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 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.

BioImaging Core VIB 9052 Ghent Belgium

Center for Plant Systems Biology VIB Ghent Belgium

Department of Biomedical Molecular Biology Ghent University Ghent Belgium

Department of Plant Biotechnology and Bioinformatics Ghent University Ghent Belgium

Institute of Experimental Botany Academy of Sciences of the Czech Republic Prague Czech Republic

Leibniz Forschungsinstitut für Molekulare Pharmakologie Berlin Germany

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Nat Plants. 2022 Dec;8(12):1339-1340. doi: 10.1038/s41477-022-01290-z. PubMed

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Nat Plants. 2023 Jan;9(1):191. doi: 10.1038/s41477-023-01344-w. PubMed

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Bitsikas V, Corrêa IR, Nichols BJ. Clathrin-independent pathways do not contribute significantly to endocytic flux. Elife. 2014;3:1–26. PubMed PMC

Kaksonen M, Roux A. Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2018;19(5):313–326. 2018. PubMed

Reynolds GD, Wang C, Pan J, Bednarek SY. Inroads into Internalization: Five Years of Endocytic Exploration. Plant Physiol. 2018;176:208–218. PubMed PMC

Gadeyne A, et al. The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants. Cell. 2014;156:691–704. PubMed

Hirst J, et al. Characterization of TSET, an ancient and widespread membrane trafficking complex. Elife. 2014;2014 PubMed PMC

Van Damme D, et al. Somatic Cytokinesis and Pollen Maturation in Arabidopsis Depend on TPLATE, Which Has Domains Similar to Coat Proteins. Plant Cell. 2006;18:3502–3518. PubMed PMC

Bashline L, Li S, Zhu X, Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis. Proc Natl Acad Sci U S A. 2015;112:12870–12875. PubMed PMC

Ohno H, et al. The Medium Subunits of Adaptor Complexes Recognize Distinct but Overlapping Sets of Tyrosine-based Sorting Signals. J Biol Chem. 1998;273:25915–25921. PubMed

Mattera R, Boehm M, Chaudhuri R, Prabhu Y, Bonifacino JS. Conservation and Diversification of Dileucine Signal Recognition by Adaptor Protein (AP) Complex Variants. J Biol Chem. 2011;286:2022. PubMed PMC

Arora D, van Damme D. Motif-based endomembrane trafficking. Plant Physiol. 2021;186:221–238. PubMed PMC

Happel N, et al. ArabidopsisμA-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1. Plant J. 2004;37:678–693. PubMed

Takano J, et al. Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways. Proc Natl Acad Sci U S A. 2010;107:5220–5225. PubMed PMC

Mravec J, et al. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature. 2009;459:1136–40. PubMed

Yoshinari A, et al. Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis. Plant Physiol. 2019;179:1569–1580. PubMed PMC

Liu D, et al. Endocytosis of BRASSINOSTEROID INSENSITIVE Is Partly Driven by a Canonical Tyr-Based Motif. Plant Cell. 2020;32:3598–3612. PubMed PMC

Yperman K, et al. Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding. Nat Commun. 2021;12(1):1–11. 2021. PubMed PMC

Robatzek S, Chinchilla D, Boller T. Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev. 2006;20:537. PubMed PMC

Wang S, et al. Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct Integr Genomics. 2010;10:533–546. PubMed

Erwig J, et al. Chitin-induced and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) phosphorylation-dependent endocytosis of Arabidopsis thaliana LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5 (LYK5) New Phytol. 2017;215:382–396. PubMed

Traub LM. Tickets to ride: selecting cargo for clathrin-regulated internalization. Nat Rev Mol Cell Biol. 2009;10(9):583–596. 2009. PubMed

Dubeaux G, Vert G. Zooming into plant ubiquitin-mediated endocytosis. Curr Opin Plant Biol. 2017;40:56–62. PubMed

Fan L, Li R, Pan J, Ding Z, Lin J. Endocytosis and its regulation in plants. Trends Plant Sci. 2015;20:388–397. PubMed

Nagel MK, et al. Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3. Proc Natl Acad Sci U S A. 2017;114:E7197–E7204. PubMed PMC

Weinberg JS, Drubin DG. Regulation of clathrin-mediated endocytosis by dynamic ubiquitination and deubiquitination. Curr Biol. 2014;24:951–959. PubMed PMC

