Eur. J. Biochem. 205, 1195-1200 (1992) Q FEBS 1992
A novel 24-kDa microtubule-associated protein purified from sea urchin eggs Shohei MAEKAWA'. Masaru TORIYAMA2 and Hikoichi SAKAI' Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Japan Department of Biology, Faculty of Liberal Arts, University of Shizuoka, Japan (Received September 20, 1991/January 29, 1992) - EJB 91 1252
Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24-kDa microtubule-associated protein (SU-MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU-MAP24 promoted tubulin polymerization in a dose-dependent manner. Immunoblotting analysis showed that SU-MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin-sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU-MAP24 is a newly identified microtubuleassociated protein.
Regulation of the organization of microtubules is an important process during the cell cycle. The formation of the mitotic apparatus and its subsequent breakdown are regulated temporally and spatially during the cell cycle. The mechanism of the dynamics of microtubule networks is, however, still unclear. Some focus on the activation of the centrosome to organize microtubules [l] and others on the physiological regulation of intracellular milieu [2, 31. Microtubule-associated proteins (MAP) are considered to be important in microtubule dynamics in vivo, although only a few proteins have so far been characterized as MAP in echinoderm eggs, which are a good model for investigating the mechanism of cell division. One example is the work by Hirokawa and Hisanaga [4]. They purified a 75-kDa MAP from sea urchin eggs and called it buttonin. Vallee and Bloom applied their microtubule-fractionation method using taxol and identified some proteins using monoclonal antibodies [5]. In the previous study [6], we identified and characterized two kinds of Ca2+-bindingproteins from sea urchin eggs using a phenyl-Sepharose column. Cell-staining experiments using specific antibodies to these proteins showed that they are localized in the mitotic-apparatus region. Cosedimentation studies of these proteins with reconstituted microtubules, however, showed no association with microtubules, suggesting the presence of an unknown structure in the mitotic apparatus in addition to the microtubules. During the study, we noticed that other proteins present in the crude eluate of the phenylSepharose column bound to microtubules. An examination of the elution conditions of these proteins from the phenylSepharose column showed that they bound tightly to the matrix and were eluted under more alkaline conditions. This Correspondence to S. Maekawa, Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Tokyo, Japan 113 Abbreviations. MAP, microtubule-associated protein; FITC, fluorescein isothiocyanate; RITC. rhodamine isothiocyanate.
paper deals with the purification and characterization of a sea urchin MAP, SU-MAP24 from its estimated molecular mass (24 kDa) in SDS/PAGE. EXPERIMENTAL PROCEDURES Preparation of proteins
Unfertilized sea urchin eggs (Heniicentrotus pulcherrimus) spawned in Caz+-free sea water (100 ml packed eggs) were washed once with 1 M glycerol containing 5 mM MgClz and extracted with 3 vol. of a solution containing 0.1 M Mes/ KOH, 10 mM p-tosyl-L-arginine methyl ester, 10 mM EGTA, 1 mM MgC12 and 1 mM phenylmethylsulfonyl fluoride (pH 6.7), and centrifuged at 100000 x g for 30 min. The recovered crude supernatant was fractionated by ammonium sulfate. Fractions obtained at 50- 80% saturation was dialyzed against 10 mM Tris/HCl containing 0.15 M NaCl, and 0.2 mM EGTA (pH 7 . 9 , followed by centrifugation at 100000 x g for 90 min. A concentrated CaClz solution was added to the supernatant to bring it to 1 mM, and the solution was applied to a phenyl-Sepharose column (3 cm x 20 cm) equilibrated with the above solution containing CaC12. After washing the column with a solution containing 10 mM Hepes/ KOH, 10 mM Tris and 0.5 mM CaC12 (PH 8.0), Ca2+-binding proteins were eluted with a solution containing 10 mM Tris/HCl and 10 mM EGTA (pH 7.8) [6]. The column was then washed with a solution containing 1 0 m M Tris/HCl and 1 mM EGTA (PH 8.8). Fractions containing proteins were collected and applied to a DEAE-cellulose column (1.4 cm x 6 cm), equilibrated with a solution containing 10 mM Tris/HCl and 0.2 mM EGTA (pH 7.8). After washing the column with the same solution, non-adsorbed fractions were collected and applied to a hydroxyapatite column (1.4cmx6cm) and eluted with a linear gradient of 0350mM phosphate buffer. Eluted proteins was assayed by SDS/PAGE from this step on. Fractions containing SU-MAP24 were pooled and concentrated using a small
1196 hydroxyapatite column after dialysis against buffer A (10 mM Mes/KOH, 0.1 M NaCl. 0.2mM EGTA and 0.1 mM dithiothreitol, pH 6.8). The concentrate was then applied to a gel-filtration column (G3000SW, 0.6 cm x 60 cm, Toyo Soda) and eluted with buffer A. Purified SU-MAP24 fractions were pooled and frozen at -80°C until use. About 0.5 mg SUMAP24 was recovered from 100 ml packed eggs. Purification of tubulin from outer fibers of starfish sperm (Asterius amurensis) Outer fibers were prepared from starfish sperm as described previously [7l by Dr. Murofushi of our laboratory. Tubulin was solubilized by sonication and further purified by one cycle of a polymerization/depolymerization process, as described previously [S]. Preparation of microtubule proteins from a crude extract of sea urchin eggs using taxol Microtubule proteins were prepared using taxol, according to Vallee and Bloom [5] with some modifications. Unfertilized cells were washed once with 1 M glycerol/5 mM MgCI2 and packed eggs were homogenized in 2 vol. buffer B (100 mM Mes/KOH, 5 mM EGTA and 1 mM MgCI2 pH 6.7) followed by centrifugation at 100000 x g for 60 min. GTP and taxol were added to the supernatant to give 0.5 mM and 0.02 mM, respectively. After a 10-min incubation at 30°C to assemble microtubules. and a successive 15-min incubation on ice, the solution was overlaid onto buffer B containing GTP, taxol and 15% sucrose. After centrifugation at 25000 x g for 30 min at 2 T, the pellet fraction was recovered and washed once in buffer B containing GTP and taxol, followed by centrifugation at 30000 x g for 20 min to collect taxol-stabilized microtubules. Binding of SU-MAP24 to microtubules Sedimentation assays were performed to assess the binding of SU-MAP24 to microtubules. In a-system with no taxol, purified SU-MAP24 (2.2 pM) was mixed with purified tubulin (20pM) and incubated at 25°C in a solution containing 10 mM Mes, 3 mM MgCI2, 1 mM EGTA, 120 mM KC1 and 0.5 mM GTP @H 6.7). In a system with taxol, outer-fiber tubulin (2 pM) was mixed with various amounts of the SUMAP24 fraction in buffer A, or SU-MAP24 (2.2 pM) was mixed with various amounts of tubulin and the medium adjusted to 10 mM Mes/KOH, 2.5 mM MgCI2, 0.5 mM GTP, 1 rnM EGTA, 50 rnM NaCl and 0.02 m M taxol (pH 6.7). In both cases, after incubation at 25°C for 30 min, the mixtures were centrifuged at 200000 x g and 25 "C for 20 min.The pellet and supernatant were recovered and subjected to SDS/PAGE. To calculate the association constant between SU-MAP24 and microtubules, the amount of cosedimented SU-MAP24 protein was determined by densitometric scanning of the gel after staining. To correct the amount of proteins recovered in the pellet fraction by binding to the wall of the centrifuge type or by aggregation itself, control incubations without added tubulin were also carried out and electrophoresed after centrifugal separation. For standards, various amounts of purified SU-MAP24 were also electrophoresed in the same gel and stained, and the color of proteins was assayed by densitometric scanning.
