The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics CAS v v i Vídeňská 1083 Prague 14200 Czech Republic

Department of Biosciences Durham University South road Durham DH1 3LE UK

Department of Plant Biotechnology and Bioinformatics Ghent University Technologiepark 71 9052 Ghent Belgium

Key Laboratory of Horticultural Plant Biology College of Horticulture and Forestry Sciences Huazhong Agricultural University Wuhan 430070 Hubei Province PR China

VIB Center for Plant Systems Biology Technologiepark 71 9052 Ghent Belgium

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Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo

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Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding

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Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1