Identifying protein-protein interactions (PPI) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.

One-sentence summary: rapamycin-dependent delocalization allows quantifying proteinprotein interactions inside plant cells. Author contributions: DVD initiated the project and designed experiments. JW, EM, ADM and PG designed and performed experiments. BP wrote the script for quantification of the data. JM performed experiments. JW, PG and DVD wrote the paper. AbstractIdentifying protein-protein interactions (PPI) is crucial to understand any type of biological process. Many PPI tools are available, yet only some function within the context of a plant cell.Narrowing down even further, only few PPI tools allow visualizing higher order interactions.Here, we present a novel and conditional in vivo PPI tool for plant research. Knocksideways in plants (KSP) uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations, which other PPI systems hold. It is an in vivo tool, it is flexible concerning the orientation of protein tagging as long as this does not interfere with the interaction and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore does not require additional controls. The interactions can be quantified and in high throughput by the scripts that we provide. Finally, we demonstrate that KSP can visualize higher-order interactions. It is therefore a versatile tool, complementing the PPI methods field with unique characteristics and applications.

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive.Here, we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this 2 evolutionary specific appendage modification allows TPC formation, but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

Endocytosis is the process by which cells internalise molecules from their cell surface via plasma membrane-derived vesicles. In plants, clathrin-mediated endocytosis requires the evolutionarily ancient TSET/TPLATE complex (TPC), which was lost in metazoan and fungal lineages. TPC is required for membrane bending, but how TPC functions in the initiation of endocytosis and clathrin assembly is unclear. Here we used live-cell imaging and biochemical approaches to investigate the function of the Arabidopsis thaliana TPC subunit AtEH1/Pan1. Using in vitro and in vivo experiments we found that AtEH/Pan1 proteins can self-assemble into condensates through phase separation, which is influenced by both structured and intrinsically disordered regions. The proteome composition of these condensates revealed many key endocytic components which are selectively recruited via prion-like- and IDR-based interactions, including the ESCRT-0 TOM-Like proteins. Furthermore, AtEH/Pan1 condensates selectively nucleate on the plasma membrane by binding specific phospholipid species that are recognised by their EH domains. Visualization of the ultrastructure of the endocytic condensates via CLEM-ET revealed that the coat protein clathrin can assemble into lattices within condensates. Our results reveal that AtEH/Pan1 proteins act as scaffolds to direct endocytic machinery to specific plasma membrane regions to initiate internalisation. These findings provide new insight into the interplay between membranes and protein condensates.

13Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane 14 (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by 15 endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein 16 complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-17 mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex 18 and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into 19 viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and 20 silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, 21 while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization 22 of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant 23 background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody 24 construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We 25 furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-26 cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex 27 cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-28 recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach 29 delocalized complexes, rather than individual adaptor complex subunits. In line with the specific 30 expression domain, we only observed minor effects on root growth and gravitropic response, yet 31 Nanobody sequestration of endocytic machinery 2 realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization 32 in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve 33 specific loss-of-function analysis of otherwise lethal mutants. 34

Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.

Clathrin-mediated endocytosis is an essential cellular internalisation pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET/TPLATE complex (TPC) plays an essential, but not well-defined role in endocytosis in plants. Here, we show that two highly disordered TPC subunits, AtEH1 and AtEH2 function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, early-, and late-stage endocytic accessory proteins. Importantly, clathrin forms ordered assemblies within the condensate environment. Biomolecular condensation therefore acts to promote dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, the disordered region sequence properties of AtEH1 regulate the material properties of the endocytic condensates in vivo. Alteration of the material properties influences endocytosis dynamics, and thereby impairs environmental adaption. In conclusion, our findings reveal how collective interactions shape endocytosis.

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