Fig. 1. Cosedimentation assay of the EGTA and the alkaline eluate from phenyl-Sepharose. 80 p1 EGTA eluate (B-E, 0.2 mg/ml) or alkaline eluate (F -J, 0.6 mg/ml) was dialyzed against buffer A or buffer alone IK, L), and mixed with (B, C, G, H, K, L) or without (D, E. I, J ) tubulin (3.3 mg/ml) and incubated in 100 111 at 25°C for 30 min. After centrifugation, each 30+1 supernatant (B, E, G, J, K) and all of the pellet (C, D, H, I, L) were electrophoresed with 30 pI of the original solution (A, F) using a 12% acrylamide gel. In lane M. molecular mass markers were also electrophoresed [indicated with black spots; from top to bottom, myosin (200 kDa). phosphorylase b (94 kDa). bovine serum albumin (68 kDa), tubulin (55 kDa), actin (42 kDa), glyceraldehyde-3-phosphatedehydrogenase (35 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (21 kDa)]. Possible microtubule-binding proteins are indicated with arrowheads (lanes C and H) and tubulin is marked by an arrow.
Assay of assembly-promoting activity of SU-MAP24 Various amounts of SU-MAP24 were mixed with tubulin (6.1 pM) in a solution containing 10 mM Mes/KOH, 0.5 niM MgC12,0.5 mM GTP, 50 mM KCI, 50 mM NaCl and 0.6 mM EGTA (PH 6.7). The turbidity change was followed at 25 "C using a Gilford 260 spectrophotometer. Preparation of antibodies Immunization of a rabbit with 0.05 mg purified protein and affinity-purification of collected sera were performed as described previously [9]. For production of monoclonal antibodies, partially purified preparation of SU-MAP24 (the eluate of the phenyl-Sepharose column) was intraperitoneally injected into three mice with complete (first) and incomplete (second and following injections) adjuvant. About 0.01 mg SU-MAP24 was injected every time. One week after the third injection, blood was collected from the tail and titers were checked. After four injections, proteins were without adjuvant were boosted into a mouse, and three days later, the spleen was dissected and spleen cells were fused with myeloma cells as described previously [lo]. Hybridoma cells were screened by immunoblotting of the culture medium of positively growing cells against partially purified SU-MAP24. Two clones termed 5B4 and 4D3 were finally obtained and, as they showed the same reactivity, clone 4D3 was used in this experiment. Cell staining Normally fertilized eggs were cultured to metaphase after the removal of the fertilization membrane. Cells were fixed
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Fig. 2. SDS/PAGE of SU-MAP24. SDS/PAGE pattern of fractions at different purification steps. (A) Crude extract (80 pg); (B) nonadsorbed fraction of the DEAE-cellulose column (6 pg); (C) eluate from hydroxyapatite (6 pg); (D) eluate from the gel-filtration column (2 pg). In lane M, molecular mass markers werc also electrophoresed, as in Fig. 1. (b) SDS/PAGE pattern of a cosedimentation experiment without taxol. Purified SU-MAP24 (0.05 mg/ml; A) was mixed with (€3, C) or without (D, E) purified outer-fiber tubulin (20 pM) and incubated at 25°C. After centrifugation, the supernatant (B, E) and pellet (C, D) were recovered and analyzed. The purified tubulin rraction was also treated and separated into supernatant (F) and pellet (G). In this case, all of the supernatant and pellet fractions were electrophorcsed.
with 3.7% formalin in Ca2+-freesea water and were embedded in paraffin. Staining of paraffin-sectioned cells was performed as described previously [l 11 using polyclonal and mouse monoclonal antibodies against SU-MAP24, rat monoclonal antibody against tubulin (YL1/2, Sera-Lab, UK) and/ or mouse monoclonal antibody against actin (Amersham, UK). Following primary antibodies, we used fluoresceinisothiocyanate (F1TC)-labeled goat anti-(rat IgG) antibodies, FITC-labeled or rhodamine-isothiocyanate(R1TC)-labeled goat anti-(mouse IgG) antibodies and RITC-labeled goat anti(rabbit IgG) antibodies as the secondary antibodies (all affinity-purified).
Others SDS/PAGE, immunoblotting, protein determination, non-equilibrium two-dimensional electrophoresis were performed as described previously [12, 131. A gel-filtration column (G3000SW, 0.8 cm x 60 cm) was used to estimate the Stokes' radius of SU-MAP24 in buffer A. Bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonuclease A were used as internal markers. Sucrose density-gradient centrifugation was done to obtain the sedimentation coefficient of SU-MAP24, in which 5 - 15% sucrose gradient in buffer A was used for centrifugation at 180000 x g and 2 "C for 40 h [t 41. Bovine serum albumin (4.2 S), ovalbumin (3.6 S), chymotrypsinogen A (2.6 S) and ribonuclease A (1.8 S) were used as marker proteins. Phenyl-Sepharose was purchased from Pharmacia (Sweden) and affinity-purified secondary antibodies were purchased from Chemicon International (USA).
Fig. 3. Association of SU-MAP24 with tubulin during polymerization/ depolymerization process. Tubulin (16.5 pM) was polymerized in the presence of SUiMAP24 (1.1 pM) at 25°C for 40 min. After centrifugation for 20 min at 200000 x g and 25"C, the supernatant ( S l ) and pellet (Pl) fractions were recovered. Thc pellet fraction was then homogenized in buffer B and incubated for 10 min at O T , followed by centrifugation at 2°C to obtain the supernatant (S2) and pellet (P2) fractions. The supernatant fraction was incubated at 25°C aftcr the addition of MgCI2 and KCI to give 2 mM and 100 mM, respectively. After centrifugation a t 25"C, the supernatant (S3) and pellet (P3) were recovered. For SDS/PAGE, sample volumes were adjusted to give the required amounts of protein in each fraction.
