Award details

Investigating the link between signal peptide peptidase and membrane protein quality control

Reference	BB/C500152/1
Principal Investigator / Supervisor	Professor Stephen High
Co-Investigators / Co-Supervisors	Mr Samuel Crawshaw
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	226,582
Status	Completed
Type	Research Grant
Start date	01/10/2004
End date	30/09/2007
Duration	36 months

Abstract

The primary objective of this project is to investigate the role of the intramembrane aspartic protease, signal peptide peptidase (SPP), in the quality control of misassembled integral membrane proteins at the endoplasmic reticulum (ER). Site-specific cross-linking experiments indicate that SPP specifically associates with transmembrane (TM) domains that have been prevented from assembling with their normal partners. The focus of this work will be to define the specific properties of a TM domain that promote its assembly with SPP and to establish whether SPP can cleave the TM domains with which it associates. An in vitro cross-linking approach will be used to generate a set of well-defined TM fragments that associate with SPP together with control TM domains that do not. A subset of these fragments will be taken forward and their association with SPP will be analysed in vivo enabling a kinetic analysis to be performed. In each case, the specificity of SPP association will be confirmed using a specific inhibitor. The ability of SPP to cleave specific fragments of polytopic membrane proteins that have been defined as potential SPP substrates will be examined both in vitro and in vivo. By using combinations of different inhibitors, the specific roles of SPP and the proteasome will be investigated, links to the process of ER associated degradation will be determined, and the order in which distinct proteolytic cleavages occur will be defined. The role of SPP in the quality control of full-length polytopic proteins will be investigated using precursors that contain clearly defined SPP substrates when fragments of the proteins are expressed in isolation. In addition to utilising well defined inhibitors, we will explore the use of RNAi based knock downs of SPP in order to precisely define its function in the quality control of membrane proteins at the ER.

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

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