Understanding the Function of TOLs in plants
Abstract
To be able to respond quickly and accurately to an ever changing environment, plants have to adjust the abundance and localization of their plasma membrane proteins in a tightly controlled manner. This project strives to unravel essential mechanisms in the intracellular trafficking of higher plants, by investigating a protein family functioning at the intersection between degradation and endocytosis. The TOL (TOM1-like) protein family are thought to be responsible for initiating the pathway of guiding ubiquitinated plasma membrane proteins to the vacuole for their final degradation. The precise starting point for this interaction is currently still unresolved. The principal aim of this project is to assess not only where but also how the TOLs localize in the endomembrane system, in other words influenced by which determinants. This will allow us to address the function of this protein family in the model plant Arabidopsis thaliana and could additionally help elucidate activities of individual TOLs, different from those we previously described. Through in depth systematic localization study of all TOLs, which will include construction of reporters and their subcellular analysis, co-localization with known endosomal markers as well as membrane fractionation experiments, we intend to find out the precise localization of the TOLs. This will allow us to assess differences between the individual TOLs, but additionally, to discriminate them from their mutagenized counterparts. Through genetic interactions with important component of the plant’s endosomal machinery we intend to learn about pathways in which the TOLs participate. Conserved elements of these endosomal pathways like ESCRT subunits, clathrin, ubiquitin and phospholipids will be analyzed by systematic in vitro binding studies to the TOLs, which will help to intensify functional links between TOL proteins and the plant sorting machinery in order to position the TOLs in particular pathways. This should reveal whether or not Arabidopsis TOLs can functionally replace the elusive plant ESCRT-0 complex. Furthermore, the TOL interactome will be extended by searching for novel binding partners by immunoprecipitation and mass spectrometry, which might reinforce already know pathways and possibly establish new one. Finally, we will investigate a potential regulatory pathway involving the UBDs, termed coupled monoubiquitination. Through different approaches, including constitutive ubiquitination and site-directed mutagenesis of the UBDs, we want to address the influence of ubiquitination on the localization and function of the TOLs. Thus, this project will not only potentially close a gap in the knowledge about the endosomal system of plants, but furthermore it will serve as important cornerstone for future studies.
Project staff
Barbara Korbei
Priv.Doz. Mag. Dr. Barbara Korbei
barbara.korbei@boku.ac.at
Tel: +43 1 47654-94230
Project Leader
01.11.2017 - 31.10.2021
Jeanette Carolina Moulinier Anzola
Dipl.-Chem. Dr. Jeanette Carolina Moulinier Anzola
jeanettemoulinier@boku.ac.at
Tel: +43 1 47654-94258
Project Staff
01.11.2017 - 31.10.2021
Rebecca Tikowsky
Rebecca Tikowsky B.Sc.
rebecca.tikowsky@boku.ac.at
Tel: +43 1 47654-94066
Project Staff
01.11.2017 - 31.10.2021