In vivo analysis of endocytic and biosynthetic transport to the plant vacuole

Abstract

The plasma membrane forms the interaction site between a cell and its environment. The proteins in the plasma membrane, namely translocators and receptors, allow for the exchange of nutrients and information. This, however, urges for a stringent regulation of these proteins. One mechanism to control their amount is to transport them into the lytic vacuole via the endocytic pathway. The degradative function of vacuoles depends on proteolytic enzymes, which reach that very organelle through a different route. They are synthesized in the endoplasmic reticulum and transported via the endomembrane system. Vacuolar transport of those soluble proteins depends on sorting receptors, separate them from secretory cargo. To gain a deeper understanding on the trafficking of membrane-bound and soluble cargo to the vacuole, we aimed at characterizing the machinery mediating those processes. For this, we employed nanobody-epitope interactions to create intra-cellular setups, which enabled us to perform transport- and interaction-analyses of proteins via confocal microscopy. We revealed that “Vacuolar Sorting Receptors” (VSRs) interact with their ligands in the endoplasmic reticulum and the Golgi apparatus, but not in the trans-Golgi network and the multivesicular body, by performing “Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer” (FRET-FLIM; Künzl et al., 2016). In order to create the reporters for the compartment-specific FRET-FLIM measurements, we linked the ligand binding domain of the VSRs to marker-proteins via a nanobody-epitope interaction. We demonstrated that VSRs do indeed recycle and identified the cis-Golgi as the destination of their retrograde transport (Früholz et al., 2018). These discoveries were based on the combination of two nanobody-epitope pairs. We used those for post-translational labelling and trapping of vacuolar sorting receptors. Concerning the machinery mediating the transport of to-be-degraded plasma membrane proteins to the vacuole, we analyzed the “Endosomal Sorting Complex Required For Transport II” (ESCRT-II). Here, we employed FRETFLIM to show that “Vacuolar Protein Sorting 22” (VPS22), 25 and 36 interact to form this specific complex. We pushed the limits of nanobody-based approaches by employing membrane-anchored nanobodies in order to import the method of co-immune precipitation into living cells. This enabled us to perform in vivo studies, which showed that ESCRT-II contains two VPS25 moieties (Fäßler et al., prepared manuscript).