When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.
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.
Nakamura
M
M
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.
Masuda
H
H
Horii
J
J
Kuma
K i
Ki
Yokoyama
N
N
Ohba
T
T
Nishitani
H
H
Miyata
T
T
Tanaka
M
M
Nishimoto
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D018385:Centrosome / Q000737:chemistry* / Q000187:drug effects; D006224:Cricetinae; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008870:Microtubules / Q000737:chemistry*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics; D020931:ran GTP-Binding Protein
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D009687:Nuclear Proteins; C116005:Ran binding protein 9; D014404:Tubulin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein