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.

Summary The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant‐specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle‐associated membrane protein‐associated proteins (VAPs). Ten VAP homologues (VAP27‐1 to 27‐10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27‐labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27‐labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27‐GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER–cytoskeleton interaction and for plant development.

The small genome of Arabidopsis contains at least nine expressed P-tubulin (TUB) genes, in contrast to the large genomes of vertebrate animals, which contain a maximum of seven expressed P-tubulin genes. In this study, we report the structures of seven new TUB genes (TUBP, TUB3, TUBB, TUB6, TUB7, TUB8, and TUB9) of Arabidopsis. The sequences of T U B l and TU64 had been reported previously. Sequence similarities and unique structural features suggest that the nine TUB genes evolved by way of three branches in the plant P-tubulin gene evolutionary tree. Two genes (TUBP and TUB3) encode the same 0-tubulin isoform; thus, the nine genes predict eight different P-tubulins. In contrast to the a-tubulin (TUA) genes with their divergent intron patterns, all nine TUB genes contain 2 introns at conserved positions. Noncoding 3'gene-specific hybridization probes have been constructed for all nine TUB genes and used in RNA gel blot analyses to demonstrate that all nine genes are transcribed. Two-dimensional protein immunoblot analyses have resolved at least seven different P-tubulin isoforms in Arabidopsis, indicating that most, if not all, of the TUB tranxripts are translated.

HighlightArabidopsis synaptotagmin 1 is localized on ER–PM contact sites distinct from VAP27-1 and plays roles in maintaining ER morphology and the dynamics of VAP27-1.

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.