Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein

Abstract

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein-class I heavy chain (EGFP-HC) chimeric molecule to study its dislocation and degradation in US2- and US11-expressing cells. The EGFP-HC fusion is stable in control cells, but is degraded rapidly in US2- or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP-HC molecules in US2- and US11-expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse-chase analysis and subcellular fractionation show that EGFP-HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

Fig. 1. The EGFP–HC reporter construct is a properly folded EGFP–class I fusion protein. U373_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min. Cells were lysed in NP-40 lysis buffer and immunoprecipitated with W6/32, a mAb that recognizes properly folded class I molecules only. The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The endogenous class I heavy chains (HC) and EGFP–HC associated with β₂m were recovered from cell lysates (lanes 1–3). Half of the immunoprecipitates were digested with Endo H (lanes 4–6). The positions of migration of class I molecules that contain an N-linked glycan (+CHO) or lack an N-linked glycan (–CHO) are indicated. Molecular weight markers are indicated on the right in kDa.

Fig. 2. Intracellular distribution of EGFP–HC is similar to that of endogenous class I molecules. U373_EGFP–HC were grown on chamber slides over night, prior to fixation, immunohistochemistry and confocal laser scanning microscopy. Double-positivity of the green fluorescent EGFP–HC molecule with W6/32 corresponds to properly folded class I complexes that contain the chimeric molecule. These are found at the cell surface (A) and in the ER. ER localization of EGFP–HC is demonstrated by staining with the ER markers calnexin (mAb AF8) (B) and PDI (anti-PDI) (C). For each antibody the green EGFP–HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown, as indicated for the individual panels.

Fig. 3. EGFP–HC is degraded in an US2- and US11-dependent manner. (A) US11_EGFP–HC and US2_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min. Cells were lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-GFP serum (αGFP) (lanes 1–9) and anti-class I heavy chain serum (αHC) (lanes 10–18). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The positions of migration of the EGFP–HC and endogenous class I heavy chain (HC) polypeptides are indicated. (B) The amount of EGFP–HC and HC polypeptides recovered from U373_EGFP–HC cells (black bar), US2_EGFP–HC cells (gray bar) and US11_EGFP–HC cells (white bar) were quantified by Phosphoimager analysis. The remaining class I molecules recovered at each chase point are given as a percentage of the class I molecules recovered at the 0 chase time.

Fig. 3. EGFP–HC is degraded in an US2- and US11-dependent manner. (A) US11_EGFP–HC and US2_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min. Cells were lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-GFP serum (αGFP) (lanes 1–9) and anti-class I heavy chain serum (αHC) (lanes 10–18). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The positions of migration of the EGFP–HC and endogenous class I heavy chain (HC) polypeptides are indicated. (B) The amount of EGFP–HC and HC polypeptides recovered from U373_EGFP–HC cells (black bar), US2_EGFP–HC cells (gray bar) and US11_EGFP–HC cells (white bar) were quantified by Phosphoimager analysis. The remaining class I molecules recovered at each chase point are given as a percentage of the class I molecules recovered at the 0 chase time.

Fig. 4. EGFP–HC is dislocated from the ER into the cytosol. (A) US11_EGFP–HC and US2_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-class I heavy chain serum (αHC). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The EGFP–HC (lanes 1–9) and endogenous class I molecules (HC) (lanes 10–15) were recovered from US2_EGFP–HC and US11_EGFP–HC cell lysates. Some class I degradation intermediates (* and **) were present in the US2 and US11 cell lysates. Half of the immunoprecipitates from US11 cells were digested with N-glycanase (PNGase) (lanes 7–9 and 16–18). (B) US11_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were homogenized and subjected to fractionation as described in Materials and methods. The EGFP–HC, endogenous HC (αHC; lanes 1–9) and transferrin receptor (αTfr; lanes 10–18), were recovered from the whole-cell lysate (Whole Cell), from the 100 000 g supernatant (100Kg-sup) and the 100 000 g pellet (100Kg-pellet). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). (C) Steady-state levels of EGFP–HC in U373_EGFP–HC and US11_EGFP–HC cells. Immunoblots of SDS lysates from equal numbers of untreated (lanes 1 and 2) and ZL₃VS-treated (3 h; 50 µM; lanes 3 and 4) cells (0.25 × 10⁶) were performed with a polyclonal anti-GFP serum. U373_EGFP–HC cells (lanes 1 and 3) express equal levels of EGFP–HC, independent of the proteasome inhibitor treatment. Inhibition of proteasomal degradation results in accumulation of EGFP–HC in US11_EGFP–HC cells (lanes 2 and 4) mostly as the deglycosylated intermediate. Molecular weight markers are indicated on the left in kDa.

