J. Cell Biol., Volume 142, Number 3, August 10, 1998 775-786

Recruitment of the -Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

Michelle Moritz,^* Yixian Zheng, Bruce M. Alberts,^§ and Karen Oegema

^* Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448;  Department of Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210; ^§ National Academy of Sciences, Washington, DC 20418; and  Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and -tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the -tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, -tubulin is found in two distinct protein complexes of 240,000 and ~3,000,000 D. The larger complex, which is analogous to the Xenopus -tubulin ring complex (TuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the TuRC is required for microtubule nucleation at the centrosome.

centrosome;  -tubulin;  microtubule nucleation;  microtubule-organizing center;  cytoskeleton

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