Dislocation of type I membrane proteins from the ER to the cytosol is sensitive to changes in redox potential - PubMed (original) (raw)
Dislocation of type I membrane proteins from the ER to the cytosol is sensitive to changes in redox potential
D Tortorella et al. J Cell Biol. 1998.
Erratum in
- J Cell Biol 1999 May 3;145(3):following 642
Abstract
The human cytomegalovirus (HCMV) gene products US2 and US11 dislocate major histocompatibility class I heavy chains from the ER and target them for proteasomal degradation in the cytosol. The dislocation reaction is inhibited by agents that affect intracellular redox potential and/or free thiol status, such as diamide and N-ethylmaleimide. Subcellular fractionation experiments indicate that this inhibition occurs at the stage of discharge from the ER into the cytosol. The T cell receptor alpha (TCR alpha) chain is also degraded by a similar set of reactions, yet in a manner independent of virally encoded gene products. Diamide and N-ethylmaleimide likewise inhibit the dislocation of the full-length TCR alpha chain from the ER, as well as a truncated, mutant version of TCR alpha chain that lacks cysteine residues. Cytosolic destruction of glycosylated, ER-resident type I membrane proteins, therefore, requires maintenance of a proper redox potential for the initial step of removal of the substrate from the ER environment.
Figures
Figure 1
(A) Diamide induces the formation of disulfide bonds (i.e., reduced glutathione [GS −] is converted to its oxidized form [_GSSG_]). (B) NEM irreversibly alkylates free sulfhydryls (− SR).
Figure 2
Intrachain -S-S- bonds in MHC class I heavy chains are reduced before deglycosylation. (A) IAA or IAM can be used as thiol-specific probes. Comparison of molecules alkylated with either IAA or IAM can be used to assess the free sulfhydryl content of the molecule of interest. Alkylation of MHC class I heavy chains (A or B) in US2+ cells with IAA introduces a negative charge for each modified free −SH group (D), but no charge is introduced upon alkylation with IAM (C). An additional negative charge is introduced when the N-linked glycan on the class I heavy chain is removed by _N_-glycanase (E and F), converting the Asn to an Asp residue. These charge differences can be resolved using IEF. Pulse-chase experiments were performed on US2+ and control cells in the presence of the proteasome inhibitor ZL3H. The cells were lysed in the presence of either IAA or IAM. MHC class I molecules were recovered by immunoprecipitation with either anti–class I heavy chain (αHC), specific for free heavy chains (B), or with W6/32, specific for assembled class I molecules composed of heavy chain and light chain (β2m) (C). The immunoprecipitates were resolved by either IEF or SDS-PAGE. The comparison of samples alkylated with IAA and IAM immediately reveals the reduced state of the free class I heavy chains (B, compare lanes 2–7 and 9–14), as judged from the more acidic isoelectric points of heavy chains (B, asterisks, lanes 9–14). Treatment of US2+ cells with tunicamycin prevents the charge shift introduced by the action of _N_-glycanase (B, arrows, lanes 3 and 4). For reference, a sample obtained at the 3-min chase point from control cells was digested with bacterial PNGase F and analyzed in parallel (lane 1). A charge shift of class I heavy chains recovered using W6/32 from US2+ and control cells is observed when these samples were alkylated with IAA (C, lanes 6–10), but not when alkylated with IAM (C, lanes 1–5).
Figure 2
Intrachain -S-S- bonds in MHC class I heavy chains are reduced before deglycosylation. (A) IAA or IAM can be used as thiol-specific probes. Comparison of molecules alkylated with either IAA or IAM can be used to assess the free sulfhydryl content of the molecule of interest. Alkylation of MHC class I heavy chains (A or B) in US2+ cells with IAA introduces a negative charge for each modified free −SH group (D), but no charge is introduced upon alkylation with IAM (C). An additional negative charge is introduced when the N-linked glycan on the class I heavy chain is removed by _N_-glycanase (E and F), converting the Asn to an Asp residue. These charge differences can be resolved using IEF. Pulse-chase experiments were performed on US2+ and control cells in the presence of the proteasome inhibitor ZL3H. The cells were lysed in the presence of either IAA or IAM. MHC class I molecules were recovered by immunoprecipitation with either anti–class I heavy chain (αHC), specific for free heavy chains (B), or with W6/32, specific for assembled class I molecules composed of heavy chain and light chain (β2m) (C). The immunoprecipitates were resolved by either IEF or SDS-PAGE. The comparison of samples alkylated with IAA and IAM immediately reveals the reduced state of the free class I heavy chains (B, compare lanes 2–7 and 9–14), as judged from the more acidic isoelectric points of heavy chains (B, asterisks, lanes 9–14). Treatment of US2+ cells with tunicamycin prevents the charge shift introduced by the action of _N_-glycanase (B, arrows, lanes 3 and 4). For reference, a sample obtained at the 3-min chase point from control cells was digested with bacterial PNGase F and analyzed in parallel (lane 1). A charge shift of class I heavy chains recovered using W6/32 from US2+ and control cells is observed when these samples were alkylated with IAA (C, lanes 6–10), but not when alkylated with IAM (C, lanes 1–5).
