Measuring Retrograde Transport to the Trans‐Golgi Network

Abstract

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.

The retromer complex and clathrin define an early endosomal retrograde exit site

“…For this, a variant of Shiga toxin B-subunit (STxB) that was specifically constructed for site-directed chemical coupling, termed STxB/Cys (Amessou et al, 2006), is covalently linked to horseradish peroxidase (HRP). Immunofluorescence and immunolectron microsocopy showed that the trafficking of STxB is not altered by this procedure (Saint-Pol et al, 2004).…”

Shiga toxin stimulates clathrin-independent endocytosis of VAMP2/3/8 SNARE proteins

“…Primary hits (Fig. 1A, green overlay) were submitted to a secondary screen for functional validation in which the corresponding VAMP proteins were depleted using small interfering RNAs (siRNAs), followed by retrograde transport analysis using the sulfation assay (Amessou et al, 2006). In this assay, retrograde transport from the plasma membrane to the TGN is measured using a STxB variant with tandem protein sulfation sites, termed STxB-Sulf 2 .…”

“…The sulfation assay was performed as previously described (Amessou et al, 2006). Briefly, cells were seeded in 24-well dishes.…”

“…The Tf endocytosis assay was performed essentially as previously described (Amessou et al, 2006). Briefly, human diferric transferrin (Tf ) was biotinylated using NHS-SS-biotin (Pierce).…”

Correlation between Shiga toxin B‐subunit stability and antigen crosspresentation: A mutational analysis

“…Transport to the ER is believed to be required for translocation of the A-subunit to the cytosol where its molecular target -ribosomal RNA -resides [12]. STxB has been used as a tool for the characterization of the retrograde route [13,14], and in biomedical research as a delivery tool for immunotherapy [3,15] and cancer targeting [16,17]. It was shown that STxB delivers exogenous peptides into the MHC class I and II pathways of human and mouse dendritic cells (DCs) and induces humoral and cell-mediated immune responses that protect mice from tumor growth [18][19][20][21][22].…”