%0 Generic
%A Pajonk, Oliver
%D 2025
%F heidok:35910
%R 10.11588/heidok.00035910
%T ESCRT function at stress-induced ER-Golgi contact sites in Saccharomyces cerevisiae
%U https://archiv.ub.uni-heidelberg.de/volltextserver/35910/
%X The endoplasmic reticulum (ER) is the major site of protein folding and lipid synthesis in eukaryotic cells. All secretory proteins are translocated into the ER, folded and modified, packed into vesicles and eventually exported towards the Golgi apparatus. These essential processes are menaced by changing environmental conditions or cellular needs. When the folding capacity in the ER lumen is overwhelmed or when lipid bilayer composition and stiffness deviate from the functional steady state, cells experience ER stress. Cells have mechanisms in place to cope with ER stress and restore homeostasis, one of which is the unfolded protein response (UPR). In this pathway, a transcriptional cascade is triggered that alleviates stress by the upregulation of chaperones and lipid synthesis genes. In the yeast Saccharomyces cerevisiae, ER stress also triggers the UPR-independent recruitment of ESCRT-III component Snf7p to specific ER domains termed ER clusters. The biological role of ESCRT recruitment to ER clusters and its contribution to ER stress relief are not known.  In this thesis, I characterized the phenotype of Snf7 recruitment to ER clusters in yeast. I showed that the ESCRT-associated protein Bro1 is essential for the recruitment of fluorescently labelled Snf7 to ER clusters and provided evidence that this recruitment reflects a physiological process. I performed genetic screens to identify genes involved in the formation of ER clusters upon ER stress and genes involved in recruiting Snf7 to these sites. I determined that glycosylphosphatidylinositol (GPI)-anchor remodeling proteins, the p24 adaptor complex for ER-to-Golgi transport and its interaction with COPII proteins, the COPII ER export machinery and the Dsl1 tethering complex are required for the formation of ER clusters. I dissected the spatial arrangement of COPI component Cop1p and COPII components at ER clusters in live cell imaging and determined that Cop1p frequently co-localizes with Snf7 puncta at ER clusters whereas COPII proteins surround Snf7 puncta in a flower-like assembly. Furthermore, I observed that the ER tethering proteins Nvj2p and Tcb3p co-localize with Snf7 puncta at ER clusters and that re- localization of Tcb3p to ER clusters depends on successful ESCRT recruitment. Both Nvj2p and Tcb3p were shown to be involved in non-vesicular ceramide transport as ER-Golgi tethers during ER stress. Given the co-localization of Tcb3p and Nvj2p with Snf7 and Cop1p at ER clusters, it is conceivable that ER clusters represent ER-Golgi contact sites that might facilitate non-vesicular transport of ceramide during ER stress and thereby increase ceramide transport efficiency.  In conclusion, I characterized ESCRT recruitment to ER clusters upon ER stress and assigned a potential physiological role to this process. This study adds to our understanding of ER stress responses in yeast. A better understanding of how different stress responses contribute to stress relief in yeast will improve our understanding of homologous processes in mammalian systems.