Nanobody-triggered lockdown of VSRs reveals ligand reloading in the Golgi.

Protein degradation in lytic compartments is crucial for eukaryotic cells. At the heart of this process, vacuolar sorting receptors (VSRs) bind soluble hydrolases in the secretory pathway and release them into the vacuolar route. Sorting efficiency is suggested to result from receptor recycling. However, how and to where plant VSRs recycle remains controversial. Here we present a nanobody-epitope interaction-based protein labeling and tracking approach to dissect their anterograde and retrograde transport routes in vivo. We simultaneously employ two different nanobody-epitope pairs: one for the location-specific post-translational fluorescence labeling of receptors and the other pair to trigger their compartment-specific lockdown via an endocytosed dual-epitope linker protein. We demonstrate VSR recycling from the TGN/EE, thereby identifying the cis-Golgi as the recycling target and show that recycled VSRs reload ligands. This is evidence that bidirectional VSR-mediated sorting of vacuolar proteins exists and occurs between the Golgi and the TGN/EE.