Characterization and reconstitution of Drosophila γ-tubulin ring complex subunits

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 Drips128 and 163 can each interact with gamma TuSC. However, this interaction is insufficient for gamma TuRC assembly.