Quality control for unfolded proteins at the plasma membrane

Abstract

Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2-E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera-accelerated internalization, impaired recycling, and endosomal sorting complex required for transport-dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.

Figure 1.

Unfolding induced down-regulation of CD4tl-λ_C at the PM. (A) Schematic picture of membrane-tethered and soluble bacteriophage λ model proteins. (B) Immunoblot (IB) analysis of CD4tl and CD4tl-λ/λ_C expression before (top) or after CHX chase at 37°C for 1.5 h (bottom). Transiently transfected COS7 cells were cultured at 37°C or 26°C before the CHX chase. (C) Indirect immunostaining of thermally unfolded (37°C) or nativelike (26°C) CD4tl-λ_C in nonpermeabilized (top) or permeabilized (bottom) COS7 cells. Calreticulin was used as an ER marker. Bars, 10 µm. (D) The PM density of chimeras was determined by ELISA as described in Materials and methods. (E) Pharmacological characterization of rescued and then unfolded CD4tl-λ_C degradation as measured by CHX chase and immunoblotting. Lactacystine (LAC), concanamycin (Con), and bafilomycin A1 (Baf) were added simultaneously with CHX for 3.5 h. NH₄Cl and chloroquin (Chl) were present during the last 2 h of the CHX chase. Equal amounts of proteins were loaded. (F and G) The PM stability of rescued and then unfolded model proteins at 37°C for 1.5 h was determined at 37°C (F) or in the absence of unfolding at 26°C (G) by ELISA. (H) Internalization of unfolded and rescued model proteins was monitored by Ab uptake at 37°C. Data are expressed as the percentage of initial Ab binding. (I) Recycling efficiency was determined by a biotin-streptavidin sandwich assay as described in Materials and methods and expressed as the percentage of CD4tl. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.

Figure 2.

Unfolding initiates ubiquitination of CD4tl-λ_C at the PM. (A) Ubiquitination of CD4 chimeras isolated by denaturing cell surface immunoprecipitation (cs-IP) was measured by immunoblotting using the HRP-conjugated P4D1 anti-Ub Ab. Three times more CD4tl-λ_C–expressing cells were harvested than CD4tl-λ–expressing cells. Molecular mass is given in kilodaltons. IB, immunoblot. (B) Approach to monitor the ubiquitination of CD4tl-RlucII-λ_C by BRET² in vivo. (C) The temperature-rescued CD4tl-RlucII-λ_C (26°C) is largely confined to the PM and shows negligible colocalization with calreticulin. Indirect immunostaining was performed before and after CHX chase for 5 h at 26°C. Bars, 5 µm. (D) 26°C temperature-rescued cells were incubated for the indicated times at 40°C, and the internalization rates of CD4tl-λ_C and CD4tl-RlucII-λ_C were determined by Ab uptake at 37°C. (E) Unfolding stimulates of CD4tl-RlucII-λ_C ubiquitination detected by BRET². HEK293 cells coexpressing rescued CD4tl-RlucII-λ_C and GFP²-Ub or GFP²-UbiAA were treated with CHX for 5 h at 26°C. BRET² was measured in the presence of 5 µM coelenterazine 400a. EPAC expression served as a positive control. (F) Summary of BRET² results of experiments shown in E. BRET² was measured on cells cultured at 26°C before or after 5-min exposure to 40°C. The dotted line indicates the background BRET² signal. epa, EPAC. The data shown represent means ± SEM.

Figure 3.

CHIP and molecular chaperones are recruited to unfolded CD4tl-λ_C at the PM. (A and B) CHIP associates with unfolded CD4tl-λ_C at the PM. Native cs-IP was performed, and precipitates were probed with anti-CHIP and anti-HA Abs (A). Exogenous CHIP-myc association was probed in HEK293 cells cotransfected with the indicated construct and cultured at 26°C or 37°C (B). (C) Molecular chaperones associate with the CD4tl-λ_C but not with CD4tl-λ at the PM. Nondenaturing cs-IP was performed and probed as described in A. Molecular mass is given in kilodaltons. IB, immunoblot.

Figure 4.

Cellular and biochemical phenotype of the CD4tl-λ_C upon inactivation of CHIP. (A) Effects of dominant-negative (DN) E3 and E2 enzymes on the PM stability of CD4tl-λ_C were measured in transiently cotransfected HEK293 cells and monitored by cell surface ELISA after 30-min chase at 37°C. Fig. S3 D depicts the CD4tl-λ PM stability (**, P < 0.01; n = 5). The dotted line indicates the chimera cell surface turnover in mock-transfected cells. (B) CHIP ablation was accomplished by lentiviral shCHIP in Flp-In T-Rex HEK293 cells expressing CD4tl-λ_C and verified by immunoblotting. CHIP-myc overexpression was detectable in the shCHIP cells. CHIP depletion had an adverse effect on tetracycline transactivation, reducing the induction level of CD4tl-λ_C in shCHIP cells. (C) Ubiquitination level of the PM CD4tl-λ_C was probed by denaturing cs-IP and immunoblotting. Three times more shCHIP cells were used. (D) Stability of internalized CD4tl-λ_C was measured by labeling the PM-resident chimera with anti-CD4 Ab at 4°C and chasing for 0–1 h at 37°C in shNT and shCHIP cells. Ab–chimera complexes were precipitated and immunoblotted. (E) The PM turnover of the CD4tl-λ_C was measured by cell surface ELISA in control, shCHIP, and CHIP-myc–transfected cells. (F) The CD4tl-λ_C and CD4cc-UbAllRΔG (UbRΔG) PM stability was determined after 1-h chase in shCHIP cells coexpressing the CHIP-myc variant. (G) The internalization rate of CD4tl-λ_C was monitored in the absence or presence of CHIP-myc at 37°C by Ab uptake in shCHIP cells for 5 min. Endocytosis of tetrameric CD4cc-UbAllRΔG (UbRΔG) and transferrin (Tf) was measured for 2.5 min. (H) Mean vesicular pH of internalized cargo was determined by FRIA in small interfering NT and small interfering CHIP (siCHIP) cells as described in Materials and methods. Anti-CD4 Ab and FITC-Fab were bound on ice, and FRIA was performed after 1-h chase at 37°C. NT, nontargeted siRNA. The lysosomal targeting of CD4tl-Ub, CD63, and dextran was not influenced by shCHIP. Molecular mass is given in kilodaltons. IB, immunoblot. The data shown represent means ± SEM.