RESULTS Purification of SU-MAP24 and demonstration of its presence in the sea-urchin MAP fraction prepared using taxol Cosedimentation assay of the EGTA eluate showed major proteins in this fraction have little or no binding capacity for polymerized tubulin, as described previously (Fig. 1, lanes C and D) [6]. In this fraction, two bands exist which cosediment with tubulin (Fig. 1, lane C, arrowheads). Preliminary studies showed that these proteins were eluted rather slowly from the column during the EGTA wash. We therefore washed the column with a more alkaline solution, which is known to be effective in removing bound proteins, after the EGTA wash. The same cosedimentation assay of this eluate showed the presence of many sedimentable bands in this fraction (Fig. 1, lane H, arrowheads). The tubuliii fraction used in this assay contains few other proteins (Fig. 1, lane L), which were judged to be candidates for microtubule-binding proteins. We tried to purify these proteins since this would be necessary for further characterization. In this paper, we describe the purification and characterization of one of these proteins. The SDS/PAGE pattern of fractions during purification is shown in Fig. 2A. The purified protein showed single band in SDS/ PAGE, with an apparent molecular mass of 24 k D a . The calculated protein recovery (about 1.6% of the original amount) is relatively low. This is partly due to the copurification of two polypeptides showing slow mobility in SDS/PAGE (Fig. 2A, lane C). Gel filtration is only partly effective in removing these polypeptides and nearly 90% of the protein was lost in this step. The cosedimentation assay showed its binding to reconstituted microtubules (Fig. 2 B), although the recovery of a large part of SU-MAP24 in the supernatant fraction suggested the
1198
Fig. 4. SDS/PAGE of purified SU-MAP24, taxol microtubule fraction and crude extract of sea urchin eggs, and their immunoblotting using a monoclonal antibody against SU-MAP24 (4D3). Each 100 pg protein of the taxol microtubule fraction (T) and crude extract (C) and 0.3 v g SU-MAP24 (24) were electrophoresed in the presence of SDS using a 12% polyacrylamide gel. Molecular mass markers were also electrophoresed in lane M (from top to bottom, 200, 94, 68, 55, 42, 35, 29 and 21 kDa) and stained with Coomassie brilliant blue (CBB). One set of proteins was transferred to a Durapore sheet after SDS/PAGE and immunoblotted using monoclonal antibodies against SU-MAP24 (A-24K).
affinity of its interaction with polymerized tubulin is not so high compared with other MAP seen in Fig. 2B (see below). We further carried out a polymerization/depolymerizationexperiment. The polymerized-microtubule fraction obtained in Fig. 2 was depolymerized in cold buffer and, after centrifugation, the soluble fraction was obtained. This fraction was then warmed and the polymerized microtubules were obtained after centrifugation. SDS/PAGE of these fractions showed the recovery of some part of SU-MAP24 in the final microtubule fraction, confirming its association with microtubules (Fig. 3). If this protein does bind microtubules in the cell, it should be recovered in the taxol-stabilized microtubule fraction prepared from the crude extract of the cell. This was confirmed with immunoblotting using specific antibodies prepared as described in Experimental Procedures. Fig. 4A shows that a protein band with the same mobility in SDS/PAGE as SUMAP24 is clearly visible both in the taxol/microtubule-protein fraction and in the crude fraction. Blotting experiments showed that these protein bands reacted with monoclonal antibodies to SU-MAP24 (Fig. 4B). A comparison of the intensities of the reacted bands showed the amount of SUMAP24 present in sea urchin eggs to be relatively high (at least 0.3% of soluble proteins). Fig. 4 also proved high specificity of the antibody. To estimate further the amount of the protein in the crude extract and to study the molecular characteristics of this protein, we analyzed the crude extract in two-dimensional gels. Equilibrium two-dimensional gels showed no corresponding spot after Coomassie-brilliant-bluestaining, and no immunoreactive band after immunoblotting (data not shown). Non-equilibrium two-dimensional gel electrophoresis to analyze basic proteins [13] was then performed, and SUMAP24 was identified as a basic protein eluted in the middle
Fig. 5. Two-dimensional SDS/PAGE using non-equilibrium isoelectric focusing. Each 100 pg protein of the crude extract (A) and taxol microtubule fraction (B) was electrophoresed according to [13]. Arrowheads show the spots of SU-MAP24, which were reacted with anti-(SU-MAP24) antibody by immunoblotting (data not shown).
of the gel (Fig. 5). Sometimes the spot was separated into several spots with different intensities (Fig. 5 B). The identification of the spots in Fig. 5 B indicated by arrows as SU-MAP24 was carried out with immunoblotting (data not shown). There were few proteins that showed the same mobility as SU-MAP24 in SDS/PAGE. The Stokes’ radius of this protein was calculated to be 2.9 nm from a gel-filtration column (G3000SW, Toyo Soda). A sedimentation coeficient of 3.3 S was obtained by sucrose density-gradient centrifugation [14]. Assuming the specific volume of this protein to be 0.75, the molecular mass was calculated to be 43 kDa, suggesting a homodimeric form of the 24-kDa subunit in solution. Cellular distribution of SU-MAP24
To study the intracellular distribution of SU-MAP24, formalin-fixed, paraffin-embedded metaphase eggs were stained with specific antibodies against SU-MAP24. Both polyclonal and monoclonal antibodies showed clear staining of the section. Fig. 6 demonstrated its localization to the mitotic apparatus. Double staining with anti-(SU-MAP24) and anti-tubulin antibodies showed its colocalization clearly. Binding of SU-MAP24 to microtubules In order to study the possible function of this protein, we next measured the ability of this protein to bind to
I199
Fig. 6. Staining of paraffin-mtiond cells with monoclonal antibody against SU-MAP24 (B, C) and with rat mono&nal anti-tubulin antibodies (A, D). FITC-labeled goat anti-(rat IgG) antibodies (A, C) and RITC-labeled goat anti-(mouse Ig) antibodies (B, D) were used as second antibodies. Little background staining and little reactivity of second antibodies with non-immunized immunoglobulins were observed in C and D. Bar, 50 vrn.
