Efficient Detection of Proteins Retro-Translocated from the ER to the Cytosol by In Vivo Biotinylation

Abstract

Retro-translocation from the ER to the cytosol of proteins within the secretory pathway takes place on misfolded molecules that are targeted for degradation by the cytosolically located 26S proteasome complex. Retro-translocation occurs also for other proteins (such as calreticulin) that, despite being synthesized and transported to the ER, are in part dislocated to the cytosol. We have taken advantage of the E. coli derived biotin-ligase (BirA) expressed in the cytosol of mammalian cells to specifically biotin-label in vivo proteins within the secretory pathway that undergo retro-translocation. We validated the method using four different proteins that are known to undergo retro-translocation upon different conditions: the human trans-membrane protein MHC class-I α chain (MHC-Iα), the Null Hong Kong mutant of the secretory α1 anti-trypsin (NHK-α1AT), the immunoglobulin heavy chain (HC) and the ER chaperone calreticulin (Crt). We observed specific mono-biotinylation of cytosolically dislocated molecules, resulting in a novel, reliable way of determining the extent of retro-translocation.

CD4 and BST-2/Tetherin Proteins Retro-translocate from Endoplasmic Reticulum to Cytosol as Partially Folded and Multimeric Molecules

“…We found that for both proteins, a fraction of the biotinylated molecules retain their glycosylation state, in agreement with previous findings (11,53) and consistent with the fact that the deglycosylating activity in mammalian cells is provided by the cytosolic PNGase (54). This confirms that deglycosylation appears to be the rate-limiting step of the process because deglycosylated biotinylated material only accumulates upon proteasomal inhibition, whereas glycosylated biotinylated molecules are also detected without inhibition.…”

“…In this assay denatured samples are run in the presence of StrAv, causing retardation only of biotinylated molecules (30). As demonstrated previously for other proteins (11), all of the biotinylated CD4 and Tetherin found in cells expressing cyt-BirA corresponds to molecules that have been retro-translocated to the cytosol.…”

“…Biotinylation of Dislocated CD4 and Tetherin-We have used our recently described method of biotinylation in living cells (11) to investigate retro-translocation of CD4 and Tetherin induced by HIV-1 Vpu. In this technique, cytosolic expression of the biotin-ligase BirA causes specific monobiotinylation of cytosolically located protein substrates tagged with the 15-amino acid-long biotin acceptor peptide BAP (GLNDIFEAQKIEWHE (27)).…”

“…We took advantage of our recently described method that allows precise and specific detection of molecules retro-translocated from the ER to the cytosol (11,47) to address the redox state of disulfide bridges in the retro-translocated fractions of CD4 and Tetherin. Biotinylation in living cells has also been used recently to study spontaneous retro-translocation of MHC class I molecules and to determine cytosolic/luminal localization of prion protein isoforms (48,49).…”

“…Here we report on the mechanism of retro-translocation of two widely studied proteins, CD4 and BST-2/Tetherin, using a novel technique to detect retro-translocation of defined proteins, based on the specific biotinylation in living cells only of molecules that from the ER reach the cytosolic compartment (11).…”