Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus
Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus
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DOI：
10.1091/mbc.e08-07-0726
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发表时间：
2009-03-01
影响因子：
3.3
通讯作者：
Canals, Josep M.
中科院分区：
文献类型：
--
作者：
del Toro, Daniel;Alberch, Jordi;Canals, Josep M.
亨廷顿蛋白调节高尔基体后分泌蛋白的运输。在这里，我们研究了突变亨廷顿蛋白损害这一过程的机制。对分离的高尔基膜的共定位研究和蛋白质印迹分析表明，表达突变亨廷顿蛋白的细胞的高尔基体中亨廷顿蛋白减少。这些发现与高尔基体中 optineurin 和 Rab8 水平的降低相关，而高尔基体中 optineurin 和 Rab8 水平的降低可以通过全长野生型亨廷顿蛋白的过度表达来恢复。此外，免疫沉淀研究表明突变亨廷顿蛋白和 optineurin/Rab8 之间的相互作用减少。电子显微镜显示，表达突变型亨廷顿蛋白的细胞在高尔基体处产生了网格蛋白接头复合物1的积累，并且在高尔基体池附近产生了网格蛋白包被的囊泡的增加。此外，对溶酶体相关膜蛋白 1 和甘露糖 6-磷酸受体进行光漂白分析后的反向荧光恢复表明，在表达突变亨廷顿蛋白或通过小干扰 RNA 降低亨廷顿蛋白水平的细胞中，optineurin/Rab8 依赖的高尔基体后转运至溶酶体受到损害。因此，这些细胞的溶酶体中组织蛋白酶 D 含量较低，导致溶酶体活性总体降低。总之，我们的结果表明，突变型亨廷顿蛋白通过使 optineurin/Rab8 复合物离域，扰乱高尔基体后向溶酶体区室的运输，从而影响溶酶体功能。
Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.