Amessou Mohamed, Popoff Vincent, Yelamos Belèn, Saint-Pol Agnès, Johannes Ludger
Institut Curie, Paris, France.
Curr Protoc Cell Biol. 2006 Oct;Chapter 15:Unit 15.10. doi: 10.1002/0471143030.cb1510s32.
The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans-Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non-toxic receptor-binding B-subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes-TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation-site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin-containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.
最近描述的逆行运输途径是一条高度选择性的通路，它允许一些内化分子从早期/再循环内体到达反式高尔基体网络，绕过再循环至质膜的途径和晚期内吞途径。细菌志贺毒素无毒的受体结合B亚基在早期/再循环内体-反式高尔基体网络界面的膜运输的发现和分子剖析中发挥了重要作用。本单元介绍了几种用于逆行运输定量生化和形态分析的最新方法。硫酸化测定法可检测和定量货物蛋白从内体到反式高尔基体网络的运输，描述了硫酸化位点肽如何化学偶联到货物蛋白上。此外，还介绍了通透细胞硫酸化测定法的一种变体。化学交联主题扩展到辣根过氧化物酶，用于通过整装分析对含志贺毒素的早期/再循环内体进行超微结构研究。最后，一种内吞测定法描述了对志贺毒素和转铁蛋白细胞摄取的伴随分析。
