Understanding the Function of TOLs in Plants

Since plants are sessile organisms, their biggest challenge is the ever changing environment to which they have to be able to respond quickly and accurately. The plasma membrane, as interface between inside and outside, is densely packed with proteins that are essential in sensing and transmitting internal and external stimuli. Thus, tight regulation of not only abundance but also localization of these proteins is pivotal to adaptive processes. In eukaryotes, this is achieved by a complex system of internal membranes that serve to transport proteins to their site of action. Proteins destined for degradation are ubiquitinated, endocytosed and transported to the vacuole for ultimate destruction by a complex machinery called the ESCRT machinery. We have recently managed to show that the initial recognition of the cargo to be degraded, is accomplished by an evolutionary conserved family, the TOL protein family. The principal aim of this project is to assess not only where but also how the TOLs localize in the endomembrane system and thus learn about determinants that control TOL distribution and abundance. This will allow us to address the function of this protein family in the model plant Arabidopsis thaliana and additionally help elucidate activities of individual TOLs. Thus this project will not only close a gap in the knowledge about the endosomal system of plants, but furthermore will serve as a cornerstone for future studies. The work suggested here will greatly enhance our understanding of the role of TOL proteins in plants, providing novel insights into the regulation of ubiquitinated membrane protein turn over in plants and thus the fine-tuning many different aspects of plant growth and development in response to the environment.

Due to accelerating climate change and the resulting rapid increase in extreme environmental conditions, the question of how plants can flexibly adapt to their constantly changing environment is a highly topical area of research. The plasma membrane serves as boundary between the inside and the outside of the cell and is densely packed with proteins that are responsible for sensing and transmitting stimuli. Responses to stresses, for example, are regulated and coordinated by phytohormones, whose transport and perception are commonly centered around plasma membrane-localized proteins. Plants thus have to be able to quickly and accurately adjust not only the localization but also the amount of their plasma membrane proteins. This is organized by endosomal trafficking, which transports membrane proteins to and from their site of action. Here, the post-translational modification of plasma membrane proteins with the small protein ubiquitin is key in signaling their removal from the plasma membrane by endocytosis and further degradation in the vacuole. This project strove to unravel the function of the TOM1-like (TOL) protein family in the recognition of ubiquitinated plasma membrane proteins destined for degradation. For this purpose, an in-depth functional characterization of the TOL protein family in the endosomal system of the model plant Arabidopsis thaliana was conducted and we found that while some TOL proteins localize to the plasma membrane, others are found in the cytoplasm and in early endosomal structures. Conserved elements of the endosomal pathways in plants were assessed for binding to the TOLs by in vitro and in vivo binding studies. This helped to intensify functional links between TOL proteins and the plant sorting machinery and thus positioned the TOLS in the early steps of the degradation pathways from the plasma membrane to the vacuole. Finally, we investigated a potential regulatory pathway involving the ubiquitin-binding domains, termed coupled ubiquitination. Through different approaches, including constitutive ubiquitination and site-directed mutagenesis of ubiquitin binding domains, we addressed the influence of ubiquitination on the localization and function of the TOLs, particularly of TOL6. Thus, this project closed a gap in the knowledge about the endosomal system of plants, and furthermore served as important cornerstone for future studies.