Figure 2
Intrachain -S-S- bonds in MHC class I heavy chains are reduced before deglycosylation. (A) IAA or IAM can be used as thiol-specific probes. Comparison of molecules alkylated with either IAA or IAM can be used to assess the free sulfhydryl content of the molecule of interest. Alkylation of MHC class I heavy chains (A or B) in US2+ cells with IAA introduces a negative charge for each modified free −SH group (D), but no charge is introduced upon alkylation with IAM (C). An additional negative charge is introduced when the N-linked glycan on the class I heavy chain is removed by _N_-glycanase (E and F), converting the Asn to an Asp residue. These charge differences can be resolved using IEF. Pulse-chase experiments were performed on US2+ and control cells in the presence of the proteasome inhibitor ZL3H. The cells were lysed in the presence of either IAA or IAM. MHC class I molecules were recovered by immunoprecipitation with either anti–class I heavy chain (αHC), specific for free heavy chains (B), or with W6/32, specific for assembled class I molecules composed of heavy chain and light chain (β2m) (C). The immunoprecipitates were resolved by either IEF or SDS-PAGE. The comparison of samples alkylated with IAA and IAM immediately reveals the reduced state of the free class I heavy chains (B, compare lanes 2–7 and 9–14), as judged from the more acidic isoelectric points of heavy chains (B, asterisks, lanes 9–14). Treatment of US2+ cells with tunicamycin prevents the charge shift introduced by the action of _N_-glycanase (B, arrows, lanes 3 and 4). For reference, a sample obtained at the 3-min chase point from control cells was digested with bacterial PNGase F and analyzed in parallel (lane 1). A charge shift of class I heavy chains recovered using W6/32 from US2+ and control cells is observed when these samples were alkylated with IAA (C, lanes 6–10), but not when alkylated with IAM (C, lanes 1–5).
Figure 3
Diamide suppresses the conversion of glycosylated class I heavy chains to its deglycosylated intermediate in US2+ cells. US2+ cells treated with or without the proteasome inhibitor ZL3H were pulsed for 10 min and chased for 0 and 25 min. Diamide was added at the onset of the chase at the indicated concentrations. Cell lysates were immunoprecipitated with either the anti–class I heavy chain monoclonal antibody HC10 (A), W6/32 (B), or anti-US2 (αUS2) (C). The immunoprecipitates were analyzed by SDS-PAGE (12.5%). While HC10 recovers free class heavy chains with (+CHO) or without (−CHO) an N-linked glycan, W6/32 immunoprecipitates only properly folded class I heavy chains (HC+CHO) associated with glycosylated-US2 (US2+CHO) and β2m. The reduced recovery of radiolabeled β2m in W6/32 precipitates from US2+ cells (compare Figs. 3 and 7) is possibly due to loss of β2m in the course of washing, replacement of β2m with US2, or the destabilization of the interaction of β2m and class I heavy chain by US2. The anti-US2 serum recognizes both nonglycosylated (−CHO) and glycosylated US2 (+CHO). The amount of the glycosylated class I heavy chains recovered from the 0- and 25-min chase points using HC10 was quantitated using a Fuji PhosphorImager. The percentage of glycosylated class I heavy chains (HC+CHO) at different diamide concentrations represents the relative amount of the class I heavy chains recovered at the 25-min chase compared with the 0-min chase point, which was used as the standard amount of glycosylated heavy chain (D).
Figure 3
Diamide suppresses the conversion of glycosylated class I heavy chains to its deglycosylated intermediate in US2+ cells. US2+ cells treated with or without the proteasome inhibitor ZL3H were pulsed for 10 min and chased for 0 and 25 min. Diamide was added at the onset of the chase at the indicated concentrations. Cell lysates were immunoprecipitated with either the anti–class I heavy chain monoclonal antibody HC10 (A), W6/32 (B), or anti-US2 (αUS2) (C). The immunoprecipitates were analyzed by SDS-PAGE (12.5%). While HC10 recovers free class heavy chains with (+CHO) or without (−CHO) an N-linked glycan, W6/32 immunoprecipitates only properly folded class I heavy chains (HC+CHO) associated with glycosylated-US2 (US2+CHO) and β2m. The reduced recovery of radiolabeled β2m in W6/32 precipitates from US2+ cells (compare Figs. 3 and 7) is possibly due to loss of β2m in the course of washing, replacement of β2m with US2, or the destabilization of the interaction of β2m and class I heavy chain by US2. The anti-US2 serum recognizes both nonglycosylated (−CHO) and glycosylated US2 (+CHO). The amount of the glycosylated class I heavy chains recovered from the 0- and 25-min chase points using HC10 was quantitated using a Fuji PhosphorImager. The percentage of glycosylated class I heavy chains (HC+CHO) at different diamide concentrations represents the relative amount of the class I heavy chains recovered at the 25-min chase compared with the 0-min chase point, which was used as the standard amount of glycosylated heavy chain (D).
