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Published online 25 December 2000. doi:10.1083/jcb.151.7.1513
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© The Rockefeller University Press, 0021-9525/2000//1513 $5.00
The Journal of Cell Biology, Volume 151, Number 7, , 2000 1513-1524

Original Article

Characterization and Reconstitution of Drosophila {gamma}-Tubulin Ring Complex Subunits



Ruwanthi N. Gunawardanea,b, Ona C. Martina, Kan Caoa,b, Lijun Zhanga, Kimberly Deja, Akihiro Iwamatsuc, and Yixian Zhenga,b

a Howard Hughes Medical Institute, Carnegie Institution of Washington, Baltimore, Maryland 21210
b Biology Department, Johns Hopkins University, Baltimore, Maryland 21218
c Central Laboratories for Key Technology, Kirin Brewery Company, Limited, Yokohama, Japan

The {gamma}-tubulin ring complex ({gamma}TuRC) is important for microtubule nucleation from the centrosome. In addition to {gamma}-tubulin, the Drosophila {gamma}TuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small {gamma}-tubulin complex ({gamma}TuSC) and the {gamma}TuRC, while the remaining subunits are found only in the {gamma}TuRC. To study {gamma}TuRC assembly and function, we first reconstituted {gamma}TuSC using the baculovirus expression system. Using the reconstituted {gamma}TuSC, we showed for the first time that this subcomplex of the {gamma}TuRC has microtubule binding and capping activities. Next, we characterized two new {gamma}TuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known {gamma}TuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with {gamma}TuSC. However, this interaction is insufficient for {gamma}TuRC assembly.

Key Words: centrosome • microtubule • {gamma}-tubulin • grip • Drosophila

© 2000 The Rockefeller University Press

Yixian Zheng, Howard Hughes Medical Institution, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210. Tel.: (410) 554-1232. Fax: (410) 243-6311. E-mail: zheng{at}ciwemb.edu

Dr. Kimberly Dej's present address is Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

Abbreviations used in this paper: β-ME, β-mercaptoethanol; Dgrip, Drosophila gamma ring protein; EGS, ethylene glycol bis-succinimidyl succinate; {gamma}TuRC, {gamma}-tubulin ring complex; {gamma}TuSC, {gamma}-tubulin small complex; MT, microtubule.


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