Yoshinari A, et al. DRP1-Dependent Endocytosis is Essential for Polar Localization and Boron-Induced Degradation of the Borate Transporter BOR1 in Arabidopsis thaliana. Plant Cell Physiol. 2016;57:1985–2000. PubMed

Leitner J, et al. Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proc Natl Acad Sci U S A. 2012;109:8322–8327. PubMed PMC

Barberon M, et al. Monoubiquitin-dependent endocytosis of the Iron-Regulated Transporter 1 (IRT1) transporter controls iron uptake in plants. Proc Natl Acad Sci U S A. 2011;108 PubMed PMC

Vert G, et al. IRT1, an Arabidopsis Transporter Essential for Iron Uptake from the Soil and for Plant Growth. Plant Cell. 2002;14:1223–1233. PubMed PMC

Liao D, et al. Arabidopsis E3 ubiquitin ligase PLANT U-BOX13 (PUB13) regulates chitin receptor LYSIN MOTIF RECEPTOR KINASE5 (LYK5) protein abundance. New Phytol. 2017;214:1646–1656. PubMed

Lu D, et al. Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science. 2011;332:1439–1442. PubMed PMC

Zhou J, et al. Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination. Proc Natl Acad Sci U S A. 2018;115:E1906–E1915. PubMed PMC

Korbei B, et al. Arabidopsis TOL Proteins Act as Gatekeepers for Vacuolar Sorting of PIN2 Plasma Membrane Protein. Curr Biol. 2013;23:2500–2505. PubMed

Moulinier-Anzola J, et al. TOLs Function as Ubiquitin Receptors in the Early Steps of the ESCRT Pathway in Higher Plants. Mol Plant. 2020;13:717–731. PubMed

Arora D, et al. Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems. Plant Cell. 2020;32:3388–3407. PubMed PMC

Wang J, et al. Conditional destabilization of the TPLATE complex impairs endocytic internalization. Proc Natl Acad Sci U S A. 2021;118 PubMed PMC

Yperman K, et al. Molecular architecture of the endocytic TPLATE complex. Sci Adv. 2021;7 PubMed PMC

Wang P, et al. Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery. Nat Commun. 2019;10 PubMed PMC

Van Damme D, et al. Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proc Natl Acad Sci U S A. 2011;108:615–620. PubMed PMC

Boutté Y, et al. Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO J. 2010;29:546–558. PubMed PMC

Mayer BJ, Hamaguchi M, Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nat. 1988;332(6161):272–275. 1988. PubMed

Kurochkina N, Guha U. SH3 domains: modules of protein-protein interactions. Biophys Rev. 2013;5:29–39. PubMed PMC

Stamenova SD, et al. Ubiquitin binds to and regulates a subset of SH3 domains. Mol Cell. 2007;25:273. PubMed PMC

Kang J, Kang S, Hyuk NK, He W, Park S. Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling. Biochim Biophys Acta. 2008;1784:1335–1341. PubMed

Bu F, Yang M, Guo X, Huang W, Chen L. Multiple Functions of ATG8 Family Proteins in Plant Autophagy. Front Cell Dev Biol. 2020;8:466. PubMed PMC

Schwihla M, Korbei B. The Beginning of the End: Initial Steps in the Degradation of Plasma Membrane Proteins. Front Plant Sci. 2020;11:680. PubMed PMC

Bashline L, Li S, Anderson CT, Lei L, Gu Y. The endocytosis of cellulose synthase in Arabidopsis is dependent on μ2, a clathrin-mediated endocytosis adaptin. Plant Physiol. 2013;163:150–160. PubMed PMC

Lian N, et al. COP1 mediates dark-specific degradation of microtubule-associated protein WDL3 in regulating Arabidopsis hypocotyl elongation. Proc Natl Acad Sci U S A. 2017;114:12321–12326. PubMed PMC

Kim JH, Kim WT. The Arabidopsis RING E3 ubiquitin ligase AtAIRP3/LOG2 participates in positive regulation of high-salt and drought stress responses. Plant Physiol. 2013;162:1733–1749. PubMed PMC

Svozil J, Hirsch-Hoffmann M, Dudler R, Gruissem W, Baerenfaller K. Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin A. Mol Cell Proteomics. 2014;13:1523–1536. PubMed PMC

Walton A, et al. It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana. Plant Cell. 2016;28:6–16. PubMed PMC