Figure 4
Diamide inhibits the conversion of the glycosylated class I heavy chains to its deglycosylated intermediate in US11+ cells. US11+ cells treated with the proteasome inhibitor ZL3VS were pulsed for 10 min and chased for 0 and 30 min. Various concentrations of diamide were added at the onset of the chase. Cell lysates were immunoprecipitated with either anti–class I heavy chains (αHC) or W6/32. The immunoprecipitates were resolved by SDS-PAGE (12.5%). The glycosylated class I heavy chains (+CHO) are recovered by αHC and W6/32, while the deglycosylated intermediate (−CHO) is recovered with only αHC. A quantitative analysis (B) of the effect of diamide on the recovery of the glycosylated free (αHC) and properly folded class I heavy chains (W6/32) in US11+ cells was performed as described in Fig. 3.
Figure 4
Diamide inhibits the conversion of the glycosylated class I heavy chains to its deglycosylated intermediate in US11+ cells. US11+ cells treated with the proteasome inhibitor ZL3VS were pulsed for 10 min and chased for 0 and 30 min. Various concentrations of diamide were added at the onset of the chase. Cell lysates were immunoprecipitated with either anti–class I heavy chains (αHC) or W6/32. The immunoprecipitates were resolved by SDS-PAGE (12.5%). The glycosylated class I heavy chains (+CHO) are recovered by αHC and W6/32, while the deglycosylated intermediate (−CHO) is recovered with only αHC. A quantitative analysis (B) of the effect of diamide on the recovery of the glycosylated free (αHC) and properly folded class I heavy chains (W6/32) in US11+ cells was performed as described in Fig. 3.
Figure 7
The interaction of class I heavy chains with a truncated form of US2 (US2-150) is not affected by diamide. Control cells infected with a recombinant vaccinia virus expressing a truncated form of US2 (US2-150) were pulsed for 10 min and chased for 0 and 20 min in the absence or presence of 1 mM diamide. Cell lysates were immunoprecipitated with either W6/32 (A) or anti-US2 (αUS2) (C). The W6/32 immunoprecipitates recovered class I heavy chains (HC+CHO) associated with the light chain β2m and US2-150. The additional bands observed in A correspond to vaccinia virus products bound to Staphylococcus aureus (used to immobilize the immune complexes). The US2-150 associated with properly folded class I molecules (W6/32-reactive material) were recovered using αUS2 (B) (see Material and Methods). Truncated US2 (US2-150) exists in a glycosylated (US2-150 (+CHO)) and nonglycosylated (_US2-150 (−_CHO)) form. The immunoprecipitates were analyzed by SDS-PAGE (12.5%). Exposure time of the autoradiograms from A and C, 2 d; B, 14 d.
Figure 5
NEM suppresses the accumulation of the deglycosylated class I heavy chain intermediate in US2+ cells. US2+ cells treated with or without the proteasome inhibitor ZL3VS were pulsed for 10 min and chased for 0 and 30 min. Various concentrations of NEM were added at the onset of the chase as indicated. Cell lysates were immunoprecipitated with either anti– class I heavy chain (αHC) (A), W6/32 (B), or anti-US2 (αUS2) (C). The immunoprecipitates were analyzed by SDS-PAGE (12.5%). The glycosylated (+CHO) and deglycosylated (−CHO) forms of the class I heavy chains and US2 are indicated. A quantitative analysis (D) of the effect of NEM on the recovery of glycosylated free class I heavy chains using αHC in US2+ cells was performed as described in Fig. 3.
Figure 5
NEM suppresses the accumulation of the deglycosylated class I heavy chain intermediate in US2+ cells. US2+ cells treated with or without the proteasome inhibitor ZL3VS were pulsed for 10 min and chased for 0 and 30 min. Various concentrations of NEM were added at the onset of the chase as indicated. Cell lysates were immunoprecipitated with either anti– class I heavy chain (αHC) (A), W6/32 (B), or anti-US2 (αUS2) (C). The immunoprecipitates were analyzed by SDS-PAGE (12.5%). The glycosylated (+CHO) and deglycosylated (−CHO) forms of the class I heavy chains and US2 are indicated. A quantitative analysis (D) of the effect of NEM on the recovery of glycosylated free class I heavy chains using αHC in US2+ cells was performed as described in Fig. 3.
