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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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Identification of human RanBPM. (A) Northern analysis. Poly (A)+ RNA (2 μg/lane) extracted from HeLa cells was  electrophoresed in 1.4% agarose gel and analyzed, using as a  probe the 32P-labeled RanBPM cDNA fragment as described in  Materials and Methods. Arrow, 3.1 kb of RanBPM mRNA. RNA  size markers are given in kilobases (kb). (B) Amino acid sequence. The amino acid sequence of human RanRPM was deduced from the nucleotide sequence of human RanBPM cDNA  deposited under GenBank/EMBL/DDBJ accession number  AB008515. Solid black arrows, amino acid sequences deduced  from the deposited nucleotide sequences of mouse cDNA fragments, the GenBank/EMBL/DDBJ accession numbers of which  are gbu-AA549267, gb-AA125423, and gb-AA427170, respectively. Dotted arrows, amino acid sequence deduced from cloned  hamster RanBPM cDNA, the nucleotide sequence of which has  been deposited under GenBank/EMBL/DDBJ accession number  AB015640. Shaded block, position of peptides used to raise the  anti-RanBPM antibodies. (C) Immunoblotting analysis. Total  cell extract of HeLa cells (lanes 1 and 3) and MRC5 cells (lanes 2  and 4) was analyzed by 12% SDS-PAGE and then transferred  to a PVDF membrane. The membrane was probed with affinity-purified anti-RanBPM antibodies in the absence (lanes 1 and  2) or the presence (lanes 3 and 4) of the peptide used for the  immunization.
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Figure 1: Identification of human RanBPM. (A) Northern analysis. Poly (A)+ RNA (2 μg/lane) extracted from HeLa cells was electrophoresed in 1.4% agarose gel and analyzed, using as a probe the 32P-labeled RanBPM cDNA fragment as described in Materials and Methods. Arrow, 3.1 kb of RanBPM mRNA. RNA size markers are given in kilobases (kb). (B) Amino acid sequence. The amino acid sequence of human RanRPM was deduced from the nucleotide sequence of human RanBPM cDNA deposited under GenBank/EMBL/DDBJ accession number AB008515. Solid black arrows, amino acid sequences deduced from the deposited nucleotide sequences of mouse cDNA fragments, the GenBank/EMBL/DDBJ accession numbers of which are gbu-AA549267, gb-AA125423, and gb-AA427170, respectively. Dotted arrows, amino acid sequence deduced from cloned hamster RanBPM cDNA, the nucleotide sequence of which has been deposited under GenBank/EMBL/DDBJ accession number AB015640. Shaded block, position of peptides used to raise the anti-RanBPM antibodies. (C) Immunoblotting analysis. Total cell extract of HeLa cells (lanes 1 and 3) and MRC5 cells (lanes 2 and 4) was analyzed by 12% SDS-PAGE and then transferred to a PVDF membrane. The membrane was probed with affinity-purified anti-RanBPM antibodies in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of the peptide used for the immunization.

Mentions: The human cDNA clone B8 was isolated by the two-hybrid method using human Ran as a bait (Yokoyama et al., 1995). Northern analysis using the B8 clone as a probe revealed the presence of a 3.1-kb mRNA in human HeLa cells (Fig. 1 A). Subsequently, the human cDNA library was screened using the cDNA clone B8 as the probe. Finally, the cDNA clone of 2.8 kb, whose nucleotide sequence has been deposited as GenBank/EMBL/DDBJ accession number AB008515, was isolated. Since there was no stop codon at the site upstream from the first methionine, we repeatedly carried out 5′ RACE using as primers the nucleotides localized either near the 5′ end or the middle of the RanBPM cDNA. But there was no cDNA clones extended from the 5′ end of RanBPM cDNA. We concluded the ORF of RanBPM (Fig. 1 B) to consist of 500 amino acid residues, encoding a protein with a calculated molecular mass of 55,079 Da.


