Award details

Establishment of signalling specificity in endoplsmic reticulum to nucleus signalling pathways

Reference	BB/C513418/1
Principal Investigator / Supervisor	Dr Martin Schroeder
Co-Investigators / Co-Supervisors
Institution	Durham University
Department	Biological and Biomedical Sciences
Funding type	Research
Value (£)	210,846
Status	Completed
Type	Research Grant
Start date	30/09/2005
End date	29/09/2008
Duration	36 months

Abstract

The unfolded protein response (UPR) is a signal transduction pathway from the endoplasmic reticulum (ER) to the nucleus. Unfolded proteins in the ER activate the UPR. The UPR increases the biosynthetic capacity of the ER through induction of molecular chaperones and foldases and decreases the influx of unfolded proteins into the ER through attenuation of transcription of genes encoding secretory proteins and attenuation of translation. In addition, the UPR increases the clearance of unfolded proteins from the ER through up-regulation of ER-associated degradation (ERAD). In yeast, the UPR is transduced through non-spliceosomal splicing of the mRNA for the bZIP transcription factor Hac1p initiated by the transmembrane kinase-endoribonuclease Ire1 Hac1p activates transcription of genes encoding ER resident molecular chaperones, foldases, and of genes involved in ERAD through the promoter element UPRE. Transcriptional activation by Hac1p is dependent on the SAGA histone acetyltransferase (HAT). I showed that Hac1p is also a transcriptional repressor of metabolic genes and of genes involved in starvation responses in unstressed cells. Transcriptional repression by Hac1p required the promoter element URS1, the URS1 binding protein Ume6p, and the RPD3 histone deacetylase (HDAC). I also showed that Hac1p interacted with the HDAC in co-immunoprecipitation experiments. I propose to test if repression of metabolic genes by Hac1p is a new arm of the UPR that decreases the biosynthetic load of the ER in periods of ER stress (hypothesis #1). I will compare survival of ER stress by strains in which repression of Ume6p targets by Hac1p is abolished to WT strains. To provide maximum sensitivity this experiment will be performed in strains in which one established arm of the protective response co-ordinated by the UPR is abolished, e.g. in strains with impaired ER luminal chaperone function and in strains in which ERAD is blocked. Further, the utilisation of two directly opposing transcriptional regulators, a HAT and a HDAC, by Hac1p implies that mechanisms that ensure signalling specificity by Hac1p exist. To demonstrate that the activating and/or repressing activity of Hac1p is regulated I will identify physiological conditions in which both activities of Hac1p are differentially regulated. I will measure the effect of ER stress on repression of Ume6p controlled genes using lacZ reporter assays, Northern blotting, and real time reverse transcriptase PCR to determine if Hac1p is an efficient repressor of Ume6p controlled genes in ER stressed cells. In addition, co-immunprecipitation experiments will establish if Hac1p interacts with the HDAC in ER stressed cells. Finally, I will determine if the proximal UPR transducer Ire1p regulates the repressing function of spliced Hac1p by comparing expression of lacZ reporters and endogenous genes between WT cells and cells deleted for IRE1 in which expression of spliced Hac1p is regulated by an inducible expression system. If so, I will use several mutants in the kinase and endoribonuclease domains of Ire1p to distinguish if the kinase or endoribonuclease function are involved in this regulation.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search