Establishment of proximity-dependent biotinylation approaches in different plant model systems

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest, enabling the covalent biotin labelling of proteins and subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes

TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in <i>Arabidopsis thaliana</i>

<p>Boron (B) is an essential micronutrient for plants; however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of B toward the root stele in <i>Arabidopsis thaliana</i>. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. To avoid over-accumulation of B, BOR1 in the plasma membrane is rapidly internalized and transported into the vacuole for proteolysis after high-B supply in an ubiquitination-dependent manner. Although BOR1 has been predicted to be transported into multi-vesicular bodies/late endosomes (MVB/LEs) via the endosomal sorting complex required for transport (ESCRT) machinery, experimental evidence was absent so far. In this study, we investigated the intracellular localization of BOR1 by visualizing endomembrane compartments, and tested the involvement of ESCRT-0-like proteins TOM1-LIKEs (TOLs) in the vacuolar sorting of BOR1. Under low-B conditions, a large portion of cytoplasmic BOR1 was localized in the <i>trans</i>-Golgi networks/early endosomes (TGN/EEs) labeled with VHA-a1 subunit. Pharmacological interference of endosomal recycling using brefeldin A-induced colocalization of BOR1 with RabA5D, which labels recycling vesicles associated with the TGN. On the other hand, under high-B conditions, BOR1 was localized in the inside of TOL5-positive MVB/LEs. To examine the roles of TOL proteins in intracellular trafficking of BOR1, we analyzed BOR1-GFP localization in the TOL quintuple mutant (<i>tolQ; tol2-1tol3-1tol5-1tol6-1tol9-1</i>) after high-B supply. In the <i>tolQ</i> mutant, vacuolar sorting of BOR1 was delayed, while the polar localization of BOR1 was not disturbed. Taken together, BOR1 is constantly transported to the TGN/EE by endocytosis and recycled to the plasma membrane likely via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, BOR1 is transported to the vacuole via TOL5-positive MVB/LEs under high-B conditions. TOL proteins are required for sorting of ubiquitinated BOR1 into MVB/LE for vacuolar degradation.</p

Endosomally Localized RGLG-Type E3 RING-Finger Ligases Modulate Sorting of Ubiquitylation-Mimic PIN2

Intracellular sorting and the abundance of sessile plant plasma membrane proteins are imperative for sensing and responding to environmental inputs. A key determinant for inducing adjustments in protein localization and hence functionality is their reversible covalent modification by the small protein modifier ubiquitin, which is for example responsible for guiding proteins from the plasma membrane to endosomal compartments. This mode of membrane protein sorting control requires the catalytic activity of E3 ubiquitin ligases, amongst which members of the RING DOMAIN LIGASE (RGLG) family have been implicated in the formation of lysine 63-linked polyubiquitin chains, serving as a prime signal for endocytic vacuolar cargo sorting. Nevertheless, except from some indirect implications for such RGLG activity, no further evidence for their role in plasma membrane protein sorting has been provided so far. Here, by employing RGLG1 reporter proteins combined with assessment of plasma membrane protein localization in a rglg1 rglg2 loss-of-function mutant, we demonstrate a role for RGLGs in cargo trafficking between plasma membrane and endosomal compartments. Specifically, our findings unveil a requirement for RGLG1 association with endosomal sorting compartments for fundamental aspects of plant morphogenesis, underlining a vital importance for ubiquitylation-controlled intracellular sorting processes

Meta-regulation of Arabidopsis Auxin Responses Depends on tRNA Maturation

Polar transport of the phytohormone auxin throughout plants shapes morphogenesis and is subject to stringent and specific control. Here, we identify basic cellular activities connected to translational control of gene expression as sufficient to specify auxin-mediated development. Mutants in subunits of Arabidopsis Elongator, a protein complex modulating translational efficiency via maturation of tRNAs, exhibit defects in auxin-controlled developmental processes, associated with reduced abundance of PIN-formed (PIN) auxin transport proteins. Similar anomalies are observed upon interference with tRNA splicing by downregulation of RNA ligase (AtRNL), pointing to a general role of tRNA maturation in auxin signaling. Elongator Protein 6 (ELP6) and AtRNL expression patterns underline an involvement in adjusting PIN protein levels, whereas rescue of mutant defects by auxin indicates rate-limiting activities in auxin-controlled organogenesis. This emphasizes mechanisms in which auxin serves as a bottleneck for plant morphogenesis, translating common cellular activities into defined developmental readouts