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)

Identification of human RanBPM. (A) Northern analysis. Poly (A)+ RNA (2 μg/lane) extracted from HeLa cells was  electrophoresed in 1.4% agarose gel and analyzed, using as a  probe the 32P-labeled RanBPM cDNA fragment as described in  Materials and Methods. Arrow, 3.1 kb of RanBPM mRNA. RNA  size markers are given in kilobases (kb). (B) Amino acid sequence. The amino acid sequence of human RanRPM was deduced from the nucleotide sequence of human RanBPM cDNA  deposited under GenBank/EMBL/DDBJ accession number  AB008515. Solid black arrows, amino acid sequences deduced  from the deposited nucleotide sequences of mouse cDNA fragments, the GenBank/EMBL/DDBJ accession numbers of which  are gbu-AA549267, gb-AA125423, and gb-AA427170, respectively. Dotted arrows, amino acid sequence deduced from cloned  hamster RanBPM cDNA, the nucleotide sequence of which has  been deposited under GenBank/EMBL/DDBJ accession number  AB015640. Shaded block, position of peptides used to raise the  anti-RanBPM antibodies. (C) Immunoblotting analysis. Total  cell extract of HeLa cells (lanes 1 and 3) and MRC5 cells (lanes 2  and 4) was analyzed by 12% SDS-PAGE and then transferred  to a PVDF membrane. The membrane was probed with affinity-purified anti-RanBPM antibodies in the absence (lanes 1 and  2) or the presence (lanes 3 and 4) of the peptide used for the  immunization.
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Figure 1: Identification of human RanBPM. (A) Northern analysis. Poly (A)+ RNA (2 μg/lane) extracted from HeLa cells was electrophoresed in 1.4% agarose gel and analyzed, using as a probe the 32P-labeled RanBPM cDNA fragment as described in Materials and Methods. Arrow, 3.1 kb of RanBPM mRNA. RNA size markers are given in kilobases (kb). (B) Amino acid sequence. The amino acid sequence of human RanRPM was deduced from the nucleotide sequence of human RanBPM cDNA deposited under GenBank/EMBL/DDBJ accession number AB008515. Solid black arrows, amino acid sequences deduced from the deposited nucleotide sequences of mouse cDNA fragments, the GenBank/EMBL/DDBJ accession numbers of which are gbu-AA549267, gb-AA125423, and gb-AA427170, respectively. Dotted arrows, amino acid sequence deduced from cloned hamster RanBPM cDNA, the nucleotide sequence of which has been deposited under GenBank/EMBL/DDBJ accession number AB015640. Shaded block, position of peptides used to raise the anti-RanBPM antibodies. (C) Immunoblotting analysis. Total cell extract of HeLa cells (lanes 1 and 3) and MRC5 cells (lanes 2 and 4) was analyzed by 12% SDS-PAGE and then transferred to a PVDF membrane. The membrane was probed with affinity-purified anti-RanBPM antibodies in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of the peptide used for the immunization.
Mentions: The human cDNA clone B8 was isolated by the two-hybrid method using human Ran as a bait (Yokoyama et al., 1995). Northern analysis using the B8 clone as a probe revealed the presence of a 3.1-kb mRNA in human HeLa cells (Fig. 1 A). Subsequently, the human cDNA library was screened using the cDNA clone B8 as the probe. Finally, the cDNA clone of 2.8 kb, whose nucleotide sequence has been deposited as GenBank/EMBL/DDBJ accession number AB008515, was isolated. Since there was no stop codon at the site upstream from the first methionine, we repeatedly carried out 5′ RACE using as primers the nucleotides localized either near the 5′ end or the middle of the RanBPM cDNA. But there was no cDNA clones extended from the 5′ end of RanBPM cDNA. We concluded the ORF of RanBPM (Fig. 1 B) to consist of 500 amino acid residues, encoding a protein with a calculated molecular mass of 55,079 Da.

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