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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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Interaction of RanBPM with GTP-Ran. pACTII carrying G19V- or T24N-Ran and a vacant vector were introduced into  the yeast Y190 strain (MATα gal4 gal80 ade2 his3 trp1 leu2  URA3::GAL1-lacZ LYS2::GAL1-HIS3) (Harper et al., 1993)  bearing pAS404-RanBPM. Transformants were selected on synthetic medium lacking tryptophan and leucine, and plated on synthetic medium either lacking histidine, tryptophan and leucine,  but in the presence of 3-aminotriazole (10 mM) (+), or lacking  tryptophan, leucine and 3-aminotriazole (−).
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Figure 8: Interaction of RanBPM with GTP-Ran. pACTII carrying G19V- or T24N-Ran and a vacant vector were introduced into the yeast Y190 strain (MATα gal4 gal80 ade2 his3 trp1 leu2 URA3::GAL1-lacZ LYS2::GAL1-HIS3) (Harper et al., 1993) bearing pAS404-RanBPM. Transformants were selected on synthetic medium lacking tryptophan and leucine, and plated on synthetic medium either lacking histidine, tryptophan and leucine, but in the presence of 3-aminotriazole (10 mM) (+), or lacking tryptophan, leucine and 3-aminotriazole (−).

Mentions: The cDNA of G19V-Ran and T24N-Ran, both of which were fused in frame with the GAL4-activation domain of pACT (Durfee et al., 1993), were introduced into cultures of the strain Y190 [pAS404-RanBPM]. Transformants were selected in synthetic medium lacking leucine and tryptophan, and plated on synthetic medium either lacking histidine, tryptophan, and leucine but containing 10 mM of 3-aminotriazole (+) or lacking tryptophan, leucine and 3-aminotriazole (−). In the presence of 3-aminotriazole, the strain Y190 [pAS404-RanBPM, pACT-G19VRan] papillated, whereas the strain Y190 [pAS404-RanBPM, pACT-T24NRan] did not (Fig. 8). This result indicates that RanBPM specifically interacts with GTP-Ran.


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)

Interaction of RanBPM with GTP-Ran. pACTII carrying G19V- or T24N-Ran and a vacant vector were introduced into  the yeast Y190 strain (MATα gal4 gal80 ade2 his3 trp1 leu2  URA3::GAL1-lacZ LYS2::GAL1-HIS3) (Harper et al., 1993)  bearing pAS404-RanBPM. Transformants were selected on synthetic medium lacking tryptophan and leucine, and plated on synthetic medium either lacking histidine, tryptophan and leucine,  but in the presence of 3-aminotriazole (10 mM) (+), or lacking  tryptophan, leucine and 3-aminotriazole (−).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Interaction of RanBPM with GTP-Ran. pACTII carrying G19V- or T24N-Ran and a vacant vector were introduced into the yeast Y190 strain (MATα gal4 gal80 ade2 his3 trp1 leu2 URA3::GAL1-lacZ LYS2::GAL1-HIS3) (Harper et al., 1993) bearing pAS404-RanBPM. Transformants were selected on synthetic medium lacking tryptophan and leucine, and plated on synthetic medium either lacking histidine, tryptophan and leucine, but in the presence of 3-aminotriazole (10 mM) (+), or lacking tryptophan, leucine and 3-aminotriazole (−).
Mentions: The cDNA of G19V-Ran and T24N-Ran, both of which were fused in frame with the GAL4-activation domain of pACT (Durfee et al., 1993), were introduced into cultures of the strain Y190 [pAS404-RanBPM]. Transformants were selected in synthetic medium lacking leucine and tryptophan, and plated on synthetic medium either lacking histidine, tryptophan, and leucine but containing 10 mM of 3-aminotriazole (+) or lacking tryptophan, leucine and 3-aminotriazole (−). In the presence of 3-aminotriazole, the strain Y190 [pAS404-RanBPM, pACT-G19VRan] papillated, whereas the strain Y190 [pAS404-RanBPM, pACT-T24NRan] did not (Fig. 8). This result indicates that RanBPM specifically interacts with GTP-Ran.

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