Characterization of stress-induced cytosolic assemblies containing components of the guided-entry of tail-anchored proteins targeting pathway

by Jonas Jennrich
Doctoral thesis
Date of Examination:2024-08-09
Date of issue:2024-09-04
Advisor:Dr. Katherine E Bohnsack
Referee:Dr. Katherine E Bohnsack
Referee:Prof. Dr. Jörg Stülke
Persistent Address: http://dx.doi.org/10.53846/goediss-10715

 

 

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Abstract

English

Up to 30% of eukaryotic proteins are integral membrane proteins and need to be targeted to their appropriate membrane locations. The guided entry of tail-anchored proteins (GET) is a key targeting route to the ER membrane for specific classes of proteins. Nascent client proteins are captured by a pre-targeting complex comprising of Sgt2, Get4, and Get5 and handed over to the ATPase Get3 that delivers them to the ER-bound GET receptor, formed by Get1 and Get2. As the last step in gene expression of membrane proteins, protein targeting needs to be adapted in different conditions. Upon cellular stress, cells mount an adaptive response to ensure viability. This can involve the regulation of transcription, translation and post-translational modifications of proteins, as well as the formation of membrane-less organelles. Sequestration of particular proteins in such compartments can contribute to the recovery of cells form the stress assault. It has been observed that the Get3 ATPase component of the GET pathway re-localizes to cytoplasmic foci upon glucose starvation. This study focused on the characterization of these foci, termed GET bodies. The distinction between acute glucose starvation-induced GET bodies as unique and other stress-induced assemblies (P-bodies and stress granules) was highlighted. The roles of the soluble GET pathway components in the formation of GET bodies was assessed revealing different effects on the GET body phenotype. The composition of GET bodies was determined by their enrichment from glucose-starved cells followed by mass spectrometry and via a high throughput microscopy-based co-localization screen. The temporal order of protein assembly into GET bodies was analyzed and interdependencies in terms of recruitment were examined. The dynamics of GET bodies were assessed with experiments focusing on their resolution and potential triggers of their formation and disassembly were uncovered. The results suggest that GET bodies might be a central hub, in which redox-state sensitive chaperones and holdases assist the folding of proteins in oxidizing conditions within yeast cells.
Keywords: chaperone; protein targeting; Get3 ATPase; membrane-less organelle; glucose metabolism; NADH; cellular stress