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

Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth

Cross-talk between plant cells and their surroundings requires tight regulation of information exchange at the plasma membrane (PM), which involves dynamic adjustments of PM protein localization and turnover to modulate signal perception and solute transport at the interface between cells and their surroundings. In animals and fungi, turnover of PM proteins is controlled by reversible ubiquitylation, which signals endocytosis and delivery to the cell’s lytic compartment, and there is emerging evidence for related mechanisms in plants. Here, we describe the fate of Arabidopsis PIN2 protein, required for directional cellular efflux of the phytohormone auxin, and identify cis- and trans-acting mediators of PIN2 ubiquitylation. We demonstrate that ubiquitin acts as a principal signal for PM protein endocytosis in plants and reveal dynamic adjustments in PIN2 ubiquitylation coinciding with variations in vacuolar targeting and proteolytic turnover. We show that control of PIN2 proteolytic turnover via its ubiquitylation status is of significant importance for auxin distribution in root meristems and for environmentally controlled adaptations of root growth. Moreover, we provide experimental evidence indicating that PIN2 vacuolar sorting depends on modification specifically by lysine63-linked ubiquitin chains. Collectively, our results establish lysine63-linked PM cargo ubiquitylation as a regulator of polar auxin transport and adaptive growth responses in higher plants