Fig.8. Cosedimentation assay of SU-MAP24 with starfish microtubules. To a constant amount of SU-MAP24 (2.2 pM), various amounts of tubulin (1,O; 2.3.3 pM, 3,6.6 pM; 4,lO pM;5 , 13.2 pM; 6, 16.5 pM) were added and polymerized. After centrifugation, the supernatant (s)and Pellet (p) were recovered and electrophoresed. as in Fig. 7.
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microtubules. For this purpose, we purified tubulin by a polymerization/depolymerization method from starfishsperm outer fibers after solubilization by sonication. Tubulin Fig. 7. Cosedimentation analysis of SU-MAP24 with microtubules prepared from this source was shown to have the same propprepared from starfish-sperm outer-fiber tubulin. To a constant amount erty as that prepared from egg extract [7]. Fig. 7 shows the binding pattern of this protein to microtubules formed in the of tubulin (2 pM), various amounts of SU-MAP24 (1.0; 2,0.33 pM; 3,0.66pM;4,0.99~1M;5,1.3pM;6,1.66pM;7,2.2pM;8,2.8pM; presence of taxol. This figure also shows the purity of the 9. 3.3 pM) were added. After tubulin polymerization in the presence tubulin fraction used in this experiment. The result showed of taxol(O.02 mM), mixtures were centrifuged and the pellet fractions a dose-dependent stoichiometric binding of SU-MAP24 to (P) were recovered. Control tubes were also incubated without tubulin microtubules. Assuming that SU-MAP24 has a molecular and treated with SU-MAP24 (P': 1,3.3 pM; 2. 2.2 pM; 3, 1.1 pM; 4, mass of 48 kDa, the Scatchard plot analysis presented an 0.55 pM). The supernatant obtained in the absence of SU-MAP24 was also electrophoresed (S) showing polymerization of tubulin and association constant of 0.9 x lo6 M - for SU-MAP24 binding little contaminating protein. Concentration of SU-MAP24 was and a stoichiometry of 1 mol SU-MAP24 dimer/6.3 mol calculated from a molecular mass of 48 kDa, estimated from its tubulin dimer. In another experiment, increasing amounts of tubulin were added to a constant amount of SU-MAP24, and physicochemical parameters.
1200 the association of SU-MAP24 to microtubules was studied with the SDSlPAGE after polymerization and centrifugation (Fig. 8). The result showed a dose-dependent recovery of SU-MAP24 in the pellet fraction, further suggesting its specific association with microtubules. Next, various amounts of SU-MAP24 were mixed with a constant arhount of tubulin to see whether or not SU-MAP24 has an ability to promote microtubule assembly. Fig. 9 shows that SU-MAP24 can promote tubulin polymerization in a dose-dependent manner. These results show that SU-MAP24 works as one of the microtubule-associated proteins of sea urchin eggs. Observations by electron microscopy, of microtubules assembled in the presence of SU-MAP24 after negative staining, revealed no clear difference from those assembled without SU-MAP24 (data not shown). DISCUSSION In this paper, we described the purification and characterization of a protein which shows microtubule binding and assembly-promoting ability in vitro. Staining of cells with the specific antibodies demonstrated its localization in the mitotic apparatus. These results satisfy some of the criteria of MAP, and we call this protein SU-MAP24 from its mobility in SDS/ PAGE. Several microtubule-associated proteins have been identified so far in eggs and oocyte. One is a high-molecular-mass MAP (XMAP of Xenopus oocyte, 21 5 kDa) reported by Gard and Kirschner [15], which regulates microtubule assembly. MAP of intermediate-molecular-mass (80 - 60 kDa) are present in echinoderm eggs. These are known to cycle with microtubules [16], localize to the mitotic spindle and bind hexagonally to microtubules [4]. These proteins seems to be identical, although a precise comparison of these proteins has not yet been performed. The presence of a 62-kDa protein which regulates microtubule polymerization through phosphorylation was also reported [17]. SU-MAP24 described here is different from these proteins in its molecular mass. Furthermore, Fig. 4 presents evidence that SU-MAP24 is not a breakdown product of a protein of higher molecular mass. Little attention has been paid to low-molecular-mass MAP so far, except for the identification of MAP using monoclonal antibodies by Vallee and Bloom [ S ] . One- and two-dimensional SDS/PAGE analysis of the crude extract showed few protein bands in the low-molecular-mass range, and this enabled us to estimate the cellular content of this protein by densitometric analysis of the gel: it was about 1.6% of the soluble protein. This means the concentration of SU-MAP24 within the cell is over 10pM. On the other hand, Raff and Kaumeyer [19] estimated the cellular tubulin content of echinoderm eggs to be 5.7 pM. A rough calculation using these values, combined with the association constant and binding ratio obtained in
Fig. 7, showed that over 60% of the binding sites for the SU-MAP24 on microtubules is occupied by this protein within the cell. The localization of this protein within the mitotic apparatus (Fig. 6) coincides well with this estimation, although we have not yet observed direct binding of SU-MAP24 to microtubules within the cell. Our preliminary experiments with microinjection of our monoclonal antibodies into sea urchin eggs resulted in inhibition of cell division. Further analysis of the effect of this protein on microtubule dynamics is important for understanding the mechanism of the regulation of microtubule networks in the sea urchin egg. We are grateful to Dr. Hiromu Murofushi in our laboratory, for supplying the outer-fiber fraction of starfish sperm. This work was partly supported by the Narishige Zoological Science Award.
