Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
PLoS One. 2009 Sep 29;4(9):e7217. doi: 10.1371/journal.pone.0007217.
Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.
METHODOLOGY/PRINCIPAL FINDINGS: Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.
CONCLUSIONS/SIGNIFICANCE: RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.
Ras 超家族中的 Ras 蛋白是最大的亚家族，主要参与囊泡靶向。Rab 家族功能的全部范围尚未被探索。已知有几种不同的 Rab 样蛋白，但只有少数被表征。在布氏锥虫中，有十六个 Rab 基因，但 RabX1、RabX2 和 RabX3 在典型序列区域内是有差异的。在已知的情况下，当可以可靠地建立同源关系时，锥虫 Rab 的功能广泛保守，但 RabX1、X2 和 X3 的特定功能尚未确定。RabX1 和 RabX2 通过串联重复产生，亚细胞定位将 RabX1 置于内质网，而 RabX2 位于高尔基体复合物，表明存在不同的功能。我们希望确定 RabX1 和 RabX2 是否参与囊泡运输或其他细胞过程。
方法/主要发现：使用比较基因组学，我们发现 RabX1 和 RabX2 仅限于动基体生物。基因敲除表明 RabX1 和 RabX2 不是必需的。同时 RNAi 敲低 RabX1 和 RabX2，虽然部分敲低，但也不是致命的，可能表明功能不冗余，这与蛋白质的不同位置一致。敲除细胞系的分析出人意料地没有发现胞吐、胞吞或多个内体系统标志物的形态或位置有缺陷，这表明 RabX1 和 RabX2 都没有在细胞内运输中发挥主要作用。然而，很明显 RabX1 和 RabX2 敲除细胞在果蝇体内的存活率有所提高。
结论/意义：尽管每个基因产物定位于高度特异的内膜系统隔室，但表明动基体亚家族的两个成员 RabX1 和 RabX2 在细胞内运输中没有主要的可检测作用。这些数据扩展了 Rab 蛋白在锥虫中的功能范围，包括在原生动物分化相关过程中非典型的作用。
