Neuronal endoplasmic reticulum architecture and roles in axonal physiology.

The endoplasmic reticulum (ER) is the largest membrane compartment within eukaryotic cells and is emerging as a key coordinator of many cellular processes. The ER can modulate local calcium fluxes and communicate with other organelles like the plasma membrane. The importance of ER in neuronal processes such as neurite growth, axon repair and neurotransmission has recently gained much attention. In this review, we highlight the importance of the ER tubular network in axonal homeostasis and discuss how the generation and maintenance of the thin tubular ER network in axons and synapses, requires a cooperative effort of ER-shaping proteins, cytoskeleton and autophagy processes.

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

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.

Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

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

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Conditional destabilization of the TPLATE complex impairs endocytic internalization.

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Molecular architecture of the endocytic TPLATE complex.

Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction. Our data therefore generate fresh insight into the differences between the hexameric TSET in Dictyostelium and the octameric TPC in plants. Structural elucidation of this ancient adaptor complex represents the missing piece in the coatomer puzzle and vastly advances our functional as well as evolutionary insight into the process of endocytosis.

Endocytosis of BRASSINOSTEROID INSENSITIVE1 Is Partly Driven by a Canonical Tyr-Based Motif.

Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles via the recognition of motifs based on Tyr or di-Leu in their cytoplasmic tails. However, in plants, very little is known about how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis (Arabidopsis thaliana), the brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) undergoes endocytosis, which depends on clathrin and AP-2. Here, we demonstrate that BRI1 binds directly to the medium AP-2 subunit (AP2M). The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed Tyr-based endocytic motifs. The Tyr-to-Phe substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2-dependent internalization of PM proteins via the recognition of functional Tyr motifs also operates in plants.

Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

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

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.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.