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S., Endo, S. & Sakai, H. (1990) J. B i d . d e m . 265, 32403247. 2. Suprynowicz, F. A. & Mazia, D. (1985) Proc. Nut1 Acad. Sci. U S A 82,2389-2393. 3. Poenie, M., Alderton, J., Steinhardt, R. & Tsien, R. (1986)Science 233,886- 889. 4. Hirokawa, N. & Hisanaga, S.-I. (1987)J. Cell Biol. 104, 15331561. 5. Vallee, R. B. & Bloom, G. S. (1983)Proc. Natl Acad. Sci. LISA 80.6253-6263. 6 . Maekawa, S., Toriyama. M. & Sakai. H. (1991)Exp. kell. Res. 194, 105-110. 7. Kuriyama. R. (1976)J. Biochem. (Tokyo) 80,153-165. 8. Nishida, E., Maekawa, S. & Sakai, H. (1983)J . Biochem. ( Tokyo) 93,1021 - 1026. 9. Maekawa, S . , Endo, S. & Sakai, H. (1987)Exp. Cell. Res. 172. 340 - 353. 10. Maekawa, S., Endo, S. & Sakai, H. (1989)Cell Struct. Funct. 14. 249 - 259. 1 1 . Ohta, K., Toriyama, M., Endo, S. & Sakai. H. (1988)Cell Moril. Cytoskeleton 10,496- 505. 12. Maekawa, S., Toriyama, M., Hisanaga. S.-I., Yonezawa, N., Hirokawa, N. & Sakai, H. (1989)J. B i d . Chem. 264, 74587465. 13. O'Farrell, P.Z., Goodman. H. & O'Farrell, P. H. (1977)Cell 12, 1133 - 1142. 14. Martin. R. G. & Ames, B. N. (1961)J . Biol. Chem. 236, 13721379. 15. Gard, D. L.& Kirschner, M. W. (1987)J. Cell Biol. 105, 22032215. 16. Keller 111, T.C. S. & Rebhum, L. I. (1982)J . Cell B i d . 83,788796. 17. Dinsmore, J. H.& Sloboda, R. D. (1988)Cell 53, 769-780. 18. Hosoya, N., Hosoya, H., Mohri, T. & Mohri, H. (1990) Cell Motil. Cyfoskeleron 15, 168 - 180. 19. Raff, R. A . & Kaumeyer. J. F. (1973)Dev. Biol. 32,309-320.
A novel 24-kDa microtubule-associated protein purified from sea urchin eggs Shohei MAEKAWA'. Masaru TORIYAMA2 and Hikoichi SAKAI' Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Japan Department of Biology, Faculty of Liberal Arts, University of Shizuoka, Japan (Received September 20, 1991/January 29, 1992) - EJB 91 1252
Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24-kDa microtubule-associated protein (SU-MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU-MAP24 promoted tubulin polymerization in a dose-dependent manner. Immunoblotting analysis showed that SU-MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin-sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU-MAP24 is a newly identified microtubuleassociated protein.
Regulation of the organization of microtubules is an important process during the cell cycle. The formation of the mitotic apparatus and its subsequent breakdown are regulated temporally and spatially during the cell cycle. The mechanism of the dynamics of microtubule networks is, however, still unclear. Some focus on the activation of the centrosome to organize microtubules [l] and others on the physiological regulation of intracellular milieu [2, 31. Microtubule-associated proteins (MAP) are considered to be important in microtubule dynamics in vivo, although only a few proteins have so far been characterized as MAP in echinoderm eggs, which are a good model for investigating the mechanism of cell division. One example is the work by Hirokawa and Hisanaga [4]. They purified a 75-kDa MAP from sea urchin eggs and called it buttonin. Vallee and Bloom applied their microtubule-fractionation method using taxol and identified some proteins using monoclonal antibodies [5]. In the previous study [6], we identified and characterized two kinds of Ca2+-bindingproteins from sea urchin eggs using a phenyl-Sepharose column. Cell-staining experiments using specific antibodies to these proteins showed that they are localized in the mitotic-apparatus region. Cosedimentation studies of these proteins with reconstituted microtubules, however, showed no association with microtubules, suggesting the presence of an unknown structure in the mitotic apparatus in addition to the microtubules. During the study, we noticed that other proteins present in the crude eluate of the phenylSepharose column bound to microtubules. An examination of the elution conditions of these proteins from the phenylSepharose column showed that they bound tightly to the matrix and were eluted under more alkaline conditions. This Correspondence to S. Maekawa, Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Tokyo, Japan 113 Abbreviations. MAP, microtubule-associated protein; FITC, fluorescein isothiocyanate; RITC. rhodamine isothiocyanate.
paper deals with the purification and characterization of a sea urchin MAP, SU-MAP24 from its estimated molecular mass (24 kDa) in SDS/PAGE. EXPERIMENTAL PROCEDURES Preparation of proteins
Unfertilized sea urchin eggs (Heniicentrotus pulcherrimus) spawned in Caz+-free sea water (100 ml packed eggs) were washed once with 1 M glycerol containing 5 mM MgClz and extracted with 3 vol. of a solution containing 0.1 M Mes/ KOH, 10 mM p-tosyl-L-arginine methyl ester, 10 mM EGTA, 1 mM MgC12 and 1 mM phenylmethylsulfonyl fluoride (pH 6.7), and centrifuged at 100000 x g for 30 min. The recovered crude supernatant was fractionated by ammonium sulfate. Fractions obtained at 50- 80% saturation was dialyzed against 10 mM Tris/HCl containing 0.15 M NaCl, and 0.2 mM EGTA (pH 7 . 9 , followed by centrifugation at 100000 x g for 90 min. A concentrated CaClz solution was added to the supernatant to bring it to 1 mM, and the solution was applied to a phenyl-Sepharose column (3 cm x 20 cm) equilibrated with the above solution containing CaC12. After washing the column with a solution containing 10 mM Hepes/ KOH, 10 mM Tris and 0.5 mM CaC12 (PH 8.0), Ca2+-binding proteins were eluted with a solution containing 10 mM Tris/HCl and 10 mM EGTA (pH 7.8) [6]. The column was then washed with a solution containing 1 0 m M Tris/HCl and 1 mM EGTA (PH 8.8). Fractions containing proteins were collected and applied to a DEAE-cellulose column (1.4 cm x 6 cm), equilibrated with a solution containing 10 mM Tris/HCl and 0.2 mM EGTA (pH 7.8). After washing the column with the same solution, non-adsorbed fractions were collected and applied to a hydroxyapatite column (1.4cmx6cm) and eluted with a linear gradient of 0350mM phosphate buffer. Eluted proteins was assayed by SDS/PAGE from this step on. Fractions containing SU-MAP24 were pooled and concentrated using a small
