Modulation of signaling pathways starting from the PM requires control over the PM proteome. While anterograde secretory pathways (exocytosis) deposit PM proteins, their removal depends on retrograde transport by endocytosis, in which PM material and extracellular ligands, together termed cargo, are predominantly internalized using coated vesicles. This mechanism provides the cell with an immediate and non-transcriptional way to react to various stimuli. Clathrin-mediated endocytosis (CME), defined by the involvement of the scaffold protein clathrin guiding the formation of a cage around the invaginating membrane, is the best characterized endocytic pathway in eukaryotes.
In contrast to other eukaryotes, knowledge of cargo recognition and the precise regulation of CME in plants is only beginning to emerge and there are still many gaps in our understanding of this process.
Although several core components of CME such as the Adaptor Protein Complex 2 (AP-2), dynamin-like proteins and clathrins are conserved across the eukaryotic Kingdoms, a broad range of effectors are absent from plants. On the other hand, plant-specific effectors of CME also exist, strongly suggesting the evolutionary divergence of a plant-specific CME mechanism.
The Adaptor Protein 2 complex (AP-2), the core adaptor complex in animal CME, was recently demonstrated to also be involved in plant CME. However, while this complex is essential in animals, loss of AP-2 subunits in plants only results in mildly aberrant phenotypes. Recently, I reported the discovery of a novel, eight-core-subunit complex (the TPLATE complex, TPC) that acts as the key plant endocytic adaptor complex. Similar to AP-2, all TPC subunits are recruited to dynamic foci at the PM. However, in contrast to AP-2, knock-outs in all tested subunits of the TPC are lethal. In line with a major role for the TPC in plant CME, induced silencing of TPC subunits impaired internalization of well-known endocytic cargo proteins as well as endocytic tracers.
The core TPC consists out of eight proteins. An extensive structural homology-based search recently did discover a similar, yet hexameric complex (TSET) in the slime mold Dictyostelium. The TPC/TSET complex represents an ancient adaptor complex which is lost completely in the lineage leading to animal and fungal cells (opisthokonts).
The fact that plants are likely the only Kingdom where this evolutionary ancient endocytic complex remained essential demonstrates fundamental differences between plant and opisthokont endocytic mechanisms. Detailed analysis of TPC ultrastructure, assembly, cargo-recognition, and its relationship with AP-2 will yield important insights into evolutionary aspects of CME in eukaryotes.
This project will resolve long-standing problems in the understanding of how plant CME operates via a multidisciplinary approach.
Firstly, a genome-wide proteomics-based approach using proximity biotinylation was optimized for plant cell cultures. This allowed to evaluate cargo-specificity of both the TPC and AP-2 adaptor complexes.
The proteomics approach did not only identify cargoes, but also several unknown interactors/modulators of these adaptor complexes. The outcome of this part of the project therefore is also be an expansion of the interaction network surrounding the both adaptor complexes operating at the PM and a substantial increase in knowledge on how CME in plant operates and the players involved.
Secondly, dynamic live-cell imaging, interaction analyses and structural biology were applied to dissect adaptor complex assembly and to determine the ultrastructure of the entire TPC as well as individual subunits/domains, yielding functional insight.
This led to a validated first structural model of the TPLATE complex which provides us with a view on endocytosis throughout eukaryotic evolution. The model currently has low overall resolution, next to high resolution of specific domains which are independently resolved via recombinant protein domain purification approaches.
This model will serve a starting point to understand its function in cargo recognition and membrane bending at the molecular level, which in turn is essential to be able to modulate agriculturally important processes such as nutrient and water uptake, biomass production and pathogen defense.
