The biogenesis of membrane proteins in the endoplasmic reticulum

A central theme in modern cell biology is the elucidation of molecular events that occur during early stages of protein folding and processing in the endoplasmic reticulum (ER). Integral to these processes is the interaction of heterologous proteins within the secretory pathway. We will use beta-amyloid presursor protein (APP) and Notch-1 as model proteins to study biogenesis and processing in the ER.

Initially, the project will examine the sequence of events and interactions occurring after translocation of APP and Notch-1 across the ER membrane, for example, the role of molecular chaperones and the dynamics of presenilin (PS) association. The project will go on to attempt to identify complex components involved in APP processing and Notch-1 cleavage. Finally, experimentation may be expanded to examine the role of mutations on the processing of both Notch-1 and APP, and, in particular, the generation of amyloid-beta peptide.

To recreate APP and Notch-1 folding and processing an in-vitro translation approach will be established that has been used previously (see below) to study the folding and assembly of multisubunit proteins in the ER. Folding and processing will be assessed by immunoprecipitation with conformation specific antibodies. Association with the secretory pathway folding machinery will be determined by co-immunoprecipitation with antibodies to chaperone proteins in a native state or following cross-linking.

This project has the potential to contribute to both the elucidation of the molecular biology of Alzheimers disease and an important area of basic cell biology. The project will be carried out in a lively and stimulating atmosphere within a vibrant neuroscience department that has extensive expertise available. The student will gain experience in the techniques of molecular biology, cell biology and protein biochemistry, including mutagenesis, SDS-PAGE separation of proteins, immunoprecipitation and cell culture.

For further background and technical information see:
Mark R. Farmery, Simon Allen, Amanda J. Allen, and Neil J. Bulleid (2000).
The Role of ERp57 in Disulfide Bond Formation during the Assembly of Major Histocompatibility Complex Class I in a Synchronized Semipermeabilized Cell Translation System. J. Biol. Chem. 275:14933-149388

Frederic Checler (2001). The multiple paradoxes of presenilins. J Neurochem 76:1621-7