1196 hydroxyapatite column after dialysis against buffer A (10 mM Mes/KOH, 0.1 M NaCl. 0.2mM EGTA and 0.1 mM dithiothreitol, pH 6.8). The concentrate was then applied to a gel-filtration column (G3000SW, 0.6 cm x 60 cm, Toyo Soda) and eluted with buffer A. Purified SU-MAP24 fractions were pooled and frozen at -80°C until use. About 0.5 mg SUMAP24 was recovered from 100 ml packed eggs. Purification of tubulin from outer fibers of starfish sperm (Asterius amurensis) Outer fibers were prepared from starfish sperm as described previously [7l by Dr. Murofushi of our laboratory. Tubulin was solubilized by sonication and further purified by one cycle of a polymerization/depolymerization process, as described previously [S]. Preparation of microtubule proteins from a crude extract of sea urchin eggs using taxol Microtubule proteins were prepared using taxol, according to Vallee and Bloom [5] with some modifications. Unfertilized cells were washed once with 1 M glycerol/5 mM MgCI2 and packed eggs were homogenized in 2 vol. buffer B (100 mM Mes/KOH, 5 mM EGTA and 1 mM MgCI2 pH 6.7) followed by centrifugation at 100000 x g for 60 min. GTP and taxol were added to the supernatant to give 0.5 mM and 0.02 mM, respectively. After a 10-min incubation at 30°C to assemble microtubules. and a successive 15-min incubation on ice, the solution was overlaid onto buffer B containing GTP, taxol and 15% sucrose. After centrifugation at 25000 x g for 30 min at 2 T, the pellet fraction was recovered and washed once in buffer B containing GTP and taxol, followed by centrifugation at 30000 x g for 20 min to collect taxol-stabilized microtubules. Binding of SU-MAP24 to microtubules Sedimentation assays were performed to assess the binding of SU-MAP24 to microtubules. In a-system with no taxol, purified SU-MAP24 (2.2 pM) was mixed with purified tubulin (20pM) and incubated at 25°C in a solution containing 10 mM Mes, 3 mM MgCI2, 1 mM EGTA, 120 mM KC1 and 0.5 mM GTP @H 6.7). In a system with taxol, outer-fiber tubulin (2 pM) was mixed with various amounts of the SUMAP24 fraction in buffer A, or SU-MAP24 (2.2 pM) was mixed with various amounts of tubulin and the medium adjusted to 10 mM Mes/KOH, 2.5 mM MgCI2, 0.5 mM GTP, 1 rnM EGTA, 50 rnM NaCl and 0.02 m M taxol (pH 6.7). In both cases, after incubation at 25°C for 30 min, the mixtures were centrifuged at 200000 x g and 25 "C for 20 min.The pellet and supernatant were recovered and subjected to SDS/PAGE. To calculate the association constant between SU-MAP24 and microtubules, the amount of cosedimented SU-MAP24 protein was determined by densitometric scanning of the gel after staining. To correct the amount of proteins recovered in the pellet fraction by binding to the wall of the centrifuge type or by aggregation itself, control incubations without added tubulin were also carried out and electrophoresed after centrifugal separation. For standards, various amounts of purified SU-MAP24 were also electrophoresed in the same gel and stained, and the color of proteins was assayed by densitometric scanning.
Fig. 1. Cosedimentation assay of the EGTA and the alkaline eluate from phenyl-Sepharose. 80 p1 EGTA eluate (B-E, 0.2 mg/ml) or alkaline eluate (F -J, 0.6 mg/ml) was dialyzed against buffer A or buffer alone IK, L), and mixed with (B, C, G, H, K, L) or without (D, E. I, J ) tubulin (3.3 mg/ml) and incubated in 100 111 at 25°C for 30 min. After centrifugation, each 30+1 supernatant (B, E, G, J, K) and all of the pellet (C, D, H, I, L) were electrophoresed with 30 pI of the original solution (A, F) using a 12% acrylamide gel. In lane M. molecular mass markers were also electrophoresed [indicated with black spots; from top to bottom, myosin (200 kDa). phosphorylase b (94 kDa). bovine serum albumin (68 kDa), tubulin (55 kDa), actin (42 kDa), glyceraldehyde-3-phosphatedehydrogenase (35 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (21 kDa)]. Possible microtubule-binding proteins are indicated with arrowheads (lanes C and H) and tubulin is marked by an arrow.
Assay of assembly-promoting activity of SU-MAP24 Various amounts of SU-MAP24 were mixed with tubulin (6.1 pM) in a solution containing 10 mM Mes/KOH, 0.5 niM MgC12,0.5 mM GTP, 50 mM KCI, 50 mM NaCl and 0.6 mM EGTA (PH 6.7). The turbidity change was followed at 25 "C using a Gilford 260 spectrophotometer. Preparation of antibodies Immunization of a rabbit with 0.05 mg purified protein and affinity-purification of collected sera were performed as described previously [9]. For production of monoclonal antibodies, partially purified preparation of SU-MAP24 (the eluate of the phenyl-Sepharose column) was intraperitoneally injected into three mice with complete (first) and incomplete (second and following injections) adjuvant. About 0.01 mg SU-MAP24 was injected every time. One week after the third injection, blood was collected from the tail and titers were checked. After four injections, proteins were without adjuvant were boosted into a mouse, and three days later, the spleen was dissected and spleen cells were fused with myeloma cells as described previously [lo]. Hybridoma cells were screened by immunoblotting of the culture medium of positively growing cells against partially purified SU-MAP24. Two clones termed 5B4 and 4D3 were finally obtained and, as they showed the same reactivity, clone 4D3 was used in this experiment. Cell staining Normally fertilized eggs were cultured to metaphase after the removal of the fertilization membrane. Cells were fixed
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Fig. 2. SDS/PAGE of SU-MAP24. SDS/PAGE pattern of fractions at different purification steps. (A) Crude extract (80 pg); (B) nonadsorbed fraction of the DEAE-cellulose column (6 pg); (C) eluate from hydroxyapatite (6 pg); (D) eluate from the gel-filtration column (2 pg). In lane M, molecular mass markers werc also electrophoresed, as in Fig. 1. (b) SDS/PAGE pattern of a cosedimentation experiment without taxol. Purified SU-MAP24 (0.05 mg/ml; A) was mixed with (€3, C) or without (D, E) purified outer-fiber tubulin (20 pM) and incubated at 25°C. After centrifugation, the supernatant (B, E) and pellet (C, D) were recovered and analyzed. The purified tubulin rraction was also treated and separated into supernatant (F) and pellet (G). In this case, all of the supernatant and pellet fractions were electrophorcsed.
with 3.7% formalin in Ca2+-freesea water and were embedded in paraffin. Staining of paraffin-sectioned cells was performed as described previously [l 11 using polyclonal and mouse monoclonal antibodies against SU-MAP24, rat monoclonal antibody against tubulin (YL1/2, Sera-Lab, UK) and/ or mouse monoclonal antibody against actin (Amersham, UK). Following primary antibodies, we used fluoresceinisothiocyanate (F1TC)-labeled goat anti-(rat IgG) antibodies, FITC-labeled or rhodamine-isothiocyanate(R1TC)-labeled goat anti-(mouse IgG) antibodies and RITC-labeled goat anti(rabbit IgG) antibodies as the secondary antibodies (all affinity-purified).
