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Significance Retrograde transport from the cell surface to intracellular compartments is essential for homeostasis and cell physiology. We developed derivatized nanobodies to follow proteins from the cell surface to endosomes and thetrans -Golgi network. Nanobodies are an emerging class of protein binders with many advantages over conventional antibodies: they are small and noncross-linking, and can be produced in bacteria. Using a nanobody against GFP, our tool is widely applicable to any GFP-tagged protein of interest. It allows the quantitative analysis of endocytic and retrograde transport biochemically, by fixed- and live-cell imaging and by electron microscopy. As proof-of-principle, we applied them to determine the contribution of adaptor protein-1/clathrin in retrograde transport of the mannose-6-phosphate receptors to thetrans -Golgi.

A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface