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Petris, Gianluca
(2013).
DOI: https://doi.org/10.21954/ou.ro.0000f05c
Abstract
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-1α chain, the non-secretory null-Hong Kong mutant of α1 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-2/Tetherin, 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-2/Tetherin 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-κ 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.