Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery

Abstract

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.

The ArathEULS3 Lectin Ends up in Stress Granules and Can Follow an Unconventional Route for Secretion

“…A peroxisome organelle marker PTS1-RFP was available from the Nottingham Arabidopsis Stock Centre (NASC), stock number CD3-983. Plasmids containing the pUBQ10::3xHA-TagRFP-AtG8a sequences were kindly provided by Prof. Dr. Daniel Van Damme (VIB-UGhent Center for Plant Systems Biology, Gent, Belgium) [57] and plasmids containing G3BP-RFP sequences were kindly provided by the lab of Prof. Dr. Peter Tompa (VIB-VUB Structural Biology Research Center, Brussels, Belgium) [58]. All vectors were introduced into Agrobacterium tumefaciens (strains C58C1 PMP90 RifR, GV3101 or UIA143) by electroporation.…”

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

“…Pan1p was also recently shown to be part of a phosphorylation-dependent mechanism connecting endocytic vesicles, endosomal compartments and actin dynamics in budding yeast (Toshima et al, 2016). The Arabidopsis AtEH/Pan1 proteins were recently shown to mediate actin-dependent autophagy in plants (Wang et al, 2019). AtEH/Pan1 and Pan1p proteins are both hallmarked by the presence of two EH domains at their N-terminus (Gadeyne et al, 2014;Wang et al, 2019).…”

“…The Arabidopsis AtEH/Pan1 proteins were recently shown to mediate actin-dependent autophagy in plants (Wang et al, 2019). AtEH/Pan1 and Pan1p proteins are both hallmarked by the presence of two EH domains at their N-terminus (Gadeyne et al, 2014;Wang et al, 2019). In animals and yeast, EH domains have been characterized in great detail due to their presence in crucial endocytic proteins like Eps15, REPS1, EHD1, etc.…”

“…Plant EHD1 and EHD2 proteins have been characterized as homologues of human EHD proteins and were suggested to play a regulatory role during plant CME, plant defense and salt stress (Bar and Avni, 2009;Bar et al, 2008Bar et al, , 2013. In contrast to the essential function of all tested TPC subunits, silencing of EHD1/2 does not result in severely aberrant phenotypes, indicating redundancy or a more specialized function (Bar et al, 2008;Gadeyne et al, 2014;Wang et al, 2019). In contrast to the single EH domain in EHD proteins, AtEH/Pan1 proteins harbor two EH domains and we asked if both EH domains serve as independent functional modules.…”

High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits

“…In Arabidopsis seedlings, AtEH1/Pan1 and AtEH2/Pan1 have so far been shown functionally associated 156 with TPLATE at autophagosomes and to be delivered to the vacuole after carbon starvation and Conc A 157 treatment (Wang et al, 2019). To address the recruitment of AtEH/Pan1 and TPLATE in endocytic foci at 158 the PM, we combined AtEH/Pan1 with TPLATE in their respective complemented mutant backgrounds.…”