## 4 Vectorial Proteomics  
Retrograde trafficking between early endosomes and the Golgi apparatus was discovered using the GL-Lect cargo protein Shiga toxin [72]. This trafficking interface has since become a hot spot of membrane biology research [73; 74; 75]. While it was initially thought that very few cargo proteins would undergo retrograde transport from the plasma membrane to the Golgi [76], this had not been addressed in a systematic manner. For this, a vectorial proteomics approach was designed [77; 78; 79]. To generate a capture reagent in the Golgi, the SNAP tag [80] was fused to the transmembrane region of Golgi-localized galactosyl transferase, which itself was fused to GFP for pull-down using GFP-trap beads. The SNAP tag reacts specifically with the small molecule compound O\({}^{6}\)-benzylguanine (BG), which is a synthetic derivative of guanine. It was thereby possible to covalently capture retrograde cargo proteins in the Golgi provided that these had first been labeled at the plasma membrane with BG. For this, a BG labeling reagent was needed with a key property: to be membrane impermeable. This was achieved by synthesizing a BG-containing molecule that had an N-hydroxy succinimide (NHS) moiety for the reaction with primary amines of plasma membrane proteins, and a PEG9 moiety to provide membrane impermeability. The first retrograde proteomics study with this system revealed that an unexpectedly large number of plasma membrane proteins underwent retrograde transport to the Golgi [81]. Proteomics results of course need to be confirmed. For this, the cell adhesion and migration factor \(\alpha\)5\(\beta\)1 integrin was chosen for which retrograde trafficking had never been described before. In experiments on cells in culture, in _C. elegans_ and in mice, it was shown that retrograde trafficking of specifically the non-ligand-bound inactive conformation of \(\alpha\)5\(\beta\)1 integrin allowed the protein to have access to the polarized secretion capacity of the Golgi. In migratory cells, retrograde transport was thereby required for the dynamic  
Figure 7: Model of how Retro-2 induces a block of Shiga toxin trafficking at the level of early endosomes. In untreated conditions (left), Shiga toxin trafficking from early endosomes to the TGN requires the interaction of GPP130 (blue) with syntaxin-5 (Syn5, yellow). From the TGN, Shiga toxin then moves on to the ER from where the catalytic A-subunit is translocated to the cytosol to inactivate ribosomes. Under Retro-2 treatment conditions (right), the anterograde transport of syntaxin-5 between ER and Golgi is slowed down by the interaction of Retro-2 with Sec16A, leading to the partial depletion of syntaxin-5 in the Golgi, a loss of its interaction with GPP130, and thereby the blockage of STxB in early endosomes. Reproduced with permission from Ref. [70]. localization of \(\alpha\)581 integrin to the leading edge toward which the Golgi is polarized in these cells. Of note, the conceptual pattern of \"retrograde transport to the Golgi to enable polarized secretion to a specialized area of the plasma membrane for the dynamic localization of proteins thereto\" may apply more widely [29]. For example, using the Golgi SNAP tag/NHS-PEG9-BG system it was also discovered that in lymphocytes the signaling protein Lat underwent retrograde trafficking and subsequent polarized secretion to be dynamically localized to a specialized plasma membrane area of T lymphocytes: the immunological synapse [82]. The SNAP tag approach has also been exploited to quantify the passage of biological or therapeutic macromolecules from the outside of cells to the cytosol [83]. Macromolecules indeed do not readily cross membranes. Cytosolic arrival, via direct translocation across the plasma membrane or endocytosis followed by endosomal escape, is currently one of the main bottlenecks for the development of macromolecular therapeutics such as nucleic acids (siRNAs, mRNAs, etc.) or peptides [84; 85; 86].