The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It is widely observed that some mitochondrial outer membrane (OM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in mistargeting of non-optimal mitochondrial OM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways

Analysis of membrane protein translocation pathways in Arabidopsis thaliana

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.Dissertation ist gesperrt bis 27.10.2023

The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER

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ER membrane receptors of the GET pathway are conserved throughout eukaryotes

Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plant Arabidopsis thaliana, most components of the pathway were identified through in silico sequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation- mass spectrometry analysis to detect in vivo interactors of AtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses with AtGET1, and binds to Arabidopsis GET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of other Atget lines, its heterologous expression together with the coreceptor AtGET1 rescues growth defects of Δget1get2 yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 in Arabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.Fil: Lisa Yasmin Asseck. Eberhard Karls Universität Tübingen.; AlemaniaFil: Mehlhorn, Dietmar Gerald. Eberhard Karls Universität Tübingen.; AlemaniaFil: Monroy, Jhon Rivera. Universität Göttingen; AlemaniaFil: Ricardi, Martiniano María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Breuninger, Holger. Eberhard Karls Universität Tübingen.; AlemaniaFil: Wallmeroth, Niklas. Eberhard Karls Universität Tübingen.; AlemaniaFil: Berendzen, Kenneth Wayne. Eberhard Karls Universität Tübingen.; AlemaniaFil: Nowrousian, Minou. Ruhr Universität Bochum; AlemaniaFil: Xing, Shuping. Eberhard Karls Universität Tübingen.; AlemaniaFil: Blanche Schwappach. Universität Göttingen; AlemaniaFil: Bayer, Martin. Institut Max Planck Fuer Gesellschaft. Institut Fur Entwicklungsbiolobie. Developmental Biology; AlemaniaFil: Grefen, Christopher. Eberhard Karls Universität Tübingen.; Alemani