Master of Research
School of Biological Sciences
ER chaperones are a critical component of cellular proteostasis that assist proteins to gain and maintain their proper functional folding state, and direct irreversibly misfolded proteins to degradation. Recent studies have demonstrated endoplasmic reticulum (ER) stress-induced release of many ER-resident chaperones from the ER into the cytosol, including binding immunoglobulin protein (BiP), calreticulin (CRT), protein disulfide isomerase (PDI), and the normally secreted chaperone clusterin (CLU). Although the physical pathway used by these chaperones to reach the cytosol from the ER is yet unknown, recently N-terminal arginylation of ER chaperone has been put forward as a mechanism of their release. Evidence is accumulating to suggest that these chaperones play cytoprotective roles in the cytosol and direct misfolded proteins for degradation. This study assessed whether it was possible to use confocal microscopy to image the ER stress-induced release of chaperones from the ER/Golgi to the cytosol. Following optimisation of cultured cell preparation, and image acquisition parameters, this was successfully achieved in both HEK293 and HeLa cells. In HeLa cells, chaperone release to the cytosol was demonstrated in response to three different inducers of ER stress - DTT, MG132, and brefeldin A. The results suggest that release of chaperones from the ER to the cytosol is a general cell response to ER stress. CNX was not released from the ER under these conditions, establishing that the results cannot be explained by a simple loss of ER membrane integrity. The results presented in this thesis are not in themselves conclusive, but when combined with future work using other techniques to interrogate the same phenomenon, should help establish the processes underpinning this potentially very important new proteostasis mechanism.
Purja, Usha, A confocal microscopy investigation of the release of chaperones from the endoplasmic reticulum during proteostasis stress, Master of Research thesis, School of Biological Sciences, University of Wollongong, 2018. https://ro.uow.edu.au/theses1/486