Secondary structure of the nascent chain as regulatory element for translocation into the endoplasmic reticulum; mechanisms and pathophysiological relevance

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Around 25-30% of all proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Membrane integration and import into the lumen of the endoplasmic reticulum (ER) is mainly mediated by a heterotrimeric membrane protein complex called Sec61 complex, which is homologous to the SecY complex in bacteria. A major fraction of secretory proteins contains N-terminal signal peptides that mediate transport of the nascent chain to the Sec61 translocon and initiate translocation into the ER lumen. Failure to deliver secretory or membrane proteins to the ER can have fatal consequences. First, these proteins do not reach their final destination (loss of their physiological function) and second, they may form cytosolic aggregates with toxic activities based on the presence of hydrophobic domains (gain of toxic function). For example, mislocalization of the cellular prion protein to the cytosol can induce neuronal cell death in transgenic mice and mammalian cell culture models. Thus, quality control pathways have evolved to eliminate mistargeted proteins. Our work revealed an intrinsic impairment of the Sec61/SecY complex of eukaryotes/bacteria to translocate secretory proteins that are entirely unstructured. The research proposal focusses on a detailed understanding of the mechanisms by which structural features of the polypeptide chain modulate the translocation process. In addition, pathophysiological consequences of a defective ER import will be addressed. Specifically, we will investigate pathways that enable the mammalian translocon to translocate client proteins with extended unstructured domains and study a deregulated translocation during stress and in cells propagating neurotoxic protein conformers. Finally, we will investigate the fate of non-translocated secretory proteins to evaluate the impact of an uncleaved signal peptide on degradation, subsequent trafficking and cytotoxic activity. The experimental approaches include in vitro assays,bacterial, yeast and mammalian cell culture models, including primary neurons, and mouse models of neurodegenerative diseases.