Kinetochores are required to fully activate secretory pathway in elevated ER stress

Abstract

The study of protein misfolding has been a major focus in many ER stress-related diseases, including type I and type II diabetes, neurodegenerative diseases, atherosclerosis and some cancers. There is a basal level of naturally occurring misfolded proteins that are corrected by the unfolded protein response (UPR) system or are degraded by the ER-associated degradation system (ERAD). In pathological conditions where there are increased and enduring amounts of misfolded proteins in the ER, an elevated level of ER stress triggers the UPR signal further. This thesis is about determining whether the same set of genes are involved in basal and elevated levels of ER stress. We developed ER stress reporter assays to measure high levels of ER stress caused by chronic misfolding in a model system, namely a mutant of carboxy peptidase Y called CPY* which does not fold.

In this dissertation, genes that are required in basal levels and elevated levels of ER stress are compared in a genome-wide manner using the yeast deletion mutant array (DMA) and modified synthetic genetic array (mSGA) analysis. ER stress reporters used in CPY* over-expression conditions included induction of a general UPR promoter element (UPRE-GFP), induction of two COPII-related proteins (Yip3p and Erv29p) and of a sphingolipid regulatory protein (Orm2p). A novel group of kinetochore genes are required for full activation of all four ER stress reporters when CPY* is over-expressed. Deletion of kinetochore gene in the presence of CPY* over-expression does not cause a general reduction in protein synthesis. Instead, it results in the down-regulation of specific groups of lipid-related and protein trafficking proteins. To explain our results, a checkpoint must be present that monitors the function of the kinetochore or cell division related to ER stress levels, resulting in down regulation of UPR and secretory pathway proteins when the checkpoint is activated.

A genome-wide GFP-tagged protein screen revealed that Bfr1p is the possible checkpoint as it is genetically linked with IRE1, a central gene in the UPR, and the spindle pole body. In conditions of CPY* over-expression in the absence of the kinetochore gene CTF19, Bfr1p as well as Scp160p are down-regulated and possibly re-localised to the cytoplasm from the ER. Both of these are mRNA ribosome binding proteins and are specifically required for transporting secretory pathway gene mRNAs to the ER. The reduction in the secretory pathway proteins in the absence of kinetochore genes might be due to reduced levels of Bfr1p and Scp160p expression. Results reported here suggested that PKA activation is another possible pathway that down-regulates the UPR when CPY* is over-expressed in the absence of kinetochore genes. It is also proposed that Bfr1p is a checkpoint that monitors kinetochores and ER stress levels and induces a secondary pathway that is able to work in parallel to IRE-HAC1-UPR. An overall novel conclusion of this thesis is that cells in a chronic misfolding ER-stress situation require intact kinetochores to maintain an elevated UPR.