RECRUITMENT OF THE GAMMA-TUBULIN RING COMPLEX TO DROSOPHILA SALT-STRIPPED CENTROSOME SCAFFOLDS

Extracting isolated Drosophila centrosomes with 2 M KI generates salt- resistant scaffolds that lack the centrosomal proteins CP190, CP60, ce ntrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional cent rosome components required for nucleation, we have developed an in vit ro complementation assay for centrosome function. Centrosome aster for mation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila emb ryo extract. The CP60 and CP190 can be removed from this extract witho ut effect, whereas removing the gamma-tubulin destroys the complementi ng activity. Consistent with these results, we find no evidence that t hese three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and similar to 3,0 00,000 D. The larger complex, which is analogous to the Xenopus gamma- tubulin ring complex (gamma TuRC) (Zheng, Y., M.L. Wong, B. Alberts, a nd T. Mitchison. 1995. Nature. 378:578-583), is necessary but not suff icient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gamma T uRC is required for microtubule nucleation at the centrosome.