Bujny Miriam V, Popoff Vincent, Johannes Ludger, Cullen Peter J
The Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
J Cell Sci. 2007 Jun 15;120(Pt 12):2010-21. doi: 10.1242/jcs.003111.
The mammalian retromer complex is a multi-protein complex that regulates retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR) from early endosomes to the trans Golgi network (TGN). It consists of two subcomplexes: a membrane-bound coat comprising sorting nexin-1 (SNX1) and possibly sorting nexin-2 (SNX2), and a cargo-selective subcomplex, composed of VPS26, VPS29 and VPS35. In addition to the retromer, a variety of other protein complexes has been suggested to regulate endosome-to-TGN transport of not only the CI-MPR but a wide range of other cargo proteins. Here, we have examined the role of SNX1 and SNX2 in endosomal sorting of Shiga and cholera toxins, two toxins that undergo endosome-to-TGN transport en route to their cellular targets located within the cytosol. By using small interfering RNA (siRNA)-mediated silencing combined with single-cell fluorescent-toxin-uptake assays and well-established biochemical assays to analyze toxin delivery to the TGN, we have established that suppression of SNX1 leads to a significant reduction in the efficiency of endosome-to-TGN transport of the Shiga toxin B-subunit. Furthermore, we show that for the B subunit of cholera toxin, retrograde endosome-to-TGN transport is less reliant upon SNX1. Overall, our data establish a role for SNX1 in the endosome-to-TGN transport of Shiga toxin and are indicative for a fundamental difference between endosomal sorting of Shiga and cholera toxins into endosome-to-TGN retrograde transport pathways.
哺乳动物的逆转录复合物是一种多蛋白复合物，它调节阳离子非依赖性甘露糖6-磷酸受体（CI-MPR）从早期内体到反式高尔基体网络（TGN）的逆向运输。它由两个亚复合物组成：一个膜结合包被，包括分选连接蛋白-1（SNX1）以及可能的分选连接蛋白-2（SNX2），还有一个货物选择性亚复合物，由VPS26、VPS29和VPS35组成。除了逆转录复合物外，还有多种其他蛋白复合物被认为不仅调节CI-MPR，还调节多种其他货物蛋白从内体到TGN的运输。在这里，我们研究了SNX1和SNX2在志贺毒素和霍乱毒素内体分选过程中的作用，这两种毒素在前往位于胞质溶胶中的细胞靶点的途中会经历从内体到TGN的运输。通过使用小干扰RNA（siRNA）介导的沉默技术，结合单细胞荧光毒素摄取试验和成熟的生化试验来分析毒素向TGN的递送，我们发现抑制SNX1会导致志贺毒素B亚基从内体到TGN的运输效率显著降低。此外，我们表明，对于霍乱毒素的B亚基，逆向的内体到TGN运输对SNX1的依赖性较小。总体而言，我们的数据确定了SNX1在志贺毒素从内体到TGN运输中的作用，并表明志贺毒素和霍乱毒素在内体分选中进入内体到TGN逆向运输途径存在根本差异。
