TOL Proteins in post-Golgi Trafficking in Plants

Endosomal vesicular transport is of central importance in many vital aspects in higher plant cells. Although these pathways are quite well defined on a molecular level in yeast and animal cells, only recently the focus has shifted to plants, with a special emphasis on plant- specific trafficking routes and unique functions of molecular players in the plant endosomal trafficking system. This research project aims to increase the understanding of the endosomal trafficking in the model plant Arabidopsis thaliana by examining the potential role of the nine different plant specific VHS-GAT domain proteins in post-Golgi vesicular transport. VHS-GAT domain proteins are present in all eukaryotic kingdoms. In higher plants, there is one highly extended family of VHS-GAT domain proteins - the TOL proteins. We have recently managed to show, that TOL protein substitute for the missing ESCRT-0 proteins (VHS-GAT domain subfamily in yeast and mammals), acting as principal gating factors for recognition of ubiquitinated cargo at the plasma membrane. Yet, while some TOL proteins function analogous to the ESCRT-0, others might perform diverse functions characteristic for other VHS-GAT domain proteins, or even plant-specific unique functions. Specifically, the proposed work should clarify aspects of endosomal trafficking in plants that require TOLs and also how this involvement is achieved. To this end we will characterize TOL interacting proteins in order to identify pathways and signaling events modulated by TOLs. Furthermore, structural and functional analysis of conserved TOL protein domains should result in insights into redundant and unique functions of the different TOLs. Finally we intend to address the dynamics of TOL expression and localization in response to diverse intrinsic and environmental stimuli. The outcome of this project will provide us with novel insights into the different roles of TOL proteins in vesicular trafficking in Arabidopsis thaliana and therefore into membrane protein trafficking and sorting pathways in higher plants at a molecular level. Furthermore it should represent the cornerstone for further studies, addressing the control of protein trafficking in the endosomal system of higher plants.

Since plants only have restricted abilities to be able to move away from unfavorable environmental conditions, they must respond quickly and accurately to an ever-changing environment. The plasma membrane of a cell is the interface between inside and outside (environment) and is interspersed with proteins that are essential for the detection and transmission of internal and external stimuli. Strict control of the amount but also the positioning of these proteins is crucial for adaptive processes. In eukaryotes, this is achieved by a complex system of membrane vesicles that serve to transport proteins to and from their site of action. Proteins destined for degradation are specifically tagged by attaching a small protein called ubiquitin. This allows them to be recognized by a large protein machinery that brings them to the vacuole for their destruction. We have recently shown that the evolutionarily conserved protein family of TOLs is needed for initial recognition of the proteins destined to be degraded. The main goal of this project was to find out not only where in the cell the TOL proteins localize but also to find determinants that control their function. TOL proteins contain two conserved ubiquitin-binding domains (UBDs) that recognize plasma membrane proteins destined for degradation. Ubiquitin receptors containing UBDs are often subject to a regulatory mechanism called coupled monoubiquitination, where their own ubiquitination prevents their ability to bind to other ubiquitins. Using TOL mutants that could no longer bind to ubiquitin and TOL mutants that consistently simulate the ubiquitinated status, we performed in vitro ubiquitin binding studies and successfully demonstrated a significant decrease in ubiquitin binding. Accordingly, we developed in planta TOL constructs to analyze the effect in the plant and were able to show that these constructs can no longer perform their function. The localization and function of the TOLs is therefore regulated by coupled monoubiquitination, which allows for fine-tuning in the degradation of plasma membrane proteins. The work accomplished here has greatly enhanced our understanding of the role of TOL proteins in plants by providing new insights into the regulation of membrane proteins in plants, thus elucidating mechanisms that allow plant growth and development to be fine-tuned to the environment.