Johnson A, Vert G. Unraveling K63 Polyubiquitination Networks by Sensor-Based Proteomics. Plant Physiol. 2016;171:1808–1820. PubMed PMC

Aguilar-Hernández V, et al. Mass Spectrometric Analyses Reveal a Central Role for Ubiquitylation in Remodeling the Arabidopsis Proteome during Photomorphogenesis. Mol Plant. 2017;10:846–865. PubMed PMC

Romero-Barrios N, et al. Advanced Cataloging of Lysine-63 Polyubiquitin Networks by Genomic, Interactome, and Sensor-Based Proteomic Analyses. Plant Cell. 2020;32:123–138. PubMed PMC

Grubb LE, et al. Large-scale identification of ubiquitination sites on membrane-associated proteins in Arabidopsis thaliana seedlings. Plant Physiol. 2021;185:1483–1488. PubMed PMC

Martins S, et al. Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination. Nat Commun. 2015;6 PubMed PMC

Winkler J, et al. Nanobody-Dependent Delocalization of Endocytic Machinery in Arabidopsis Root Cells Dampens Their Internalization Capacity. Front Plant Sci. 2021;12 PubMed PMC

Fan L, et al. Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development. Dev. 2013;140:3826–3837. PubMed

Wendrich JR, et al. Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions. Science. 2020;370 PubMed PMC

Berrío RT, et al. Single-cell transcriptomics sheds light on the identity and metabolism of developing leaf cells. Plant Physiol. 2022;188:898–918. PubMed PMC

Song Q, Ando A, Jiang N, Ikeda Y, Chen ZJ. Single-cell RNA-seq analysis reveals ploidy-dependent and cell-specific transcriptome changes in Arabidopsis female gametophytes. Genome Biol. 2020;21 PubMed PMC

Lin D, et al. Rho GTPase Signaling Activates Microtubule Severing to Promote Microtubule Ordering in Arabidopsis. Curr Biol. 2013;23:290–297. PubMed

Abas L, et al. Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol. 2006;8(3):249–256. 2006. PubMed

Leitner J, Retzer K, Korbei B, Luschnig C. Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots. 2012;7:1271–1273. doi: 10.4161/psb.21715. PubMed DOI PMC

Yoshinari A, et al. Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation. Plant Cell. 2021;33:420–438. PubMed PMC

Luo Y, et al. Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1. EMBO Rep. 2022 doi: 10.15252/embr.202153354. PubMed DOI PMC

Hicke L, Schubert HL, Hill CP. Ubiquitin-binding domains. Nat Rev Mol Cell Biol. 2005;6(8):610–621. 2005. PubMed

Hawryluk MJ, et al. Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein. Traffic. 2006;7:262–281. PubMed

Sorkina T, et al. RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis. J Neurosci. 2006;26:8195–8205. PubMed PMC

Haglund K, Dikic I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J Cell Sci. 2012;125:265–275. PubMed

Mayers JR, et al. Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc Natl Acad Sci U S A. 2013;110:11857–11862. PubMed PMC

Schuh AL, Audhya A. The ESCRT machinery: From the plasma membrane to endosomes and back again. 2014;49:242–261. doi: 10.3109/10409238.2014.881777. PubMed DOI PMC

Adamowski M, Matijević I, Narasimhan M, Friml J. SH3Ps recruit auxilin-like vesicle uncoating factors into clathrin-mediated endocytosis. doi: 10.1101/2022.01.07.475403. PubMed DOI

Pashkova N, et al. Article WD40 Repeat Propellers Define a Ubiquitin-Binding Domain that Regulates Turnover of F Box Proteins. Mol Cell. 2010;40:433–443. PubMed PMC

Isono E, et al. The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis in Arabidopsis thaliana. Plant Cell. 2010;22:1826–1837. PubMed PMC

Katsiarimpa A, et al. The Arabidopsis Deubiquitinating Enzyme AMSH3 Interacts with ESCRT-III Subunits and Regulates Their Localization. Plant Cell. 2011;23:3026–3040. PubMed PMC

Katsiarimpa A, et al. The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis. Plant Cell. 2013;25:2236. PubMed PMC

Katsiarimpa A, et al. The ESCRT-III-Interacting Deubiquitinating Enzyme AMSH3 is Essential for Degradation of Ubiquitinated Membrane Proteins in Arabidopsis thaliana. Plant Cell Physiol. 2014;55:727–736. PubMed

