Mechanisms of vacuolar protein transport in plants

Abstract

Protein transport to plant vacuoles can occur by the biosynthetic pathway, leading from the endoplasmic reticulum (ER) via the Golgi apparatus, or by the endocytic pathway from the plasma membrane (PM). Both routes converge in the trans-Golgi network/early endosome (TGN/EE), from where cargo passes a multivesicular late endosome (MVB/LE) before reaching the vacuole. PM proteins destined for vacuolar degradation are internalized into the intraluminal vesicles of MVBs/LEs in a process involving recognition and sorting by the ESCRT machinery. Soluble vacuolar proteins from the biosynthetic pathway have to interact with vacuolar sorting receptors (VSRs) to be diverted from the secretory pathway and sent into the vacuolar route. Regarding these transport events, several aspects have remained unclear: how are proteins transported from the TGN/EE to the MVB/LE, and how do they arrive in the vacuole? Where does ESCRT recognize its cargo, and when does it initiate intraluminal sorting? What is the sorting signal for entry into the ESCRT-mediated vacuolar pathway? Where in the endomembrane system do VSRs bind soluble cargo ligands and where do they release them? In this thesis, results from two publications and a currently submitted manuscript are presented. We established that “multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis” (Scheuring et al., 2011) by capturing the moment in which nascent MVBs/LEs bud off from the TGN/EE. Mature MVBs/LEs were found to be non-persistent transport carriers that are steadily consumed by fusion with the vacuole. We showed that ESCRT acts at the TGN/EE and that maturation of MVBs/LEs and vacuolar transport fail if ESCRT functionality is blocked. ESCRT was also found to localize to the Golgi apparatus, indicating vacuolar sorting in the stack. Translational ubiquitin fusions of a Golgi marker indeed revealed that “ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway” (Scheuring et al., 2012). We then addressed the question as to where in the endomembrane system soluble vacuolar proteins are sorted. In an in vivo analysis, all compartments of the vacuolar route were tested individually for the ability to promote VSR-ligand interactions. We showed that VSRs bind ligands in the ER and the Golgi, but neither in the TGN/EE nor in the MVB/LE, indicating that “vacuolar sorting receptors transport ligands from the ER and the Golgi to the TGN/EE” (Künzl et al., submitted manuscript). Accordingly, post-TGN/EE trafficking of ligands is VSR-independent, being supported by the observation that non-VSR-ligands are delivered from the TGN/EE to the vacuole by default.