Analysis of GET pathway receptors in Arabidopsis thaliana

Tail-anchored (TA) proteins are a class of integral membrane proteins that are involved in diverse cellular processes, including protein translocation (SEC61), vesicle trafficking (SNAREs) and apoptosis (BCL-2). A transmembrane domain (TMD) at the extreme C-terminus prevents TA proteins access to the classical co-translational route into the endoplasmic reticulum (ER); instead, they require post-translational insertion via the Guided Entry of Tail-anchored proteins (GET) pathway that was recently described in yeast and mammals. Here, the cytosolic targeting factor GET3 (in yeast; TRC40 in mammals) chaperones newly synthesized TA proteins from the ribosome to the ER where the GET1-GET2 (in yeast; WRB-CAML in mammals) receptor complex facilitates membrane insertion. However, so far hardly anything is known about the conservation and function of the GET pathway in higher plants. We identified several GET components in Arabidopsis thaliana through in silico analysis, including an orthologue of GET1, and found a role of the GET pathway in root hair elongation. Additionally, direct in planta interaction analysis using immunoprecipitation-mass spectrometry of AtGET1-GFP expressing lines identified a promising candidate for the membrane receptor GET2, which has no sequence orthologue in plants. Remarkably, this protein resembles a similar TMD architecture as GET2 and CAML and carries a conserved positively charged motif in its N-terminus. We provide here experimental evidence that this protein of unknown function might indeed represent the functional GET2 orthologue in Arabidopsis thaliana

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