Rab4 is an essential regulator of lysosomal trafficking in trypanosomes
Rab4 is an essential regulator of lysosomal trafficking in trypanosomes
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DOI：
10.1074/jbc.m407271200
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发表时间：
2004-10-22
影响因子：
4.8
通讯作者：
Field, MC
中科院分区：
文献类型：
--
作者：
Hall, BS;Pal, A;Field, MC
表面蛋白的快速内吞作用和再循环是大多数有核真核细胞所共有的重要过程。最典型的膜回收途径是由小型 GTP 酶 Rab4 和 Rab11 介导的，但这些途径所发挥的确切作用尚未完全阐明。原生动物布氏锥虫具有高度发达的内吞系统，与后生动物中的内吞系统相似，尽管膜周转率加快。我们利用这种生物体，通过 RNA 干扰、显微镜检查和定量运输分析的组合来研究 Rab4 锥虫直系同源物 (TbRAB4) 的功能。 RNA 干扰介导的 TbRAB4 表达抑制抑制锥虫的生长，而不影响受体介导的内吞作用或配体再循环。超微结构分析表明膜转运事件存在重大缺陷。在缺乏 TbRAB4 蛋白的细胞中，荧光右旋糖酐（一种液相标记物）的积累被阻断。由于大多数液相标记物被转运至 T. brucei 的溶酶体，因此研究了 TbRAB4 RNA 干扰对溶酶体功能的影响。通过免疫荧光，主要溶酶体蛋白 p67 逐渐分散在缺乏 TbRAB4 蛋白的细胞中。脉冲追踪分析表明，p67 的初始蛋白水解切割和聚糖加工不受影响，但细胞未能积累与溶酶体相关的后期 p67 蛋白水解产物。为了证实 TbRAB4 在溶酶体运输中的作用，表达了一种组成型活性突变体 TbRAB4(QL)。 TbRAB4(QL)与含有p67的增大的多泡体密切相关。此外，与亲本系相比，表达 TbRAB4(QL) 的细胞显示出增加的液相摄取。总而言之，这些数据表明 TbRAB4 参与调节 T. brucei 中溶酶体的液相运输，但不参与受体介导的内吞作用或再循环。这些数据对 Rab4 在其他细胞系统中的作用具有影响。
Rapid endocytosis and recycling of surface proteins are important processes common to most nucleated eukaryotic cells. The best characterized membrane recycling routes are mediated by the small GTPases Rab4 and Rab11, but the precise roles that these pathways play have not been fully elucidated. The protozoan Trypanosoma brucei has a highly developed endocytic system that is similar to that found in metazoans, albeit with an accelerated rate of membrane turnover. We have used this organism to investigate the function of the trypanosome orthologue of Rab4 (TbRAB4) by a combination of RNA interference, microscopy, and quantitative trafficking assays. RNA interference-mediated suppression of TbRAB4 expression inhibited the growth of trypanosomes without affecting receptor-mediated endocytosis or ligand recycling. Ultrastructural analysis indicated a major defect in membrane transport events. The accumulation of fluorescent dextran, a fluid-phase marker, was blocked in cells lacking TbRAB4 protein. Since most fluid-phase markers are transported to the lysosome in T. brucei, the effects of TbRAB4 RNA interference on lysosomal function were investigated. By immunofluorescence, the major lysosomal protein p67 became progressively dispersed in cells lacking the TbRAB4 protein. Pulse-chase analysis demonstrated that initial proteolytic cleavage and glycan processing of p67 were unaffected but that cells failed to accumulate the later p67 proteolyzed products associated with the lysosome. To confirm the role of TbRAB4 in lysosomal trafficking, a constitutively active mutant, TbRAB4(QL), was expressed. TbRAB4(QL) was closely associated with an enlarged multivesicular body that contained p67. In addition, cells expressing TbRAB4(QL) showed increased fluid-phase uptake when compared with the parental line. Taken together, these data suggest that TbRAB4 is involved in regulation of fluid-phase traffic to the lysosome in T. brucei but not in receptor-mediated endocytosis or recycling. These data have implications for the role of Rab4 in other cell systems.