Nanobody-based VSR7 tracing shows clathrin-dependent TGN to Golgi recycling

Receptor-mediated transport of soluble proteins is nature’s key to empowering eukaryotic cells to access a plethora of macromolecules, either by direct accumulation or as products from resulting biochemical pathways. The transport efficiency of these mechanisms results from the receptor’s capability to capture, transport, and release ligands on the one hand and the cycling ability that allows for performing multiple rounds of ligand transport on the other. However, the plant VACUOLAR SORTING RECEPTOR (VSR) protein family is diverse, and their ligand-specificity and bidirectional trafficking routes and transport mechanisms remain highly controversial. Here we employ nanobody-epitope interaction-based molecular tools to assess the function of the VSR 7 in vivo. We demonstrate the specificity of the VSR7 for sequence-specific vacuolar sorting signals, and we trace its anterograde transport and retrograde recycling route. VSR7 localizes at the cis-Golgi apparatus at steady state conditions and transports ligands downstream to release them in the trans-Golgi network/early endosome (TGN/EE) before undergoing clathrin-dependent recycling from the TGN/EE back to the cis-Golgi.