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

Abstract

Background: CD4 and Tetherin are stabilized through intrachain and interchain disulfide bonds, respectively. Results: CD4 and Tetherin retro-translocate from ER to cytosol with oxidized disulfide bridges as folded and multimeric molecules. Conclusion: Cysteines reduction is not a prerequisite for ER to cytosol dislocation. Significance: Our observations challenge the requirements of reduction and unfolding before dislocation.

The VCP/p97 and YOD1 Proteins Have Different Substrate-dependent Activities in Endoplasmic Reticulum-associated Degradation (ERAD)

“…Consistently, cell fractionation of MHC-I␣ and CD4 found them to be associated to the pelleted/microsomal fraction. In addition, the presence of small amounts of biotinylated material found with p97QQ remained associated to the pellet as we have demonstrated previously for CD4, which becomes soluble only after deglycosylation (39). In contrast, as shown below, three other different ERAD models, NS1, NHK-␣1AT, and BST-2/Tetherin, follow retro-translocation pathways where p97 and YOD1 activities are dispensable for substrate exposure to the cytosolic environment.…”

“…MG132 treatment stabilized deglycosylated CD4 (Fig. 1, E and F, arrows) (39). In the presence of p97QQ (Fig.…”

“…Tetherin is targeted to degradation by the HIV-1 accessory protein Vpu. It has, however, an intrinsic capacity to be degraded through ERAD when expressed in the absence of Vpu (39).…”

“…Plasmids encoding the N-terminal SV5-BAP tagged version of human MHC-I␣ and the same tag at the C-terminal domain of NHK-␣1AT have been described previously (38). N terminus BAP-tagged CD4 has been described previously (39). US2 and US11 plasmids were provided by Domenico Tortorella.…”

“…US2 and US11 plasmids were provided by Domenico Tortorella. Human Tetherin was tagged with SV5 and BAP immediately upstream of the GPI anchor signal after Tyr-154 (Tetherin-BAP) or with BAP inserted after Ala-48 (12 amino acids distant from the transmembrane domain, BAPTetherin) as described previously (39). The CCHFV-L OTU (ovarian tumor deubiquitination domain, OTU, derived from the Crimean-Congo hemorrhagic fever virus protein L) plasmid was provided by Adolfo García-Sastre and the N-terminal FLAG-tagged human YOD1 and YOD-C160S plasmids by Christian Schlieker.…”