Figure 5.

Unfolded CD4tl-λ_C undergoes mixed-chain polyubiquitination at the PM. (A) Ub-chain configuration of native and unfolded CD4tl-λ_C at the PM and endosomes. Denaturing cs-IP of the CD4tl-λ_C was performed after (a) temperature rescue at 26°C (native), (b) unfolding the rescued chimera at 37°C for 1.5 h, and (c) culturing the cells at 37°C. Anti-CD4 Ab was bound at 4°C, except in b, in which Ab labeling was performed at 37°C for 1.5 h. Precipitates were probed with anti-Ub Abs. Denaturing IP of EGFP-λ_C and EGFP was accomplished with anti-EGFP Ab. Ab specificity was probed by 2 µg mono-Ub and 0.2 µg K63- and K48-linked poly-Ub chains. Asterisks indicate nonspecific Ab reactivity. (B) Ubiquitination of soluble EGFP-λ and -λ_C was detected after denaturing IP and immunoblotting of the cells exposed to 26°C or 37°C. (C) Association of the EGFP-λ and -λ_C chimeras with endogenous and exogenous CHIP was examined by nondenaturing IP and immunoblotting. (D) Turnover rates of the EGFP-λ and -λ_C were measured in control and shCHIP cells by CHX chase and immunoblotting. Molecular mass is given in kilodaltons. IB, immunoblot.

Figure 6.

Lysosomal delivery of unfolded CD4tl-λ_C is ESCRT dependent. (A) Indirect immunolocalization of internalized CD4 chimeras with transferrin (Tf) and LAMP2 by laser confocal fluorescence microscopy. CD4 chimeras were labeled with anti-CD4 Ab capture for 20 min at 37°C and chased in Ab-free medium for 1 h. Bars, 5 µm. (B–D) Luminal pH of vesicles containing endocytosed CD4 chimeras and control cargoes was measured by FRIA. Internalized chimeras were labeled with anti-CD4 Ab and FITC-Fab on ice and chased for 1 h (B) or for the indicated time (C and D) at 37°C. In B, the Gaussian distribution (curved lines) and mean vesicular pH (bold numbers) are indicated. The chimera was rescued at 26°C before FRIA (D). (E) Verification of Tsg101 (Tsg), Hrs, and Stam1 depletion in siRNA-treated cells. (F) The stability of internalized CD4tl-λ_C, labeled by anti-CD4 Ab capture, was measured in shNT, shHrs, and shStam cells as described in Fig. 4 D. IB, immunoblot. (G) The mean vesicular pH of CD4tl-λ_C and other cargoes was determined after 1-h chase in cells depleted for Tsg101, Hrs, and Stam1. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.

Figure 7.

The disposal of mutant V2R and DRD4 from the PM is CHIP and ESCRT dependent. (A) Cellular (top) and PM (bottom) expression of Flag-DRD4 and myc-V2R variants in transiently transfected HEK-293 cells. Immunoblotting and cell surface density were measured as described in Materials and methods. Molecular mass is given in kilodaltons. Open arrowhead, complex glycosylated forms. Closed arrowhead, core glycosylated forms. Circle, nonglycosylated form. (B) Turnover of GPCRs that accumulate constitutively at the PM was determined by using anti-Flag or anti-myc Ab and ELISA assay at 37°C. (C) The internalization of DRD4s and V2Rs was measured by the Ab uptake assay. (D and E) Postendocytic sorting of DRD4 and V2R variants was followed by FRIA after 15–60-min chase in control, shStam-, or shHrs-expressing cells as described in Fig. 6 B. The data shown represent means ± SEM.

Figure 8.

CHIP-dependent ubiquitination contributes to mutant V2R and DRD4 disposal from the PM. (A) Ubiquitination of wt and M345T-DRD4 was measured by denaturing cs-IP as described in Fig. 2 A using anti-Flag and P4D1 anti-Ub Abs. Open arrowhead, complex glycosylated form. (B) Effect of CHIP depletion (shCHIP) on the ubiquitination of wt and M345T-DRD4 at the PM as measured in A. (C) Metabolic stability of internalized DRD4 was determined after in vivo labeling with anti-Flag Ab in control and CHIP-ablated cells as in Fig. 4 D. (D) The impeded internalization and PM turnover of the M345T-DRD4 and W164S-V2R in shCHIP cells were reverted to the mutant phenotype by overexpressing the wt but not the catalytically inactive or TPR mutant CHIP-myc. The internalization and PM stability of GPCRs were monitored with cell surface ELISA. Western blots show the expression of CHIP-myc variants. Molecular mass is given in kilodaltons. IB, immunoblot. The data shown represent means ± SEM.