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© The Rockefeller University Press, 0021-9525/1998//1041 $5.00
The Journal of Cell Biology, Volume 143, Number 4, , 1998 1041-1052

Regular Articles

When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to {gamma}-Tubulin



Masafumi Nakamura*,{ddagger}, Hirohisa Masuda§,**, Johji Horii*, Kei-ichi Kuma||, Nobuhiko Yokoyama*,¶, Tomoyuki Ohba*, Hideo Nishitani*, Takashi Miyata||, Masao Tanaka{ddagger}, and Takeharu Nishimoto*

* Department of Molecular Biology, {ddagger} Department of Surgery, and Department of Neurosurgery, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan; § Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01, Japan; ** Inheritance and Variation Group, PRESTO, Japan Science and Technology Corporation, Kyoto 619-0237, Japan; and || Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

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 GTP{gamma}S-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.

Key Words: centrosome • {gamma}-tubulin • Ran • RanBPM • YGL227w

Abbreviations used in this paper: {gamma}-TuRC, {gamma}-tubulin ring complex; ORF, open reading frame; PCM, pericentriolar material; RACE, rapid amplification of cDNA ends; SPB, spindle pole body; ts, temperature sensitive.


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