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.
Highlights
The Arabidopsis EH proteins (AtEH1/Pan[1] and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis
Proteins that regulate actin polymerization are found to be associated with the endoplasmic reticulum (ER) and the plasma membrane (PM)[7,8,9], where they have been found to regulate the biogenesis of autophagosomes: the mechanical forces produced by actin filament assembly are utilized to drive phagophore membrane expansion and the engulfment of autophagy cargo[10,11,12,13]
We show here that AtEH/Pan[1] proteins are able to interact with F-actin and VAP27-1, which is a protein that localizes to the ER-PM contact site[24,25,26], and regulate the formation of autophagosomes
Summary
The failure to transfer the mutation to the generation via the pollen can be complemented by expressing the respective AtEH/Pan[1] proteins fused to mRUBY3 and driven by the Histon[3] promotor in the mutant background, indicating that the fusions are functional (Table 1) Both AtEH1/ Pan1-mRuby[3] and AtEH2/Pan1-mRuby[3] localise to the plasma membrane in Arabidopsis roots and hypocotyl cells where they can be visualized localizing to dynamic endocytic foci (Fig. 2d, e). Some discrete cytosolic punctate structures can be observed with low frequency in root cells (Fig. 2b, c) To further confirm this result, we performed immunofluorescence studies using an antibody which recognizes both the AtEH/ Pan[1] proteins.
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