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When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

Nakamura M, Masuda H, Horii J, Kuma Ki, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M, Nishimoto T - J. Cell Biol. (1998)

Bottom Line: Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.

ABSTRACT
A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

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Cosedimentation of RanBPM with the  centrosomes. The centrosome extracts prepared  from HeLa cells (2.5 × 107 cells) which had been  incubated in the presence of nocodazole (10 μg/ ml) and cytochalasin B (5 μg/ml) for 2 h, as described in Materials and Methods, were set into a  tube for SRP28-SA rotor (Hitachi) with 3.5 ml of  60% and 3.5 ml of 40% sucrose to be fractionated by discontinuous sucrose-density gradient  centrifugation at 14,500 rpm for 1 h. Fractions  collected from the bottom upwards were analyzed for the ability to form microtubule asters  (A), and for the presence of RanBPM and  γ-tubulin by immunoblotting using the antibodies to RanBPM and the mAb to γ-tubulin (B).  On the horizontal axis, the fraction numbers  from the bottom to the top are shown.
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Figure 5: Cosedimentation of RanBPM with the centrosomes. The centrosome extracts prepared from HeLa cells (2.5 × 107 cells) which had been incubated in the presence of nocodazole (10 μg/ ml) and cytochalasin B (5 μg/ml) for 2 h, as described in Materials and Methods, were set into a tube for SRP28-SA rotor (Hitachi) with 3.5 ml of 60% and 3.5 ml of 40% sucrose to be fractionated by discontinuous sucrose-density gradient centrifugation at 14,500 rpm for 1 h. Fractions collected from the bottom upwards were analyzed for the ability to form microtubule asters (A), and for the presence of RanBPM and γ-tubulin by immunoblotting using the antibodies to RanBPM and the mAb to γ-tubulin (B). On the horizontal axis, the fraction numbers from the bottom to the top are shown.

Mentions: We then determined the relationship between RanBPM and microtubule nucleation, using the isolated centrosome. To achieve this, the centrosome extracts were prepared from HeLa cells by sucrose-density gradient centrifugation as described (Mitchison and Kirschner, 1984; Bornes et al., 1987). Fractions containing the centrosomes were determined by microtubule nucleation ability (Fig. 5 A). Immunoblotting analysis revealed that both RanBPM and γ-tubulin were cosedimented with the centrosome fractions (Fig. 5 B). The protein bands higher than 55 kD which were fractionated into the top fractions were recognized by the affinity-purified anti-RanBPM antibodies. The major band of these corresponds to the band of 57 kD recognized in the total extract of HeLa cells (Fig. 1 C). We do not know whether they are modified forms of RanBPM or proteins cross-reactive to the affinity-purified anti-RanBPM antibodies as mentioned above. These proteins were concentrated into the top fractions, suggesting that they were localized in the cytoplasm. Similarly, a majority of γ-tubulin was fractionated into the top fraction, being consistent with the previous report that the majority of γ-tubulin is localized in the cytoplasm (Stearns and Kirschner, 1994).


When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

Nakamura M, Masuda H, Horii J, Kuma Ki, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M, Nishimoto T - J. Cell Biol. (1998)

Cosedimentation of RanBPM with the  centrosomes. The centrosome extracts prepared  from HeLa cells (2.5 × 107 cells) which had been  incubated in the presence of nocodazole (10 μg/ ml) and cytochalasin B (5 μg/ml) for 2 h, as described in Materials and Methods, were set into a  tube for SRP28-SA rotor (Hitachi) with 3.5 ml of  60% and 3.5 ml of 40% sucrose to be fractionated by discontinuous sucrose-density gradient  centrifugation at 14,500 rpm for 1 h. Fractions  collected from the bottom upwards were analyzed for the ability to form microtubule asters  (A), and for the presence of RanBPM and  γ-tubulin by immunoblotting using the antibodies to RanBPM and the mAb to γ-tubulin (B).  On the horizontal axis, the fraction numbers  from the bottom to the top are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132962&req=5

Figure 5: Cosedimentation of RanBPM with the centrosomes. The centrosome extracts prepared from HeLa cells (2.5 × 107 cells) which had been incubated in the presence of nocodazole (10 μg/ ml) and cytochalasin B (5 μg/ml) for 2 h, as described in Materials and Methods, were set into a tube for SRP28-SA rotor (Hitachi) with 3.5 ml of 60% and 3.5 ml of 40% sucrose to be fractionated by discontinuous sucrose-density gradient centrifugation at 14,500 rpm for 1 h. Fractions collected from the bottom upwards were analyzed for the ability to form microtubule asters (A), and for the presence of RanBPM and γ-tubulin by immunoblotting using the antibodies to RanBPM and the mAb to γ-tubulin (B). On the horizontal axis, the fraction numbers from the bottom to the top are shown.
Mentions: We then determined the relationship between RanBPM and microtubule nucleation, using the isolated centrosome. To achieve this, the centrosome extracts were prepared from HeLa cells by sucrose-density gradient centrifugation as described (Mitchison and Kirschner, 1984; Bornes et al., 1987). Fractions containing the centrosomes were determined by microtubule nucleation ability (Fig. 5 A). Immunoblotting analysis revealed that both RanBPM and γ-tubulin were cosedimented with the centrosome fractions (Fig. 5 B). The protein bands higher than 55 kD which were fractionated into the top fractions were recognized by the affinity-purified anti-RanBPM antibodies. The major band of these corresponds to the band of 57 kD recognized in the total extract of HeLa cells (Fig. 1 C). We do not know whether they are modified forms of RanBPM or proteins cross-reactive to the affinity-purified anti-RanBPM antibodies as mentioned above. These proteins were concentrated into the top fractions, suggesting that they were localized in the cytoplasm. Similarly, a majority of γ-tubulin was fractionated into the top fraction, being consistent with the previous report that the majority of γ-tubulin is localized in the cytoplasm (Stearns and Kirschner, 1994).

Bottom Line: Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.

ABSTRACT
A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Show MeSH
Related in: MedlinePlus