Abstract
CD4 and BST-2/Tetherin are cellular membrane proteins targeted to degradation by the HIV-1 protein Vpu. In both cases proteasomal degradation following recruitment into the ERAD pathway has been described. CD4 is a type I transmembrane glycoprotein, with four extracellular immunoglobulin-like domains containing three intrachain disulfide bridges. BST-2/Tetherin is an atypical type II transmembrane glycoprotein with an N-terminal transmembrane domain and a C-terminal glycophosphatidylinositol anchor, which dimerizes through three interchain bridges. We investigated spontaneous and Vpu-induced retro-translocation of CD4 and BST-2/Tetherin using our novel biotinylation technique in living cells to determine ER-to-cytosol retro-translocation of proteins. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and then becomes deglycosylated and reduced to monomers. These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation.
Highlights
CD4 and Tetherin are stabilized through intrachain and interchain disulfide bonds, respectively
Our results show that both CD4 and Tetherin are dislocated with intact disulfide bridges and as partially folded species
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
Summary
CD4 and Tetherin are stabilized through intrachain and interchain disulfide bonds, respectively. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and becomes deglycosylated and reduced to monomers These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation. Tetherin can be in part degraded following the autophagy/lysosomal pathway, most of it is retro-translocated and degraded by the cytoplasmic proteasome These data offer new insights on the mechanism of dislocation and raise questions on the required features of the putative retro-translocon
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