. 1995), ZYG-9 (Matthews et al. 1998) and MSPS (Cullen et al. 1999) cause mitotic defects. Cells expressing GFP-DdCP224 fusion protein show defects in centrosome duplication and cytokinesis (Gräf et al. 2000). XMAP215 was initially identified as a factor that promoted microtubule assembly at plus ends in Xenopus egg extracts (Gard and Kirschner 1987, Vasquez et al. 1994). More recent studies have demonstrated that the assembly-promoting activity is counterbalanced by a disassembly-promoting activity of XKCM1, possibly as a means to regulate microtubule dynamics (Tournebize et al. 2000). Since phragmoplast microtubules assemble at plus ends (Vantard et al. 1990, Asada et al. 1991), TMBP200 might participate not only in the formation of crossbridges between phragmoplast microtubules, but also in the regulation of phragmoplast microtubule assembly. To know whether or not TMBP200, a member of the XMAP215 family, plays a role in plant cell division, we are now engaged in overexpression experiments of truncated TMBP200 that are expected to affect the function of endogenous TMBP200. Materials and Methods Plant material Tobacco BY-2 cells (Nicotiana tabacum ‘Bright Yellow 2’) were cultured in suspension using modified Linsmaire and Skoog’s medium (modified LS medium) at 27°C as described by Nagata et al. (1981). Isolation of miniprotoplasts at telophase Protoplasts at telophase were isolated by the procedure described by Kakimoto and Shibaoka (1992) except for modifications described below. An enzyme solution [1% Sumizyme C (Shin-nihon Kagaku Kougyou Co. Ltd., Aichi, Japan), 0.1% Sumizyme AP2 (Shin-nihon Kagaku Kougyou Co. Ltd.), 0.03% Pectolyase Y23 (Seishin Pharmaceutical Co., Nagareyama, Chiba, Japan), 0.3 M mannitol dissolved in modified LS medium, pH 5.5] was used for digestion of cell walls. Protoplasts, the majority (>80%) of which were in telophase, were harvested 120 min after the termination of treatment with propyzamide. Miniprotoplasts were isolated by the procedure described by Jiang et al. (1992) with some modifications. About 30 ml of protoplast pellet in packed volume was suspended in 170 ml of cooled (4°C) miniprotoplast preparation solution (MPS; 22.5% sucrose, 175 mM mannitol, 10 mM MgCl2, 10 mM MES-KOH, pH 5.8) and 25 ml aliquots were 602 A MOR1 homologue from telophase tobacco cells transferred to centrifuge tubes containing a 1 ml percoll cushion (0.8 M glycerol, 65% percoll, pH 5.8). After centrifugation at 15,000´g for 30 min, miniprotoplasts were collected from the layer near the bottom of each centrifuge tube and washed once with a cooled (4°C) miniprotoplast washing solution (MWS; 0.6 M mannitol, 0.1 M NaCl, 10 mM MgCl2, MES-KOH, pH 5.8). About 4 ml of miniprotoplasts (packed volume) was usually obtained by this procedure. Preparation of microtubule proteins Miniprotoplasts were washed once with ice-cold homogenization buffer (80 mM PIPES, 2 mM EGTA, 1 mM MgCl2, 1 mM MgGTP, 2 mM DTT, 1 mM PMSF, 50 mg ml–1 leupeptin, pH 6.9) that contained 12.5% sucrose. Washed miniprotoplasts were suspend in the same volume of homogenization buffer and ruptured with a glass homogenizer. Homogenates were centrifuged at 10,000´g for 10 min and the resulting supernatants were diluted with a 40% volume of homogenization buffer and then centrifuged at 200,000´g for 30 min. The supernatant obtained was designated telophase extract. Taxol was added to the telophase extract to a final concentration of 20 mM and the extract was incubated at 27°C for 20 min to polymerize microtubules. Microtubules and their associated proteins were pelletted through a sucrose cushion (homogenization buffer containing 25% sucrose and 20 mM taxol) by centrifugation at 100,000´g for 30 min. The resultant pellets, termed microtubule proteins (MTPs), were frozen in liquid nitrogen and stored at –85°C. Isolation of microtubule bundling polypeptides Aliquots of MTPs were suspended in (1) PME buffer (80 mM PIPES, 2 mM EGTA, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 20 mM taxol, pH 6.9), PME buffer that contained (2) 150 mM KCl, (3) 300 mM KCl or (4) 300 mM KCl and 5 mM MgAMPPNP, an unhydrolysable ATP analogue, and centrifuged at 100,000´g for 15 min. KCl and AMPPNP were used to dissociate MAPs from microtubules and to strengthen the association of kinesin-related proteins such as TKRP125 (Asada and Shibaoka 1994, Asada et al. 1997) to microtubules, respectively. The supernatants and the pellets were analyzed by SDS-PAGE (Laemmli 1970). Each pellet was suspended in PME buffer and applied to a formvar-coated microgrid, negatively stained with 1% uranyl acetate and examined with an electron microscope (JEM-1210; JEOL, Tokyo, Japan) for the presence of microtubule bundles. Since release of the 120-kDa polypeptide from microtubules by 300 mM KCl was inhibited in the presence of 5 mM AMPPNP (Fig. 2), the 200-kDa polypeptide and the 120-kDa polypeptide were separated as follows. MTPs from 12–15 ml of miniprotoplasts in packed volume were suspended in PME buffer that contained 300 mM KCl and 5 mM MgAMPPNP and centrifuged at 100,000´g for 15 min. The supernatant