Fig. 4. EGFP–HC is dislocated from the ER into the cytosol. (A) US11_EGFP–HC and US2_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-class I heavy chain serum (αHC). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The EGFP–HC (lanes 1–9) and endogenous class I molecules (HC) (lanes 10–15) were recovered from US2_EGFP–HC and US11_EGFP–HC cell lysates. Some class I degradation intermediates (* and **) were present in the US2 and US11 cell lysates. Half of the immunoprecipitates from US11 cells were digested with N-glycanase (PNGase) (lanes 7–9 and 16–18). (B) US11_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were homogenized and subjected to fractionation as described in Materials and methods. The EGFP–HC, endogenous HC (αHC; lanes 1–9) and transferrin receptor (αTfr; lanes 10–18), were recovered from the whole-cell lysate (Whole Cell), from the 100 000 g supernatant (100Kg-sup) and the 100 000 g pellet (100Kg-pellet). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). (C) Steady-state levels of EGFP–HC in U373_EGFP–HC and US11_EGFP–HC cells. Immunoblots of SDS lysates from equal numbers of untreated (lanes 1 and 2) and ZL₃VS-treated (3 h; 50 µM; lanes 3 and 4) cells (0.25 × 10⁶) were performed with a polyclonal anti-GFP serum. U373_EGFP–HC cells (lanes 1 and 3) express equal levels of EGFP–HC, independent of the proteasome inhibitor treatment. Inhibition of proteasomal degradation results in accumulation of EGFP–HC in US11_EGFP–HC cells (lanes 2 and 4) mostly as the deglycosylated intermediate. Molecular weight markers are indicated on the left in kDa.

Fig. 4. EGFP–HC is dislocated from the ER into the cytosol. (A) US11_EGFP–HC and US2_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were lysed in 1% SDS, then diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-class I heavy chain serum (αHC). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). The EGFP–HC (lanes 1–9) and endogenous class I molecules (HC) (lanes 10–15) were recovered from US2_EGFP–HC and US11_EGFP–HC cell lysates. Some class I degradation intermediates (* and **) were present in the US2 and US11 cell lysates. Half of the immunoprecipitates from US11 cells were digested with N-glycanase (PNGase) (lanes 7–9 and 16–18). (B) US11_EGFP–HC cells were pulse-labeled with [³⁵S]methionine for 15 min and chased up to 90 min in the presence of the proteasome inhibitor, ZL₃VS. Cells were homogenized and subjected to fractionation as described in Materials and methods. The EGFP–HC, endogenous HC (αHC; lanes 1–9) and transferrin receptor (αTfr; lanes 10–18), were recovered from the whole-cell lysate (Whole Cell), from the 100 000 g supernatant (100Kg-sup) and the 100 000 g pellet (100Kg-pellet). The immunoprecipitates were analyzed by SDS–PAGE (12.5%). (C) Steady-state levels of EGFP–HC in U373_EGFP–HC and US11_EGFP–HC cells. Immunoblots of SDS lysates from equal numbers of untreated (lanes 1 and 2) and ZL₃VS-treated (3 h; 50 µM; lanes 3 and 4) cells (0.25 × 10⁶) were performed with a polyclonal anti-GFP serum. U373_EGFP–HC cells (lanes 1 and 3) express equal levels of EGFP–HC, independent of the proteasome inhibitor treatment. Inhibition of proteasomal degradation results in accumulation of EGFP–HC in US11_EGFP–HC cells (lanes 2 and 4) mostly as the deglycosylated intermediate. Molecular weight markers are indicated on the left in kDa.

