Functional Analysis of the TOM1 like (TOL) Protein Family

A coordinated control of intracellular protein distribution is of vital importance for all organisms. In fungi and animals, the ESCRT (Endosomal Sorting Complex Required for Transport) is responsible for the endosomal sorting of plasma membrane proteins for degradation at the lysosome/vacuole. For the most part, the ESCRT machinery appears to be conserved in plants, but so far only a limited number of studies have addressed a few aspects of its biological role. ESCRT activity has been implicated in the recognition of ubiquitinated membrane protein cargo, which is then passed on via early endosomes to the multivesicular body (MVB). In animals and fungi, initial recognition of ubiquitinated cargo depends on the ESCRT-0 complex, which -surprisingly- has not been identified in higher plants` genomes. Thus in plants recognition of membrane protein cargo at the plasma membrane, might depend on mechanisms dissimilar from those described in other organisms. A possible role has been attributed to an Arabidopsis gene family encoding TOM1-like (TOL) proteins, which share functionally important domains with the ESCRT-0 complex proteins and might thus substitute for ESCRT-0 tasks. This research project should increase our knowledge on the control of membrane protein fate in the model plant Arabidopsis thaliana by examining a possible role of the nine different TOL proteins in endosomal trafficking. The proposed work should clarify whether or not TOLs are principally involved in an ESCRT pathway in plants. A combination of state-of-the art cell biological and genetic approaches will be put forward for a concise functional TOL analysis. Specifically, by using a set of well-characterized potential ESCRT cargo proteins, the role of TOLs in membrane protein sorting will be determined. Moreover it is planned to perform a range of in vitro and in vivo TOL-domain binding studies, in order to determine a potential function of TOLs in endosomal trafficking in general and in the recognition of ubiquitinated membrane protein cargo in particular. Finally, large scale in vivo pull down experiments together with mass spectrometry should result in the identification of novel TOL interaction partners, which should help to determine the biological role of this protein family and will provide a foundation for a further analysis of the endocytic pathway in plants. Collectively, the outcome of this project is expected to result in novel insights into endosomal vesicular trafficking and protein sorting in Arabidopsis thaliana and thus, should represent a relevant contribution to our understanding of crosstalk between plants and an ever-changing environment.

This project unraveled essential mechanisms in the intracellular trafficking of plants by investigated a novel protein family, the TOL proteins, which function at the intersection between degradation and endocytosis and thus contribute to adaption mechanisms of plants to their variable surroundings. Plants, with their sessile life style, have evolved a plethora of mechanism to be able to respond quickly and accurately to an ever-changing environment. Since the plasma membrane functions an interface between the cellular compartment and the outside it is not surprising that several regulatory pathways are involved in the tight regulation of localization and activity of plasma membrane proteins. This underlines the essential role of the spatiotemporal control of plasma membrane protein turnover in plant development and responses.Proteins, which are removed from the plasma membrane, in other words endocytosed, either cycle between plasma membrane domains and endosomes or are transported for their irreversible destruction to the vacuoles. A key function in this sorting process has been attributed to ubiquitination. Recognition and transport of the ubiquitinated cargo proteins towards the vacuole is achieved by the conserved ESCRT (Endosomal Sorting Complex Required for Transport) machinery. The ESCRT-0, which functions in the initial recognition and concentration of the ubiquitinated cargo, serves as a central hub in the degradation of plasma membrane proteins. As no orthologs for the ESCRT-0 subunits in other eukaryotes have been identified in plants, this raised questions about proteins that could substitute for these in the otherwise highly conserved protein sorting machinery.In this project we could show that in the model plant Arabidopsis thaliana TOL proteins represent such determinants in terms of cargo recognition and sorting of ubiquitinated plasma membrane proteins. In the absence of TOLs, these proteins are no longer correctly transported to the vacuole for their degradation. The resulting defects lead to massive alterations in the development of the plants and its adaption processes. Our experiments identify TOL proteins as gatekeepers at the plasma membrane and moreover provide essential insights into the evolution of the eukaryotic sorting machinery, supporting a model, in which TOL proteins represent ancesteral cargo recognition determinants. Thus, life without ESCRT-0 could be accounted for by TOL proteins, which are seemingly sufficient for orchestration of the multifaceted growth responses that characterize the development of higher plants.