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

doi:10.1083/jcb.151.7.1513

Published online 27 December 2000. doi:10.1083/jcb.151.7.1513

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© The Rockefeller University Press, 0021-9525/2000/12/1513/ $5.00
The Journal of Cell Biology, Volume 151, Number 7, December 25, 2000 1513-1524

Original Article

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

Ruwanthi N. Gunawardane^a,b, Ona C. Martin^a, Kan Cao^a,b, Lijun Zhang^a, Kimberly Dej^a, Akihiro Iwamatsu^c, and Yixian Zheng^a,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 microtubulenucleation from the centrosome. In addition to ${gamma}$ -tubulin, theDrosophila ${gamma}$ 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 presentin both the small ${gamma}$ -tubulin complex ( ${gamma}$ TuSC) and the ${gamma}$ TuRC, whilethe remaining subunits are found only in the ${gamma}$ TuRC. To study ${gamma}$ TuRC assembly and function, we first reconstituted ${gamma}$ TuSC usingthe baculovirus expression system. Using the reconstituted ${gamma}$ TuSC,we showed for the first time that this subcomplex of the ${gamma}$ TuRChas microtubule binding and capping activities. Next, we characterizedtwo new ${gamma}$ TuRC subunits, Dgrips128 and 163, and showed that theyare centrosomal proteins. Sequence comparisons among all known ${gamma}$ TuRC subunits revealed two novel sequence motifs, which we namedgrip motifs 1 and 2. We found that Dgrips128 and 163 can eachinteract with ${gamma}$ TuSC. However, this interaction is insufficientfor ${gamma}$ TuRC assembly.

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

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Original Article

Characterization and Reconstitution of Drosophila -Tubulin Ring Complex Subunits

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