Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome.
Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome.
复制标题
DOI：
10.1083/jcb.141.1.163
复制
发表时间：
1998-04-06
期刊：
影响因子：
--
通讯作者：
Doxsey SJ
中科院分区：
文献类型：
--
作者：
Dictenberg JB;Zimmerman W;Sparks CA;Young A;Vidair C;Zheng Y;Carrington W;Fay FS;Doxsey SJ
周蛋白和γ-微管蛋白是在微管核形成和组织过程中起作用的完整的中心体蛋白。在这项研究中，我们研究了这些蛋白质在细胞质和中心体之间的关系。经蔗糖梯度沉淀、凝胶过滤和免疫共沉淀分析表明，从非洲爪蛙卵中提取的蛋白质是一个大的复合体的一部分。周中心蛋白-γ-微管蛋白复合体不同于先前描述的γ-微管蛋白环复合体(γ-TURC)，因为纯化的γ-TURC组分不包含可检测到的周中心蛋白。荧光共振能量转移显示，当在中心体组装时，这两种蛋白质仍然非常接近。用一种改进的免疫荧光方法确定了这些蛋白质中心体相关部分的三维结构。这一分析揭示了一种新的网状晶格，它从哺乳动物到两栖动物都是保守的，并且是独立于中心粒组织的。在细胞周期中，晶格发生了巨大的变化，从G1期扩大到有丝分裂，然后随着细胞退出有丝分裂而迅速解体。在被标记以检测中心体和有核微管的细胞中，晶格元素似乎接触有核微管的负端。我们的结果表明，周中心素和γ-微管蛋白组装成一个独特的中心体晶格，代表了中心体上微管成核位置的更高级别的组织。
Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.