Fig. 5. Inhibition of proteasomal degradation in US11_EGFP–HC and US2_EGFP–HC cells induces accumulation of fluorescent EGFP–HC. (A) Flow cytometric quantification of the induction of green reporter fluorescence. Cells were incubated with ZL₃VS (50 µM) for the indicated time periods. The mean fluorescence intensity was measured by FACS: untransfected U373 cells (open squares), U373_EGFP–HC (closed squares), US11_EGFP–HC (open circles) and US2_EGFP–HC (open diamonds). The results shown are representative of three experiments. (B) Fluorimetric emission quantification of NP-40 lysates from U373 cells, U373_EGFP–HC and US11_EGFP–HC cells. Fluorescent units [490 nm(excitation)/515 nm(emission)] of EGFP–HC were measured from cell lysates of cells treated for 5 h with Zl₃VS (black bars) or without Zl₃VS (white bars). (C) Immunofluorescence of US11_EGFP–HC cells. Immunostaining followed by confocal laser scanning microscopy was carried out as described in Materials and methods. For each antibody the green EGFP–HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown, as indicated for the individual panels. Double-positivity of EGFP–HC with W6/32 shows the localization and the co-localization of properly folded class I complexes and their presence at the cell surface (top row). Almost complete co-localization of EGFP–HC with HC10, the monoclonal antibody reactive with class I independent of its folding, shows that this fluorescence is indeed derived from a green class I fusion protein (middle row). Staining with the αGFP reagent depicts no additive GFP-tagged population that for some reason (unfolding) failed to acquire green fluorescence (anti-GFP; bottom row).

Fig. 5. Inhibition of proteasomal degradation in US11_EGFP–HC and US2_EGFP–HC cells induces accumulation of fluorescent EGFP–HC. (A) Flow cytometric quantification of the induction of green reporter fluorescence. Cells were incubated with ZL₃VS (50 µM) for the indicated time periods. The mean fluorescence intensity was measured by FACS: untransfected U373 cells (open squares), U373_EGFP–HC (closed squares), US11_EGFP–HC (open circles) and US2_EGFP–HC (open diamonds). The results shown are representative of three experiments. (B) Fluorimetric emission quantification of NP-40 lysates from U373 cells, U373_EGFP–HC and US11_EGFP–HC cells. Fluorescent units [490 nm(excitation)/515 nm(emission)] of EGFP–HC were measured from cell lysates of cells treated for 5 h with Zl₃VS (black bars) or without Zl₃VS (white bars). (C) Immunofluorescence of US11_EGFP–HC cells. Immunostaining followed by confocal laser scanning microscopy was carried out as described in Materials and methods. For each antibody the green EGFP–HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown, as indicated for the individual panels. Double-positivity of EGFP–HC with W6/32 shows the localization and the co-localization of properly folded class I complexes and their presence at the cell surface (top row). Almost complete co-localization of EGFP–HC with HC10, the monoclonal antibody reactive with class I independent of its folding, shows that this fluorescence is indeed derived from a green class I fusion protein (middle row). Staining with the αGFP reagent depicts no additive GFP-tagged population that for some reason (unfolding) failed to acquire green fluorescence (anti-GFP; bottom row).

Fig. 6. Inhibition of proteasomal degradation in US11_EGFP–HC cells does not induce the formation of aggresomes. Immunostaining with anti-vimentin mAb V9 and confocal laser scanning microscopy was performed to study the distribution of vimentin filaments in untreated U373_EGFP–HC cells (A) and US11_EGFP–HC cells treated with ZL₃VS (50 µM; 3 h) (B). No significant difference between the vimentin distribution was detected for either cell type. The green EGFP–HC (left column), the anti-vimentin staining in red (middle column) and a merge image (right column) are shown for each panel.

Fig. 7. Direct visualization of EGFP–HC dislocation with intact cells. (A) A significant subpopulation of EGFP–HC is detected in the endoplasmic reticulum. Immunostaining followed by confocal laser scanning microscopy was used to analyze subcellular distribution of the accumulated EGFP–HC. US11_EGFP–HC cells were incubated with ZL₃VS (50 µM) for 3 h, fixed, and stained. ER localization of EGFP–HC is demonstrated by co-staining with the ER markers concanavalin A (top row), calnexin (anti-calnexin, mAb AF8; middle row) and PDI (anti-PDI; bottom row), as indicated. For each stain the green EGFP–HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown. Similar results were obtained in four independent experiments. (B) EGFP–HC traffics from the ER to the cytosol. The co-localization program from Bio-Rad was used for evaluation of the confocal laser scanning microscopy. The program depicts colocalization green and red signals from the merge picture (data not shown) and reproduces them as yellow pixels. Single color signals are shown in gray. Stainings were performed as in (A). In the presence of inhibitor, a large proportion of EGFP–HC colocalizes with concanavalin A (top row, left image), calnexin (top row, middle image) and PDI (top row, right image). Following wash-out of the proteasome inhibitor and a 90 min chase in the presence of cycloheximide (CHX) (bottom row), EGFP–HC is no longer detected in concanavalin A- (bottom row, left image), calnexin- (bottom row, middle image) and PDI-positive (bottom row, right image) compartments, yet is readily visible in the cytosol (see also Figure 8). Similar results were obtained in four independent experiments.