Ingouff M, et al. Live-cell analysis of DNA methylation during sexual reproduction in arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM. Genes Dev. 2017;31:72–83. PubMed PMC

Karimi M, Depicker A, Hilson P. Recombinational Cloning with Plant Gateway Vectors. Plant Physiol. 2007;145:1144. PubMed PMC

Lampropoulos A, et al. GreenGate-A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis. PLoS One. 2013;8:e83043. PubMed PMC

Waadt R, et al. Dual-Reporting Transcriptionally Linked Genetically Encoded Fluorescent Indicators Resolve the Spatiotemporal Coordination of Cytosolic Abscisic Acid and Second Messenger Dynamics in Arabidopsis. Plant Cell. 2020;32:2582–2601. PubMed PMC

Waadt R, Krebs M, Kudla J, Schumacher K. Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis. New Phytol. 2017;216:303–320. PubMed

Decaestecker W, et al. CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis. Plant Cell. 2019;31:2868–2887. PubMed PMC

Mertens N, Remaut E, Fiers W Versatile. multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines. Gene. 1995;164:9–15. PubMed

Kim SY, et al. Adaptor Protein Complex 2-Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis. Plant Cell. 2013;25:2970–2985. PubMed PMC

Mravec J, et al. Report Cell Plate Restricted Association of DRP1A and PIN Proteins Is Required for Cell Polarity Establishment in Arabidopsis. Curr Biol. 2011;21:1055–1060. PubMed

Reichardt I, et al. Plant Cytokinesis Requires De Novo Secretory Trafficking but Not Endocytosis. Curr Biol. 2007;17:2047–2053. PubMed

Dejonghe W, et al. Disruption of endocytosis through chemical inhibition of clathrin heavy chain function. Nat Chem Biol. 2019;15:641–649. PubMed PMC

Xu T, et al. Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling. Science. 2014;343:1025–8. PubMed PMC

Di Rubbo S, et al. The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis. Plant Cell. 2013;25:2986–2997. PubMed PMC

Belkhadir Y, et al. Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc Natl Acad Sci U S A. 2012;109:297–302. PubMed PMC

Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006;1(4):2019–2025. 2006. PubMed

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. PubMed PMC

Altschul SF, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. PubMed PMC

Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018;46:D493–D496. PubMed PMC

Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019;20:1160–1166. PubMed PMC

Guindon S, et al. New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst Biol. 2010;59:307–321. PubMed

Lefort V, Longueville JE, Gascuel O. SMS: Smart Model Selection in PhyML. Mol Biol Evol. 2017;34:2422–2424. PubMed PMC

Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–W296. PubMed PMC

Varadi M, et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50:D439–D444. PubMed PMC

Mirdita M, et al. ColabFold-Making protein folding accessible to all. 2021 doi: 10.21203/rs.3.rs-1032816/v1. PubMed DOI PMC

Desta IT, Porter KA, Xia B, Kozakov D, Vajda S. Performance and Its Limits in Rigid Body Protein-Protein Docking. Structure. 2020;28:1071–1081.:e3. PubMed PMC

Yan Y, Tao H, He J, Huang SY. The HDOCK server for integrated proteinprotein docking. Nat Protoc. 2020;15:1829–1852. PubMed

Pierce BG, et al. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics. 2014;30:1771–1773. PubMed PMC

Pettersen EF, et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 2021;30:70–82. PubMed PMC

Ashkenazy H, et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 2016;44:W344–W350. PubMed PMC

Kapust RB, et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng Des Sel. 2001;14:993–1000. PubMed

Dahhan DA, et al. Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components. Plant Cell. 2022 doi: 10.1093/PLCELL/KOAC071. PubMed DOI PMC

Wendrich JR, Boeren S, Möller BK, Weijers D, De Rybel B. In Vivo Identifi cation of Plant Protein Complexes Using IP-MS/MS. Methods Mol Biol. 1497 PubMed

Sauer M, Paciorek T, Benkovó E, Friml J. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc. 2006;1:98–103. PubMed

von Wangenheim D, et al. Live tracking of moving samples in confocal microscopy for vertically grown roots. Elife. 2017;6 PubMed PMC

Johnson A, Vert G. Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy. Front Plant Sci. 2017;8 PubMed PMC

Narasimhan M, et al. Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. Elife. 2020;9 PubMed PMC

Winkler J, et al. Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization. Plant Cell. 2021;33:1101–1117. PubMed PMC

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants