Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
PLoS One. 2012;7(1):e29919. doi: 10.1371/journal.pone.0029919. Epub 2012 Jan 3.
γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity.
γ-微管蛋白是微管核形成的关键蛋白。γ-微管蛋白基因在进化过程中发生了多次复制，在哺乳动物中，γ-微管蛋白基因编码的蛋白质具有约 97%的序列同一性。先前对 Tubg1 和 Tubg2 敲除小鼠的分析表明，γ-微管蛋白并非功能等同。Tubg1 敲除小鼠在胚泡阶段死亡，而 Tubg2 敲除小鼠发育正常且具有生育能力。有人提出，γ-微管蛋白 1 代表普遍存在的 γ-微管蛋白，而 γ-微管蛋白 2 可能具有一些特定的功能，并且不能替代胚泡中 γ-微管蛋白 1 的缺失。γ-微管蛋白之间建议的功能差异的分子基础仍然未知。在这里，我们表明外源性 γ-微管蛋白 2 靶向中心体并与 γ-微管蛋白复合物蛋白 2 和 4 相互作用。U2OS 细胞中 γ-微管蛋白 1 的 RNAi 耗尽导致微管核形成受损和中期停滞。外源性鼠或人 γ-微管蛋白 2 的表达可恢复 γ-微管蛋白 1 耗尽细胞的野生型表型。此外，我们使用 RT-qPCR 和 2D-PAGE 分别在 mRNA 和蛋白质水平上显示，与 Tubg1 相反，Tubg2 在小鼠胚泡中的表达显著降低。这表明由于其极低的量，γ-微管蛋白 2 不能挽救敲除胚泡中 γ-微管蛋白 1 的缺失。综合数据表明，γ-微管蛋白 2 能够核形成微管并替代 γ-微管蛋白 1。我们提出，哺乳动物 γ-微管蛋白在核形成活性方面具有功能冗余性。
