Characterization of an organelle integrity checkpoint

Lead Research Organisation: Durham University
Department Name: Biological and Biomedical Sciences

Abstract

Cellular 'checkpoints' create a hierarchy between otherwise unrelated cellular events. For example, a delay in DNA replication results in a delay in nuclear division due to the 'DNA replication checkpoints'. They are signal transduction pathways that link these unrelated events, e.g. sensing of a block in DNA replication is transduced by a signalling pathway to inhibit nuclear division. The majority of checkpoints have been identified in the cell cycle. However, additional checkpoints exist, e.g. the morphogenesis checkpoint that responds to perturbations in the cytoskeleton. Based on my previous work I propose that a checkpoint exists that links integrity of an eukaryotic organelle, the endoplasmic reticulum, to differentiation programs that down the line will require an intact endoplasmic reticulum. The purpose of this checkpoint is that only cells that can successfully complete the differentiation process will initiate it. Cells with compromised ER function would die trying to complete the differentiation program. The checkpoint consists of two signalling events, one of which I already characterized in some detail. The second one is less well understand at the moment. The only thing we currently know is that cells lacking upstream elements of the checkpoint are defective in initiating the differentiation program. Further, genes involved in the first signalling activity of this checkpoint are not involved in the second signalling event. From a molecular perspective this gives me the opportunity to uncover a novel signal transduction pathway by studying this second signalling event. I will use a range of genetic experiments in Baker's yeast to identify components of this second signalling pathway of this endoplasmic reticulum integrity checkpoint. Biochemical experiments will then be performed to further understand the role of these genes and proteins in this signalling pathway.

Technical Summary

This proposal addresses two new concepts: 1. I hypothesize that organelle integrity checkpoints exist. 2. I hypothesize that a new signal transduction pathway in the eukaryotic unfolded protein response (UPR) exists. This work will advance our understanding of mechanisms of signal integration. The UPR is activated when protein folding in the endoplasmic reticulum (ER) is inhibited. Regulation of all known adaptive responses to ER stress by the UPR requires two genes, IRE1 and HAC1. Ire1 is a bifunctional kinase-endoribonuclease that initiates non-spliceosomal splicing of HAC1 mRNA. I identified two events through which the UPR regulates entry of yeast into meiosis. Meiosis is governed by a transcriptional cascade consisting of IME1, early, middle and late meiotic genes. Ime1p activates transcription of early meiotic genes (EMGs). I showed that Hac1p represses EMG transcription, but had no effect on transcription of IME1. Cells deleted for IRE1 were defective in transcription of IME1. Expression of spliced Hac1p in IRE1 deletion cells had no effect on IME1 mRNA levels, arguing that Ire1p regulates IME1 independent of HAC1. I propose that these two signalling activities of the UPR are an organelle integrity checkpoint. Meiosis requires synthesis of phospholipids and cell wall proteins in the ER. The function of this checkpoint is to delay entry into meiosis when ER function, phospholipid, and cell wall synthesis are perturbed. To gain insight into how Ire1p regulates IME1 and to identify new factors downstream of Ire1p I will use three independent but converging genetic approaches: 1. I will determine if the kinase, RNase, or unknown activity of Ire1p is required for regulation of IME1 using chemical genetics. 2. I will determine the minimal region in the IME1 promoter that is responsive to Ire1p. 3. I will determine if IRE1 signals through a pathway that was already implicated in regulation of IME1, or if the pathway originating at Ire1p is novel.

Publications

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Cox DJ (2011) Measuring signaling by the unfolded protein response. in Methods in enzymology

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Parmar V (2012) Self and Nonself

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Schröder M (2008) Engineering eukaryotic protein factories. in Biotechnology letters

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Schröder M (2008) Endoplasmic reticulum stress responses. in Cellular and molecular life sciences : CMLS

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Schröder M (2007) Recent advances in understanding the unfolded protein response. in Antioxidants & redox signaling

 
Description Identification of events in signal transduction by the ER stress sensor IRE1. Once the work is published a more detailed description of the results will be made available.
Exploitation Route Results from this work allow the separation of prosurvival and pro-apoptotic signaling of human IRE1alpha. This knowledge can be exploited, for example, in mammalian cell lines used for industrial protein production. To do this I entered into a collaboration with Lonza Biologics. Initial work toward exploitation of this knowledge was funded through a BBSRC CASE studentship award. Results from this work allow the separation of prosurvival and pro-apoptotic signaling of human IRE1alpha. This knowledge can be exploited, for example, in mammalian cell lines used for industrial protein production. To do this I entered into a collaboration with Lonza Biologics. Initial work toward exploitation of this knowledge was funded through a BBSRC CASE studentship award.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Identification of signal transduction activities by IRE1 helps protein manufscturers to improve their cellular expression systems.
First Year Of Impact 2010
Sector Chemicals,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Chemical genetic dissection of efferent IRE1alpha signaling
Amount £83,000 (GBP)
Funding ID BB/D526188/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2012
 
Title Ire1 Plasmids 
Description Plasmids for expression of wild type (WT) and mutant yeast Ire1 in Escherichia coli and Saccharomyces cerevisiae (single and multiple copy). The plasmids for E. coli encode N-terminal GST fusion proteins. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title PIME1-lacZ reporter plasmids 
Description Reporter plasmids in which thye IME1 promoter is fused to lacZ. Several truncations have been made. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title Yeast strains 
Description Yeast strains deleted for IRE1. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Description Collaboration/Partnership with Lonza Biologics (Slough, Berkshire) 
Organisation Lonza Group
Country Global 
Sector Private 
PI Contribution Collaboration with Lonza Biologics (Slough, Berkshire) to exploit knowledge gain on the unfolded protein response to improve heterolgous protein production in mammalian cells.
Start Year 2008
 
Description Slovak Academy of Sciences 
Organisation Slovak Academy of Sciences
Country Slovakia 
Sector Public 
PI Contribution Collaboration with Dr. Sergej Sestak
Start Year 2009
 
Description A role for the unfolded protein response in nutrient sensing and control of differentiation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A role for the unfolded protein response in nutrient sensing and control of differentiation

no actual impacts realised to date
Year(s) Of Engagement Activity 2007