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The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate.

Shamu CE, Story CM, Rapoport TA, Ploegh HL - J. Cell Biol. (1999)

Bottom Line: We find that heavy chains are ubiquitinated before they are degraded.Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane.Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. shamu@bcmp.med.harvard.edu

ABSTRACT
The human cytomegalovirus protein, US11, initiates the destruction of MHC class I heavy chains by targeting them for dislocation from the ER to the cytosol and subsequent degradation by the proteasome. We report the development of a permeabilized cell system that recapitulates US11-dependent degradation of class I heavy chains. We have used this system, in combination with experiments in intact cells, to identify and order intermediates in the US11-dependent degradation pathway. We find that heavy chains are ubiquitinated before they are degraded. Ubiquitination of the cytosolic tail of heavy chain is not required for its dislocation and degradation, suggesting that ubiquitination occurs after at least part of the heavy chain has been dislocated from the ER. Thus, ubiquitination of the heavy chain does not appear to be the signal to start dislocation. Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane. Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

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Ubiquitin-conjugated and deglycosylated heavy chains accumulate at the same rate. (A) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. αHC immunoprecipitates of each lysate were split and part was reimmunoprecipitated with αUb antibodies, as described for the experiments shown in Fig. 6 B. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. Note that the exposure of the αHC gel is 12 h whereas the exposure of the αUb gel is 3 wk. (B) The data in A were quantitated on a PhosphorImager and graphed. Note the different scales on the two y axes.
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Figure 7: Ubiquitin-conjugated and deglycosylated heavy chains accumulate at the same rate. (A) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. αHC immunoprecipitates of each lysate were split and part was reimmunoprecipitated with αUb antibodies, as described for the experiments shown in Fig. 6 B. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. Note that the exposure of the αHC gel is 12 h whereas the exposure of the αUb gel is 3 wk. (B) The data in A were quantitated on a PhosphorImager and graphed. Note the different scales on the two y axes.

Mentions: As noted in the figure legends, lysates were made in three different ways, depending on the experiment. In all cases, 1–2 × 106 cells (or cell equivalents) were used to make 1–1.5 ml of lysate for each immunoprecipitation. The type of lysate did not affect the overall outcome of any of the immunoprecipitation experiments (data not shown), although the yield of immunoprecipitated heavy chain was greater when SDS was present. Nondenaturing NP-40 lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions so that the final buffer was 0.5% NP-40 (or Igepal CA-630; Sigma Chemical Co.), 50 mM Tris, pH 8, and 10 mM MgCl2. Samples were agitated for 20 min at 4°C and then clarified by centrifuging in a microfuge at full speed for 10 min. The resulting supernatant was used for immunoprecipitation. Denaturing SDS lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions in 100–150 μl of 1% SDS and 2 mM DTT. Samples were heated to 95°C for 5 min, cooled to room temperature, agitated vigorously, and diluted into NP-40 buffer so that the final lysate used for immunoprecipitation was 0.1% SDS, 0.2 mM DTT, 0.5% Igepal, 50 mM Tris, pH 8, and 10 mM MgCl2. In some experiments (Fig. 7, Fig. 9C and Fig. D, Fig. 11, and Fig. 12), nondenaturing NP-40 lysates were made and then SDS and DTT were added after the clarifying spin to final concentrations of 0.1% and 0.2 mM, respectively. All immune complexes were recovered by precipitation with fixed Staphylococcus aureus bacteria (Staph A).


The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate.

Shamu CE, Story CM, Rapoport TA, Ploegh HL - J. Cell Biol. (1999)

Ubiquitin-conjugated and deglycosylated heavy chains accumulate at the same rate. (A) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. αHC immunoprecipitates of each lysate were split and part was reimmunoprecipitated with αUb antibodies, as described for the experiments shown in Fig. 6 B. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. Note that the exposure of the αHC gel is 12 h whereas the exposure of the αUb gel is 3 wk. (B) The data in A were quantitated on a PhosphorImager and graphed. Note the different scales on the two y axes.
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Related In: Results  -  Collection

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Figure 7: Ubiquitin-conjugated and deglycosylated heavy chains accumulate at the same rate. (A) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. αHC immunoprecipitates of each lysate were split and part was reimmunoprecipitated with αUb antibodies, as described for the experiments shown in Fig. 6 B. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. Note that the exposure of the αHC gel is 12 h whereas the exposure of the αUb gel is 3 wk. (B) The data in A were quantitated on a PhosphorImager and graphed. Note the different scales on the two y axes.
Mentions: As noted in the figure legends, lysates were made in three different ways, depending on the experiment. In all cases, 1–2 × 106 cells (or cell equivalents) were used to make 1–1.5 ml of lysate for each immunoprecipitation. The type of lysate did not affect the overall outcome of any of the immunoprecipitation experiments (data not shown), although the yield of immunoprecipitated heavy chain was greater when SDS was present. Nondenaturing NP-40 lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions so that the final buffer was 0.5% NP-40 (or Igepal CA-630; Sigma Chemical Co.), 50 mM Tris, pH 8, and 10 mM MgCl2. Samples were agitated for 20 min at 4°C and then clarified by centrifuging in a microfuge at full speed for 10 min. The resulting supernatant was used for immunoprecipitation. Denaturing SDS lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions in 100–150 μl of 1% SDS and 2 mM DTT. Samples were heated to 95°C for 5 min, cooled to room temperature, agitated vigorously, and diluted into NP-40 buffer so that the final lysate used for immunoprecipitation was 0.1% SDS, 0.2 mM DTT, 0.5% Igepal, 50 mM Tris, pH 8, and 10 mM MgCl2. In some experiments (Fig. 7, Fig. 9C and Fig. D, Fig. 11, and Fig. 12), nondenaturing NP-40 lysates were made and then SDS and DTT were added after the clarifying spin to final concentrations of 0.1% and 0.2 mM, respectively. All immune complexes were recovered by precipitation with fixed Staphylococcus aureus bacteria (Staph A).

Bottom Line: We find that heavy chains are ubiquitinated before they are degraded.Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane.Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. shamu@bcmp.med.harvard.edu

ABSTRACT
The human cytomegalovirus protein, US11, initiates the destruction of MHC class I heavy chains by targeting them for dislocation from the ER to the cytosol and subsequent degradation by the proteasome. We report the development of a permeabilized cell system that recapitulates US11-dependent degradation of class I heavy chains. We have used this system, in combination with experiments in intact cells, to identify and order intermediates in the US11-dependent degradation pathway. We find that heavy chains are ubiquitinated before they are degraded. Ubiquitination of the cytosolic tail of heavy chain is not required for its dislocation and degradation, suggesting that ubiquitination occurs after at least part of the heavy chain has been dislocated from the ER. Thus, ubiquitination of the heavy chain does not appear to be the signal to start dislocation. Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane. Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

Show MeSH
Related in: MedlinePlus