Use this URL to cite or link to this record in EThOS:
Title: The mechanism of ER to cytosol retrotranslocation of proteins targeted to ERAD (ER association degradation)
Author: Petris, Gianluca
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2013
Availability of Full Text:
Access from EThOS:
Retro-translocation from the endoplasmic reticulum (ER) to the cytosol of secretory and membrane proteins takes place on misfolded molecules targeted for proteasomal degradation, in a process called ER associated degradation (ERAD), Because of the difficulties in clearly discriminating the fraction of molecules already retro-translocated from the ones in the ER, we took advantage of the E coli biotin-ligase (BirA) expressed in the cytosol of mammalian cells, to specifically biotin-label proteins that undergo retro-translocation, The method was validated using four different 'model proteins, known to undergo retro-translocation upon different conditions; the MHC-Iα chain, the non-secretory null-Hong Kong mutant of 01 antitrypsin, the immunoglobulin γH chain and calreticulin, The -- specific mono-biotinylation of only cytosolically dislocated molecules resulted in a novel quantitative method to determine the extent of retro-translocation. The method was used to study dislocation of CD4 and BST-2ITetherin, two membrane proteins targeted to degradation by the HIV-1 protein Vpu, It was found that CD4 retro-translocates with oxidised intra-chain disulphide bridges that only upon proteasomal inhibition accumulates in the cytosol in reduced and de-glycosylated form, Similarly, BST-2lTetherin is first exposed to the cytosol as a dimeric-oxidised complex, which then becomes de-glycosylated and reduced to monomers, Experiments with the non-secreted NS1 Ig -K light chain showed that also this ERAD model protein is retro-translocated with oxidised cysteines, The role of VCP/p97-ATPase in retro-translocation was investigated, In contrast to what previously reported , it was found that it is not required to dislocate ERAD substrates from the ER lumen to the cytosol, while it is required for efficient de-glycosylation and proteasomal degradation of ERAD substrates, The results obtained indicate that complete cysteine reduction and unfolding is not strictly required for retro-translocation, suggesting alternative mechanisms of the ERAD pathway, In addition, the role of VCP/p97-ATPase was found associated to stages downstream of membrane crossing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available