Analysis of membrane protein translocation pathways in Arabidopsis thaliana

Abstract

Insertion of proteins in the membrane of the endoplasmic reticulum is a pivotal process during their biogenesis. Tail-anchored (TA) proteins, a specific class of membrane proteins, play key roles in many vital cellular processes in almost all cellular membranes. Proper translocation is therefore pivotal. Their characteristic single carboxy-terminal transmembrane domain (TMD) dictates a post-translational translocation, as translation termination occurs concurrent with release from the ribosome. For that reason, TA proteins are prone to aggregation requiring accurate guidance to the destined membranes, and dedicated insertion pathways. In this thesis, orthologues of one such pathway, the Guided entry of TA protein (GET) pathway was identified in Arabidopsis thaliana. Subcellular localisation, extensive interaction studies and characterization of T-DNA insertion lines revealed the conservation and importance of AtGET1, AtGET3a and AtGET4 in a TA protein translocation pathway. Abolishing their function resulted in reduced root hair growth which coincided with reduced protein levels of the TA protein SYP123, which is important for root hair tip growth. However, AtGET seemed to be dispensable and less than 5% of predicted TA proteins in Arabidopsis were shown to interact with AtGET-proteins raising the question about alternative routes evolved in plants by which TA proteins can be targeted to the ER. The investigation of AtGET1-GFP interaction by an immunoprecipitation-mass spectrometry approach led to the discovery of a potential GET-receptor component, G1IP (AtGET1-interacting protein). It did not share sequence homology with yeast GET2 or mammalian CAML yet its subcellular localisation and a functional analysis associated G1IP as an AtGET1 co-receptor structurally and functionally related to GET2/CAML. Furthermore, in this thesis one functional orthologue of the SRP-independent (SND) pathway was identified in Arabidopsis thaliana, that exists as two homologs. Investigation of T-DNA insertion lines and interaction screens implicated both proteins, AtSND2a and AtSND2b, as components of a sophisticated system for translocation and/or stress response. In this study we investigated similarities to the yeast/mammal SND2 but highlighted and discussed contrasts to these as well. Large parts of this study were based on extensive protein-protein interaction (PPI) analyses. We made use of ratiometric Bimolecular Fluorescence Complementation (rBiFC), Förster Resonance Energy Transfer Acceptor Photobleaching (FRET-AB) and Fluorescent Lifetime Imaging (FRET-FLIM) and reported here on their recent improvements by incorporating a 2in1-cloning approach. Putative components of the Arabidopsis SEC61 translocon served as examples.