Analysis of the bidirectional VSR-mediated transport in the plant endomembrane system

Abstract

Post-translational regulation of membrane proteins is pivotal for all eukaryotic cells. In particular, the regulation of transporter proteins at the plasma membrane. They are post-translationally regulated by vacuolar degradation after their endocytic removal. For this to occur, the vacuole needs a constant supply of soluble hydrolyzing enzymes. At the heart of this process operate vacuolar sorting receptors (VSRs) mediating the transport of these enzymes towards the lytic vacuole. Based on the research on mammalian cells, it is assumed that VSR transport occurs bidirectional and follows the common principle of receptor-mediated transport: Receptors bind ligands in the donor compartment, thereby forming a receptor-ligand complex that is transported to the acceptor compartment. Upon arrival, ligands are released and receptors recycle back to the donor compartment to reload ligands. It is presumed that this transport occurs between the trans-Golgi network/early endosome (TGN/EE) and the multivesicular bodies/late endosomes (MVBs/LEs) in plants. It now became clear that VSR bind ligand in the early secretory pathway and transport them to the TGN/EE were they are released (Künzl et al., 2016).

To analyze the post TGN/EE transport of soluble proteins, we generated a nanobody-based system to follow the fate of soluble proteins lacking vacuolar sorting signals that were placed in the TGN/EE via the endocytic pathway. This enabled us to demonstrated that post TGN/EE transport of ligands to the vacuole occurs independently of VSRs (Künzl et al., 2016). Usage of this system, however, required testing that nanobody-triggered protein-protein interactions between two soluble proteins can occur in the endomembrane system (Früholz and Pimpl, 2017). With the demonstration that VSRs release ligands in the TGN/EE (Künzl et al.,2016) it became clear that if VSRs do recycle, then the TGN/EE would be the starting point for such a recycling. To identify the target compartment of the VSR recycling route, we devised an approach where we employ simultaneously two different nanobody-epitope pairs. One to label VSRs fluorescently in the TGN/EE and a second to trigger the lockdown of recycled VSRs via an endocytosed dual epitope linker to block their further anterograde transport. Using this approach, we demonstrate that VSRs recycle from the TGN/EE to the cis-Golgi and show that recycled VSRs reload ligands there (Früholz et al., in press). Together, we proof that the bidirectional VSR-mediated transport exists and occurs between the TGN/EE and the cis-Golgi.