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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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Ubiquitination of the heavy chain cytoplasmic tail is not required for its dislocation from the ER. The stability of HA-tagged MHC class I allele A2 (HA/A2), with either a wt cytoplasmic tail (wt) or with a mutant cytoplasmic tail (K→R), was analyzed in US11 and control cells. Astrocytoma cells stably expressing the HA/A2 constructs were pulse-labeled and chased in the absence (A) or in the presence (C) of ZL3VS, and NP-40 lysates were made as described for Fig. 1 A. HA-tagged heavy chains were immunoprecipitated specifically using monoclonal antibody 12CA5. (B) The data in A were quantitated by PhosphorImager.
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Figure 10: Ubiquitination of the heavy chain cytoplasmic tail is not required for its dislocation from the ER. The stability of HA-tagged MHC class I allele A2 (HA/A2), with either a wt cytoplasmic tail (wt) or with a mutant cytoplasmic tail (K→R), was analyzed in US11 and control cells. Astrocytoma cells stably expressing the HA/A2 constructs were pulse-labeled and chased in the absence (A) or in the presence (C) of ZL3VS, and NP-40 lysates were made as described for Fig. 1 A. HA-tagged heavy chains were immunoprecipitated specifically using monoclonal antibody 12CA5. (B) The data in A were quantitated by PhosphorImager.

Mentions: With heavy chain ubiquitination occurring at the ER membrane, it seemed possible that ubiquitination might be the initiating signal for heavy chain dislocation. To determine whether ubiquitination of the heavy chain is required for its dislocation, we prevented ubiquitination of the heavy chain cytosolic tail by removing all lysines from the tail domain. Two different HA epitope-tagged heavy chain constructs, based on the HLA allele A2, were made, one with a wild-type (wt) cytosolic tail and one with the three cytosolic tail lysines mutated to arginine (K→R). Cell lines stably expressing the HA-tagged heavy chains (HA/A2) were selected. We found that K→R HA/A2 is degraded at approximately the same rate as wt HA/A2 in US11 cell lines, while both HA/A2 proteins are stable in control cells (Fig. 10A and Fig. B, and data not shown). When pulse–chase experiments were conducted in the presence of proteasome inhibitor, deglycosylated HA-tagged wt and K→R heavy chain intermediates accumulated at roughly the same rate (Fig. 10 C). In all cases, the HA/A2 heavy chains were dislocated and degraded more slowly than endogenous heavy chain in cells not expressing HA/A2 constructs. We attribute this to the overexpression of the HA/A2 degradation substrates, which may saturate the degradation machinery (Story et al. 1999). These results were reproducible in multiple, independently derived cell lines. Ubiquitination of the heavy chain cytosolic tail, therefore, is not required to initiate US11-dependent destruction of the protein.


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)

Ubiquitination of the heavy chain cytoplasmic tail is not required for its dislocation from the ER. The stability of HA-tagged MHC class I allele A2 (HA/A2), with either a wt cytoplasmic tail (wt) or with a mutant cytoplasmic tail (K→R), was analyzed in US11 and control cells. Astrocytoma cells stably expressing the HA/A2 constructs were pulse-labeled and chased in the absence (A) or in the presence (C) of ZL3VS, and NP-40 lysates were made as described for Fig. 1 A. HA-tagged heavy chains were immunoprecipitated specifically using monoclonal antibody 12CA5. (B) The data in A were quantitated by PhosphorImager.
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Figure 10: Ubiquitination of the heavy chain cytoplasmic tail is not required for its dislocation from the ER. The stability of HA-tagged MHC class I allele A2 (HA/A2), with either a wt cytoplasmic tail (wt) or with a mutant cytoplasmic tail (K→R), was analyzed in US11 and control cells. Astrocytoma cells stably expressing the HA/A2 constructs were pulse-labeled and chased in the absence (A) or in the presence (C) of ZL3VS, and NP-40 lysates were made as described for Fig. 1 A. HA-tagged heavy chains were immunoprecipitated specifically using monoclonal antibody 12CA5. (B) The data in A were quantitated by PhosphorImager.
Mentions: With heavy chain ubiquitination occurring at the ER membrane, it seemed possible that ubiquitination might be the initiating signal for heavy chain dislocation. To determine whether ubiquitination of the heavy chain is required for its dislocation, we prevented ubiquitination of the heavy chain cytosolic tail by removing all lysines from the tail domain. Two different HA epitope-tagged heavy chain constructs, based on the HLA allele A2, were made, one with a wild-type (wt) cytosolic tail and one with the three cytosolic tail lysines mutated to arginine (K→R). Cell lines stably expressing the HA-tagged heavy chains (HA/A2) were selected. We found that K→R HA/A2 is degraded at approximately the same rate as wt HA/A2 in US11 cell lines, while both HA/A2 proteins are stable in control cells (Fig. 10A and Fig. B, and data not shown). When pulse–chase experiments were conducted in the presence of proteasome inhibitor, deglycosylated HA-tagged wt and K→R heavy chain intermediates accumulated at roughly the same rate (Fig. 10 C). In all cases, the HA/A2 heavy chains were dislocated and degraded more slowly than endogenous heavy chain in cells not expressing HA/A2 constructs. We attribute this to the overexpression of the HA/A2 degradation substrates, which may saturate the degradation machinery (Story et al. 1999). These results were reproducible in multiple, independently derived cell lines. Ubiquitination of the heavy chain cytosolic tail, therefore, is not required to initiate US11-dependent destruction of the protein.

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