containing the 200-kDa polypeptide, and the pellet containing microtubules and the 120-kDa polypeptide, were designated S3 and P3, respectively. The P3 was suspended in PME buffer that contained 300 mM KCl and 10 mM MgATP to release the 120-kDa polypeptide from microtubules and again centrifuged at 100,000´g for 15 min to obtain a supernatant (S4) and a pellet (P4). Each aliquot of the S3 and the S4 was dialyzed against PME buffer and assayed for their abilities to bind to, and/or bundle, microtubules. Microtubule binding and bundling assays Tubulin was prepared from bovine brains as described elsewhere (Shelanski et al. 1973), followed by purification on a phosphocellulose column. Microtubules were assembled from an 8 mg ml–1 solution of the purified brain tubulin in the presence of 2.5 mM taxol and 1 mM GTP. Samples were incubated at room temperature for 20 min with the microtubules and centrifuged at 100,000´g for 15 min. Super- natants and pellets were analyzed by SDS-PAGE. The pellets were also examined with an electron microscope (JEM-1210) for microtubule bundling activity after negatively staining the samples with 1% uranyl acetate. Purification of the 200-kDa polypeptide The S3 supernatant containing the 200-kDa polypeptide was diluted with an equal volume of double-distilled water to decrease the concentration of KCl and applied to a FPLC-Mono S column (Amersham Pharmacia Biotech, Uppsala, Sweden). After washing the column with elution buffer (20 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, pH 7.0) that contained 180 mM NaCl, proteins absorbed onto the column were eluted with the same buffer that contained linear gradient concentrations (180–300 mM) of NaCl. Eluted fractions were analyzed by SDS-PAGE for the presence of the 200-kDa polypeptide. Fractions that contained the 200-kDa polypeptide were pooled and dialyzed against PME buffer. The dialyzed sample was centrifuged at 100,000´g for 10 min. The supernatant was designated purified 200-kDa polypeptide. Preparation of ultra-thin sections of microtubule bundles About 4 mg of the purified 200-kDa polypeptide and 14 mg of in vitro-assembled bovine brain microtubules were incubated at room temperature for 20 min. The concentration of the 200-kDa polypeptide was estimated from the density of Coomassie blue staining of the polypeptide on an SDS-PAGE gel. Microtubules and microtubule bundles were sedimented by centrifugation at 100,000´g for 15 min and fixed with 2.5% glutaraldehyde in PME buffer followed by 1% OsO4. Fixed samples were dehydrated and embedded in Spurr’s resin (Spurr 1969). Sections were cut, stained with uranyl acetate and lead citrate and examined with an electron microscope (JEM-1210). Sequencing of the 200-kDa polypeptide The supernatant S3 was separated by SDS-PAGE and blotted on a PVDF membrane. After staining of the membrane with Ponceau S, the 200-kDa polypeptide band was excised and treated with CNBr. The CNBr fragments were again separated by SDS-PAGE and blotted on PVDF membrane. After staining the membrane with Coomassie blue, two bands of peptide fragment p200-2 and p200-3 were excised and were sequenced with a protein sequencer (model 610A, Applied Biosystems, Inc., CA, U.S.A.) Cloning the cDNA encoding the 200-kDa polypeptide Minus strand cDNA was synthesized with AMV Reverse Transcriptase XL (Takara Shuzou Co., Ltd, Shiga, Japan) from poly(A)+ RNA, isolated from synchronously cultured BY-2 cells. A partial cDNA that encoded the region between p200-2 and p200-3 was amplified by TaKaRa LA Taq (Takara Shuzou Co., Ltd.). The sequence of primers were 5¢ GCNGTNGAYGAYTTYGGNGT 3¢ (sp2-1) corresponding to TAVDDFGV in p200-2, 5¢ AARGAYYTNATHGAYTTYTGY 3¢ (sp2-2) corresponding to KDLIDFC 3¢ in p200-2 and 5¢ ARIGCIGGICCYTGDATRTC 3¢ (ap3-3) corresponding to KDIQGPAL in p200-3. A nested PCR was performed using a primer pair of sp2-1 and ap3-3 in the primary PCR and using a primer pair of sp2-1 and ap3-3 in the secondary PCR. PCR products were subcloned into the TA-cloning vector pCR2.1 (Invitrogen Corporation, CA, U.S.A.) and were sequenced by the cycle sequencing method with fluorescently labelled primers. The 5¢- and 3¢- ends of the cDNA were obtained using a SMART RACE cDNA amplification Kit (Clontech, CA, U.S.A.) according to the supplier’s instructions. Minus strand cDNA for RACE PCR was synthesized with SuperScript II reverse transcriptase (Invitrogen Corporation) from poly (A)+ RNA of premitotic BY-2 cells. Primers for A MOR1 homologue from telophase tobacco cells the RACE PCR were designed from the sequence of the partial cDNA fragment. Both 5¢- and 3¢- RACE PCR products were subcloned into pCR2.1 vector and about 500 bp of both ends of the RACE PCR products were sequenced. The cDNA was completely sequenced by primer walking. Acknowledgments The authors gratefully acknowledge Professor H. Shibaoka of the Osaka University for his encouragement throughout this work and for critical reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Scientific and Culture to H. Y. (#06740608 and #08740626). 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