Figure 6
Diamide and NEM only moderately impede the folding of MHC class I molecules. Control cells were pulsed for 10 min and chased for 0, 15, and 30 min as described in Materials and Methods. Diamide (1 mM) or NEM (0.75 mM) were added at the onset of the chase. Lysates from untreated, diamide-treated, or NEM-treated cells were immunoprecipitated with either αHC or W6/32. The immunoprecipitates were analyzed by SDS-PAGE (12.5%). Free class I heavy chains (HC) associate with β2m to form properly folded class I molecules (W6/32-reactive material).
Figure 8
Diamide and NEM block the dislocation of class I heavy chains from the ER to the cytosol in US2+ cells. Subcellular fractionation was performed on pulse-chased US2+ cells treated with diamide or NEM in the presence or absence of the proteasome inhibitor ZL3VS. The class I heavy chains (A), US2 molecules (B), and transferrin receptor (C) were immunoprecipitated from nonfractionated cells or 100,000-g supernatant (cytosol) of fractionated cells using their respective antibodies (see Materials and Methods). The immunoprecipitates were analyzed by SDS-PAGE (12.5%). The glycosylated (+CHO) and deglycosylated (−CHO) forms of the class I heavy chains or the US2 molecules are indicated.
Figure 9
Diamide inhibits the dislocation of the TCR α chain from the ER into the cytosol. COS-1 cells transiently transfected with TCR α in the absence or presence of the proteasome inhibitor ZL3H were pulsed for 10 min and chased for 1.5 h (A). Diamide (1 mM) was added at the onset of the chase. TCR α chain was immunoprecipitated from cell lysates and analyzed by SDS-PAGE (12.5%). The number of glycans attached to the TCR α chain is indicated. COS-1 cells transiently transfected with TCR α chain were pulsed for 10 min and chased for 2 h (B). Cell homogenates were subjected to differential centrifugation as described in Materials and Methods. TCR α chains were immunoprecipitated from the particulate fractions (P) and the 100,000-g supernatant (S) and analyzed by SDS-PAGE (12.5%). A 20-kD fragment of the TCR α chain has been described as a degradation intermediate (22).
Figure 10
Diamide inhibits the degradation of a truncated form of the TCR α chain (VαTMΔC) lacking cysteines. A truncated form of the TCR α chain (VαTMΔC) devoid of all cysteines was generated as described in Materials and Methods (A). Both the full-length TCR α chain and its truncated counterpart, VαTMΔC, were recovered using a rabbit anti–TCR α chain serum from metabolically labeled COS cells transiently transfected with the respective DNA and digested with Endo H (B). Degradation of VαTMΔC was examined in transiently transfected COS cells that were metabolically labeled for 10 min and chased for 0, 45, 90, and 180 min. Cells were either untreated or treated with ZL3H or 2 mM diamide. VαTMΔC was recovered by immunoprecipitation using the anti–TCR α chain serum and digested with endoglycosidase H (Endo H). The precipitates were analyzed by SDS-PAGE (12.5%) and quantitated by densitometry. The amount of VαTMΔC recovered under different conditions was expressed as the percent of VαTMΔC recovered at the different chase times relative to the 0-min chase point.
Figure 10
Diamide inhibits the degradation of a truncated form of the TCR α chain (VαTMΔC) lacking cysteines. A truncated form of the TCR α chain (VαTMΔC) devoid of all cysteines was generated as described in Materials and Methods (A). Both the full-length TCR α chain and its truncated counterpart, VαTMΔC, were recovered using a rabbit anti–TCR α chain serum from metabolically labeled COS cells transiently transfected with the respective DNA and digested with Endo H (B). Degradation of VαTMΔC was examined in transiently transfected COS cells that were metabolically labeled for 10 min and chased for 0, 45, 90, and 180 min. Cells were either untreated or treated with ZL3H or 2 mM diamide. VαTMΔC was recovered by immunoprecipitation using the anti–TCR α chain serum and digested with endoglycosidase H (Endo H). The precipitates were analyzed by SDS-PAGE (12.5%) and quantitated by densitometry. The amount of VαTMΔC recovered under different conditions was expressed as the percent of VαTMΔC recovered at the different chase times relative to the 0-min chase point.
Figure 11
Diamide and NEM prevents the degradation of the MHC class I heavy chains in Daudi cells. Daudi cells treated with or without the proteasome inhibitor ZL3VS were pulsed for 10 min and chased for 0, 1, and 2 h. To untreated Daudi cells, diamide (1 mM) or NEM (1 mM) was added at the onset of the chase. Cell lysates from the above experiments were immunoprecipitated with αHC. The immunoprecipitates were analyzed by SDS-PAGE (12.5%).
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