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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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Related in: MedlinePlus

Deglycosylated heavy chain from permeabilized US11 cells is soluble. US11 and control cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (A) Fractionation after homogenization. After 30 min of chase at 37°C, the cells were homogenized mechanically and the homogenates were fractionated by centrifugation. Fractions were diluted into NP-40 lysis buffer and immunoprecipitation was carried out with antibodies to HC, transferrin receptor (TfR), β2m, or US11. (B) Fractionation by squeeze-out centrifugation (see Materials and Methods). Total starting material (T), pellet (P), and supernatant (S) fractions are labeled. Denaturing SDS lysates were made and immunoprecipitations were carried out with αHC serum.
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Figure 3: Deglycosylated heavy chain from permeabilized US11 cells is soluble. US11 and control cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (A) Fractionation after homogenization. After 30 min of chase at 37°C, the cells were homogenized mechanically and the homogenates were fractionated by centrifugation. Fractions were diluted into NP-40 lysis buffer and immunoprecipitation was carried out with antibodies to HC, transferrin receptor (TfR), β2m, or US11. (B) Fractionation by squeeze-out centrifugation (see Materials and Methods). Total starting material (T), pellet (P), and supernatant (S) fractions are labeled. Denaturing SDS lysates were made and immunoprecipitations were carried out with αHC serum.

Mentions: As in intact cells, the deglycosylated heavy chains in the permeabilized cells were soluble and cytosolic (Fig. 3 A). The glycosylated heavy chains from both permeabilized US11 and control cells fractionated mostly with the particulate fractions, as did the control membrane proteins transferrin receptor (TfR) and US11. The light chain β2m, a soluble secretory protein, fractionated with membrane pellets in permeabilized control cells, as expected from its tight association with the MHC class I heavy chain. In US11 cells, because there is little heavy chain, most of the β2m is soluble in the ER lumen (Wiertz et al. 1996a). We found β2m in both the 10-K pellet and the 100-K supernatant fractions in permeabilized US11 cells (Fig. 3 A), indicating that a portion of the ER content was released during homogenization.


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)

Deglycosylated heavy chain from permeabilized US11 cells is soluble. US11 and control cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (A) Fractionation after homogenization. After 30 min of chase at 37°C, the cells were homogenized mechanically and the homogenates were fractionated by centrifugation. Fractions were diluted into NP-40 lysis buffer and immunoprecipitation was carried out with antibodies to HC, transferrin receptor (TfR), β2m, or US11. (B) Fractionation by squeeze-out centrifugation (see Materials and Methods). Total starting material (T), pellet (P), and supernatant (S) fractions are labeled. Denaturing SDS lysates were made and immunoprecipitations were carried out with αHC serum.
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Related In: Results  -  Collection

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Figure 3: Deglycosylated heavy chain from permeabilized US11 cells is soluble. US11 and control cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (A) Fractionation after homogenization. After 30 min of chase at 37°C, the cells were homogenized mechanically and the homogenates were fractionated by centrifugation. Fractions were diluted into NP-40 lysis buffer and immunoprecipitation was carried out with antibodies to HC, transferrin receptor (TfR), β2m, or US11. (B) Fractionation by squeeze-out centrifugation (see Materials and Methods). Total starting material (T), pellet (P), and supernatant (S) fractions are labeled. Denaturing SDS lysates were made and immunoprecipitations were carried out with αHC serum.
Mentions: As in intact cells, the deglycosylated heavy chains in the permeabilized cells were soluble and cytosolic (Fig. 3 A). The glycosylated heavy chains from both permeabilized US11 and control cells fractionated mostly with the particulate fractions, as did the control membrane proteins transferrin receptor (TfR) and US11. The light chain β2m, a soluble secretory protein, fractionated with membrane pellets in permeabilized control cells, as expected from its tight association with the MHC class I heavy chain. In US11 cells, because there is little heavy chain, most of the β2m is soluble in the ER lumen (Wiertz et al. 1996a). We found β2m in both the 10-K pellet and the 100-K supernatant fractions in permeabilized US11 cells (Fig. 3 A), indicating that a portion of the ER content was released during homogenization.

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