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, centrosomin, and gamma-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 gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (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 gammaTuRC is required for microtubule nucleation at the centrosome.
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448, USA.
M
M
Moritz
Y
Y
Zheng
B M
BM
Alberts
K
K
Oegema
D000818:Animals; D018797:Cell Cycle Proteins; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D007452:Iodates / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D017680:Potassium Compounds / Q000494:pharmacology; D012492:Salts / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D014981:Xenopus
C107964:CP190 protein, Drosophila; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D007452:Iodates; C098388:Map60 protein, Drosophila; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D017680:Potassium Compounds; D012492:Salts; D014404:Tubulin; C039693:potassium iodate