Others SDS/PAGE, immunoblotting, protein determination, non-equilibrium two-dimensional electrophoresis were performed as described previously [12, 131. A gel-filtration column (G3000SW, 0.8 cm x 60 cm) was used to estimate the Stokes' radius of SU-MAP24 in buffer A. Bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonuclease A were used as internal markers. Sucrose density-gradient centrifugation was done to obtain the sedimentation coefficient of SU-MAP24, in which 5 - 15% sucrose gradient in buffer A was used for centrifugation at 180000 x g and 2 "C for 40 h [t 41. Bovine serum albumin (4.2 S), ovalbumin (3.6 S), chymotrypsinogen A (2.6 S) and ribonuclease A (1.8 S) were used as marker proteins. Phenyl-Sepharose was purchased from Pharmacia (Sweden) and affinity-purified secondary antibodies were purchased from Chemicon International (USA).
Fig. 3. Association of SU-MAP24 with tubulin during polymerization/ depolymerization process. Tubulin (16.5 pM) was polymerized in the presence of SUiMAP24 (1.1 pM) at 25°C for 40 min. After centrifugation for 20 min at 200000 x g and 25"C, the supernatant ( S l ) and pellet (Pl) fractions were recovered. Thc pellet fraction was then homogenized in buffer B and incubated for 10 min at O T , followed by centrifugation at 2°C to obtain the supernatant (S2) and pellet (P2) fractions. The supernatant fraction was incubated at 25°C aftcr the addition of MgCI2 and KCI to give 2 mM and 100 mM, respectively. After centrifugation a t 25"C, the supernatant (S3) and pellet (P3) were recovered. For SDS/PAGE, sample volumes were adjusted to give the required amounts of protein in each fraction.
RESULTS Purification of SU-MAP24 and demonstration of its presence in the sea-urchin MAP fraction prepared using taxol Cosedimentation assay of the EGTA eluate showed major proteins in this fraction have little or no binding capacity for polymerized tubulin, as described previously (Fig. 1, lanes C and D) [6]. In this fraction, two bands exist which cosediment with tubulin (Fig. 1, lane C, arrowheads). Preliminary studies showed that these proteins were eluted rather slowly from the column during the EGTA wash. We therefore washed the column with a more alkaline solution, which is known to be effective in removing bound proteins, after the EGTA wash. The same cosedimentation assay of this eluate showed the presence of many sedimentable bands in this fraction (Fig. 1, lane H, arrowheads). The tubuliii fraction used in this assay contains few other proteins (Fig. 1, lane L), which were judged to be candidates for microtubule-binding proteins. We tried to purify these proteins since this would be necessary for further characterization. In this paper, we describe the purification and characterization of one of these proteins. The SDS/PAGE pattern of fractions during purification is shown in Fig. 2A. The purified protein showed single band in SDS/ PAGE, with an apparent molecular mass of 24 k D a . The calculated protein recovery (about 1.6% of the original amount) is relatively low. This is partly due to the copurification of two polypeptides showing slow mobility in SDS/PAGE (Fig. 2A, lane C). Gel filtration is only partly effective in removing these polypeptides and nearly 90% of the protein was lost in this step. The cosedimentation assay showed its binding to reconstituted microtubules (Fig. 2 B), although the recovery of a large part of SU-MAP24 in the supernatant fraction suggested the
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Fig. 4. SDS/PAGE of purified SU-MAP24, taxol microtubule fraction and crude extract of sea urchin eggs, and their immunoblotting using a monoclonal antibody against SU-MAP24 (4D3). Each 100 pg protein of the taxol microtubule fraction (T) and crude extract (C) and 0.3 v g SU-MAP24 (24) were electrophoresed in the presence of SDS using a 12% polyacrylamide gel. Molecular mass markers were also electrophoresed in lane M (from top to bottom, 200, 94, 68, 55, 42, 35, 29 and 21 kDa) and stained with Coomassie brilliant blue (CBB). One set of proteins was transferred to a Durapore sheet after SDS/PAGE and immunoblotted using monoclonal antibodies against SU-MAP24 (A-24K).
affinity of its interaction with polymerized tubulin is not so high compared with other MAP seen in Fig. 2B (see below). We further carried out a polymerization/depolymerizationexperiment. The polymerized-microtubule fraction obtained in Fig. 2 was depolymerized in cold buffer and, after centrifugation, the soluble fraction was obtained. This fraction was then warmed and the polymerized microtubules were obtained after centrifugation. SDS/PAGE of these fractions showed the recovery of some part of SU-MAP24 in the final microtubule fraction, confirming its association with microtubules (Fig. 3). If this protein does bind microtubules in the cell, it should be recovered in the taxol-stabilized microtubule fraction prepared from the crude extract of the cell. This was confirmed with immunoblotting using specific antibodies prepared as described in Experimental Procedures. Fig. 4A shows that a protein band with the same mobility in SDS/PAGE as SUMAP24 is clearly visible both in the taxol/microtubule-protein fraction and in the crude fraction. Blotting experiments showed that these protein bands reacted with monoclonal antibodies to SU-MAP24 (Fig. 4B). A comparison of the intensities of the reacted bands showed the amount of SUMAP24 present in sea urchin eggs to be relatively high (at least 0.3% of soluble proteins). Fig. 4 also proved high specificity of the antibody. To estimate further the amount of the protein in the crude extract and to study the molecular characteristics of this protein, we analyzed the crude extract in two-dimensional gels. Equilibrium two-dimensional gels showed no corresponding spot after Coomassie-brilliant-bluestaining, and no immunoreactive band after immunoblotting (data not shown). Non-equilibrium two-dimensional gel electrophoresis to analyze basic proteins [13] was then performed, and SUMAP24 was identified as a basic protein eluted in the middle
Fig. 5. Two-dimensional SDS/PAGE using non-equilibrium isoelectric focusing. Each 100 pg protein of the crude extract (A) and taxol microtubule fraction (B) was electrophoresed according to [13]. Arrowheads show the spots of SU-MAP24, which were reacted with anti-(SU-MAP24) antibody by immunoblotting (data not shown).
