Original Article

Characterization and Reconstitution of Drosophila -Tubulin Ring Complex Subunits

The -tubulin ring complex (TuRC) is important for microtubule nucleation from the centrosome. In addition to -tubulin, the Drosophila 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 -tubulin complex (TuSC) and the TuRC, while the remaining subunits are found only in the TuRC. To study TuRC assembly and function, we first reconstituted TuSC using the baculovirus expression system. Using the reconstituted TuSC, we showed for the first time that this subcomplex of the TuRC has microtubule binding and capping activities. Next, we characterized two new TuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known 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 TuSC. However, this interaction is insufficient for TuRC assembly.

Key Words: centrosome, microtubule, -tubulin, grip, Drosophila

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