CHAPERONE-ASSISTED PROTEIN FOLDING AT THE ER MEMBRANE

PhD student project 3
(Early Stage Researcher 3, ESR3)

CHAPERONE-ASSISTED PROTEIN FOLDING AT THE ER MEMBRANE

Supervision: Ineke Braakman

Early Stage Researcher: Daniel Fonseca

   

Aim:
To determine the role of Hsp90 and its co-chaperones on folding of CFTR and the disease mutant F508del.

Methodology:
The aim is to determine the role of Hsp90 and its co-chaperones on folding of the cystic fibrosis-related CFTR protein and the disease mutant F508del. We will use established methodology in our lab, which employs radioactive labelling of cells expressing wt/F508del CFTR or part of it (a single domain for instance). Alternative is an in-vitro translation in the presence of ER membranes and 35S-labeled amino acids. We will examine the effect of Hsp90 and its functional mutants (affected in ATP binding or hydrolysis or in client binding or release) and the effect of co-chaperones on folding of CFTR. Folding will be probed with limited concentrations of proteases, if necessary followed by isolation of domain-specific fragments using domain-specific antibodies.

Collaborators:
Rüdiger; Thomas (McGill); Nyken

Key publications:

1. I. Braakman, N. Bulleid (2011) ‘Cellular Protein Folding and Modification in the Endoplasmic Reticulum.’ Annu. Rev. Biochem. 80: 9.1-9.29.
2. *H. Hoelen, *B. Kleizen, A. Schmidt, J. Richardson, P. Charitou, P.J. Thomas, I. Braakman (2010) 'The primary folding defect and rescue of ∆F508 CFTR emerge during translation of the mutant domain.' PLoS ONE 5: e15458. *joint first authors
3. B. Kleizen, B., T. van Vlijmen, H. de Jonge, I. Braakman (2005) CFTR folds predominantly co-translational. Mol. Cell. 20: 277-287.