Fig. 7. Direct visualization of EGFP–HC dislocation with intact cells. (A) A significant subpopulation of EGFP–HC is detected in the endoplasmic reticulum. Immunostaining followed by confocal laser scanning microscopy was used to analyze subcellular distribution of the accumulated EGFP–HC. US11_EGFP–HC cells were incubated with ZL₃VS (50 µM) for 3 h, fixed, and stained. ER localization of EGFP–HC is demonstrated by co-staining with the ER markers concanavalin A (top row), calnexin (anti-calnexin, mAb AF8; middle row) and PDI (anti-PDI; bottom row), as indicated. For each stain the green EGFP–HC (left column), the respective second staining in red (middle column) and a merge image (right column) are shown. Similar results were obtained in four independent experiments. (B) EGFP–HC traffics from the ER to the cytosol. The co-localization program from Bio-Rad was used for evaluation of the confocal laser scanning microscopy. The program depicts colocalization green and red signals from the merge picture (data not shown) and reproduces them as yellow pixels. Single color signals are shown in gray. Stainings were performed as in (A). In the presence of inhibitor, a large proportion of EGFP–HC colocalizes with concanavalin A (top row, left image), calnexin (top row, middle image) and PDI (top row, right image). Following wash-out of the proteasome inhibitor and a 90 min chase in the presence of cycloheximide (CHX) (bottom row), EGFP–HC is no longer detected in concanavalin A- (bottom row, left image), calnexin- (bottom row, middle image) and PDI-positive (bottom row, right image) compartments, yet is readily visible in the cytosol (see also Figure 8). Similar results were obtained in four independent experiments.

Fig. 8. Dislocated EGFP–HC is a cytosolic green fluorescent protein and does not, once unfolded, reacquire fluorescence. (A) FACS analysis shows that accumulated EGFP–HC induced by proteasome inhibition can be degraded. U373_EGFP–HC (closed squares) and US11_EGFP–HC (open circles) were incubated for 3 h with the reversible proteasome inhibitor MG132 (50 µM). After removal of MG132, the accumulated EGFP–HC was chased for an additional 3 h in the presence of cycloheximide (CHX). Similar results were obtained in three independent experiments. (B) Fluorimetric emission quantification of EGFP–HC from cytosolic fractions of U373_EGFP–HC, US2_EGFP–HC and US11_EGFP–HC cells treated with the proteasome inhibitor, ZL₃VS (white and black bars). The fluorescence emitted by cytosolic EGFP–HC molecules was examined for US2_EGFP–HC and US11_EGFP–HC cells after addition of proteasome inhibitor and cycloheximide (CHX) for 1 h (black bars). Cytosolic fractions were prepared as described in Materials and methods. The fluorescence emission of the cytosolic population of EGFP–HC was plotted as a percentage of signal calculated for unfractionated homogenates. (C) Fluorimetric quantification of the EGFP–HC molecules after exposure to the denaturant guanidine hydrochloride (GuHCl). The fluorescence emission of lysates from U373_EGFP–HC (open squares), ZL₃VS-treated US2_EGFP–HC (open diamonds), ZL₃VS-treated US11_EGFP–HC (asterisks) and U373_EGFP (open circles) cells incubated with increasing concentrations of GuHCl were examined. The percentage of EGFP–HC fluorescence intensity was plotted against GuHCl concentration. The mean fluorescence of EGFP–HC at 0 M GuHCl was used as 100% fluorescence. (D) Renaturation of denatured EGFP–HC and EGFP molecules. EGFP–HC molecules from U373_EGFP–HC cells and EGFP from U373_EGFP cells were denatured with 5.0 M GuHCl (open square and circle, respectively) followed by dilution with PBS to 0.3 M GuHCl (closed square and circle, respectively). The percentage of green fluorescent signal was plotted against GuHCl concentration. The mean green fluorescence signal at 0 M GuHCl was used as 100% fluorescence.