Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.
Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.
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DOI：
10.1083/jcb.142.3.775
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发表时间：
1998-08-10
期刊：
影响因子：
--
通讯作者：
Oegema K
中科院分区：
文献类型：
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作者：
Moritz M;Zheng Y;Alberts BM;Oegema K
用2 M KI提取分离的果蝇中心体产生缺乏中心体蛋白CP 190、CP 60、中心体蛋白和γ-微管蛋白的耐盐支架。为了澄清这些蛋白质在微管成核的中心体的作用，并确定额外的中心体成核所需的组件，我们已经开发了一种体外互补测定中心体功能。当这些无活性的、盐剥离的中心体支架补充有果蝇胚胎提取物的可溶性部分时，中心体星形体形成被重建。CP 60和CP 190可以从该提取物中去除而没有影响，而去除γ-微管蛋白破坏了互补活性。与这些结果一致，我们没有发现这三种蛋白质一起形成复合物的证据。相反，γ-微管蛋白存在于两种不同的蛋白质复合物中，分子量分别为240，000和13，000，000 D。较大的复合物类似于非洲爪蟾γ-微管蛋白环复合物（γTuRC）（Zheng，Y.，M.L.王，B。Alberts和T.米奇森1995.自然378：578-583），是必要的，但不足以补充。需要在提取物中发现一个额外的因素。这些结果提供了第一个证据，表明γTuRC是微管在中心体成核所必需的。
Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-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 γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-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 γ-tubulin ring complex (γTuRC) (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 γTuRC is required for microtubule nucleation at the centrosome.