of the gel (Fig. 5). Sometimes the spot was separated into several spots with different intensities (Fig. 5 B). The identification of the spots in Fig. 5 B indicated by arrows as SU-MAP24 was carried out with immunoblotting (data not shown). There were few proteins that showed the same mobility as SU-MAP24 in SDS/PAGE. The Stokes’ radius of this protein was calculated to be 2.9 nm from a gel-filtration column (G3000SW, Toyo Soda). A sedimentation coeficient of 3.3 S was obtained by sucrose density-gradient centrifugation [14]. Assuming the specific volume of this protein to be 0.75, the molecular mass was calculated to be 43 kDa, suggesting a homodimeric form of the 24-kDa subunit in solution. Cellular distribution of SU-MAP24
To study the intracellular distribution of SU-MAP24, formalin-fixed, paraffin-embedded metaphase eggs were stained with specific antibodies against SU-MAP24. Both polyclonal and monoclonal antibodies showed clear staining of the section. Fig. 6 demonstrated its localization to the mitotic apparatus. Double staining with anti-(SU-MAP24) and anti-tubulin antibodies showed its colocalization clearly. Binding of SU-MAP24 to microtubules In order to study the possible function of this protein, we next measured the ability of this protein to bind to
I199
Fig. 6. Staining of paraffin-mtiond cells with monoclonal antibody against SU-MAP24 (B, C) and with rat mono&nal anti-tubulin antibodies (A, D). FITC-labeled goat anti-(rat IgG) antibodies (A, C) and RITC-labeled goat anti-(mouse Ig) antibodies (B, D) were used as second antibodies. Little background staining and little reactivity of second antibodies with non-immunized immunoglobulins were observed in C and D. Bar, 50 vrn.
Fig.8. Cosedimentation assay of SU-MAP24 with starfish microtubules. To a constant amount of SU-MAP24 (2.2 pM), various amounts of tubulin (1,O; 2.3.3 pM, 3,6.6 pM; 4,lO pM;5 , 13.2 pM; 6, 16.5 pM) were added and polymerized. After centrifugation, the supernatant (s)and Pellet (p) were recovered and electrophoresed. as in Fig. 7.
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microtubules. For this purpose, we purified tubulin by a polymerization/depolymerization method from starfishsperm outer fibers after solubilization by sonication. Tubulin Fig. 7. Cosedimentation analysis of SU-MAP24 with microtubules prepared from this source was shown to have the same propprepared from starfish-sperm outer-fiber tubulin. To a constant amount erty as that prepared from egg extract [7]. Fig. 7 shows the binding pattern of this protein to microtubules formed in the of tubulin (2 pM), various amounts of SU-MAP24 (1.0; 2,0.33 pM; 3,0.66pM;4,0.99~1M;5,1.3pM;6,1.66pM;7,2.2pM;8,2.8pM; presence of taxol. This figure also shows the purity of the 9. 3.3 pM) were added. After tubulin polymerization in the presence tubulin fraction used in this experiment. The result showed of taxol(O.02 mM), mixtures were centrifuged and the pellet fractions a dose-dependent stoichiometric binding of SU-MAP24 to (P) were recovered. Control tubes were also incubated without tubulin microtubules. Assuming that SU-MAP24 has a molecular and treated with SU-MAP24 (P': 1,3.3 pM; 2. 2.2 pM; 3, 1.1 pM; 4, mass of 48 kDa, the Scatchard plot analysis presented an 0.55 pM). The supernatant obtained in the absence of SU-MAP24 was also electrophoresed (S) showing polymerization of tubulin and association constant of 0.9 x lo6 M - for SU-MAP24 binding little contaminating protein. Concentration of SU-MAP24 was and a stoichiometry of 1 mol SU-MAP24 dimer/6.3 mol calculated from a molecular mass of 48 kDa, estimated from its tubulin dimer. In another experiment, increasing amounts of tubulin were added to a constant amount of SU-MAP24, and physicochemical parameters.
1200 the association of SU-MAP24 to microtubules was studied with the SDSlPAGE after polymerization and centrifugation (Fig. 8). The result showed a dose-dependent recovery of SU-MAP24 in the pellet fraction, further suggesting its specific association with microtubules. Next, various amounts of SU-MAP24 were mixed with a constant arhount of tubulin to see whether or not SU-MAP24 has an ability to promote microtubule assembly. Fig. 9 shows that SU-MAP24 can promote tubulin polymerization in a dose-dependent manner. These results show that SU-MAP24 works as one of the microtubule-associated proteins of sea urchin eggs. Observations by electron microscopy, of microtubules assembled in the presence of SU-MAP24 after negative staining, revealed no clear difference from those assembled without SU-MAP24 (data not shown). DISCUSSION In this paper, we described the purification and characterization of a protein which shows microtubule binding and assembly-promoting ability in vitro. Staining of cells with the specific antibodies demonstrated its localization in the mitotic apparatus. These results satisfy some of the criteria of MAP, and we call this protein SU-MAP24 from its mobility in SDS/ PAGE. Several microtubule-associated proteins have been identified so far in eggs and oocyte. One is a high-molecular-mass MAP (XMAP of Xenopus oocyte, 21 5 kDa) reported by Gard and Kirschner [15], which regulates microtubule assembly. MAP of intermediate-molecular-mass (80 - 60 kDa) are present in echinoderm eggs. These are known to cycle with microtubules [16], localize to the mitotic spindle and bind hexagonally to microtubules [4]. These proteins seems to be identical, although a precise comparison of these proteins has not yet been performed. The presence of a 62-kDa protein which regulates microtubule polymerization through phosphorylation was also reported [17]. SU-MAP24 described here is different from these proteins in its molecular mass. Furthermore, Fig. 4 presents evidence that SU-MAP24 is not a breakdown product of a protein of higher molecular mass. Little attention has been paid to low-molecular-mass MAP so far, except for the identification of MAP using monoclonal antibodies by Vallee and Bloom [ S ] . One- and two-dimensional SDS/PAGE analysis of the crude extract showed few protein bands in the low-molecular-mass range, and this enabled us to estimate the cellular content of this protein by densitometric analysis of the gel: it was about 1.6% of the soluble protein. This means the concentration of SU-MAP24 within the cell is over 10pM. On the other hand, Raff and Kaumeyer [19] estimated the cellular tubulin content of echinoderm eggs to be 5.7 pM. A rough calculation using these values, combined with the association constant and binding ratio obtained in
Fig. 7, showed that over 60% of the binding sites for the SU-MAP24 on microtubules is occupied by this protein within the cell. The localization of this protein within the mitotic apparatus (Fig. 6) coincides well with this estimation, although we have not yet observed direct binding of SU-MAP24 to microtubules within the cell. Our preliminary experiments with microinjection of our monoclonal antibodies into sea urchin eggs resulted in inhibition of cell division. Further analysis of the effect of this protein on microtubule dynamics is important for understanding the mechanism of the regulation of microtubule networks in the sea urchin egg. We are grateful to Dr. Hiromu Murofushi in our laboratory, for supplying the outer-fiber fraction of starfish sperm. This work was partly supported by the Narishige Zoological Science Award.
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