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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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Appearance of deglycosylated heavy chain in permeabilized US11 cells requires ATP. (Lanes 1–4) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (Lanes 5–16) Prepared as in lanes 1–4, except that the ATP regenerating system was omitted from the permeabilization buffers and with the additions as indicated. Glucose was at 10 mM, hexokinase was at 0.1 unit/μl, and AMPPNP and magnesium acetate were each at 3 mM. Note that some degradation of heavy chain occurs in the absence of ATP. This degradation is not inhibitable by the addition of proteasome inhibitor (ZL3VS) or standard protease inhibitors (aprotinin, leupeptin, pepstatin, chymostatin, or elastatinal) and has not been characterized further.
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Figure 5: Appearance of deglycosylated heavy chain in permeabilized US11 cells requires ATP. (Lanes 1–4) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (Lanes 5–16) Prepared as in lanes 1–4, except that the ATP regenerating system was omitted from the permeabilization buffers and with the additions as indicated. Glucose was at 10 mM, hexokinase was at 0.1 unit/μl, and AMPPNP and magnesium acetate were each at 3 mM. Note that some degradation of heavy chain occurs in the absence of ATP. This degradation is not inhibitable by the addition of proteasome inhibitor (ZL3VS) or standard protease inhibitors (aprotinin, leupeptin, pepstatin, chymostatin, or elastatinal) and has not been characterized further.

Mentions: In intact astrocytomas expressing US11 or US2, accumulation of deglycosylated heavy chain requires ATP (Wiertz et al. 1996b; Tortorella, D., unpublished observation). To test whether this also applies to the permeabilized cell system, we depleted ATP from permeabilization reactions carried out in the presence of proteasome inhibitor. Simply omitting the ATP-regenerating system from the permeabilization reactions significantly reduced the amount of deglycosylated heavy chain that appeared (Fig. 5, lanes 5–8). When remaining ATP was depleted by the addition of hexokinase and glucose, no deglycosylated heavy chain was detectable (Fig. 5, lanes 9–12). Moreover, ATP could not be substituted with the nonhydrolyzable ATP analogue AMPPNP (Fig. 5, lanes 13–16). Thus, as in intact cells, the US11-dependent accumulation of deglycosylated heavy chain in permeabilized cells requires ATP. Taken together, these data demonstrate that the permeabilized cell system faithfully reproduces the US11-dependent degradation of MHC class I heavy chain seen in intact cells.


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)

Appearance of deglycosylated heavy chain in permeabilized US11 cells requires ATP. (Lanes 1–4) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (Lanes 5–16) Prepared as in lanes 1–4, except that the ATP regenerating system was omitted from the permeabilization buffers and with the additions as indicated. Glucose was at 10 mM, hexokinase was at 0.1 unit/μl, and AMPPNP and magnesium acetate were each at 3 mM. Note that some degradation of heavy chain occurs in the absence of ATP. This degradation is not inhibitable by the addition of proteasome inhibitor (ZL3VS) or standard protease inhibitors (aprotinin, leupeptin, pepstatin, chymostatin, or elastatinal) and has not been characterized further.
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Related In: Results  -  Collection

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Figure 5: Appearance of deglycosylated heavy chain in permeabilized US11 cells requires ATP. (Lanes 1–4) US11 cells were labeled, permeabilized, and chased in the presence of the proteasome inhibitor ZL3VS as described for Fig. 1 D. (Lanes 5–16) Prepared as in lanes 1–4, except that the ATP regenerating system was omitted from the permeabilization buffers and with the additions as indicated. Glucose was at 10 mM, hexokinase was at 0.1 unit/μl, and AMPPNP and magnesium acetate were each at 3 mM. Note that some degradation of heavy chain occurs in the absence of ATP. This degradation is not inhibitable by the addition of proteasome inhibitor (ZL3VS) or standard protease inhibitors (aprotinin, leupeptin, pepstatin, chymostatin, or elastatinal) and has not been characterized further.
Mentions: In intact astrocytomas expressing US11 or US2, accumulation of deglycosylated heavy chain requires ATP (Wiertz et al. 1996b; Tortorella, D., unpublished observation). To test whether this also applies to the permeabilized cell system, we depleted ATP from permeabilization reactions carried out in the presence of proteasome inhibitor. Simply omitting the ATP-regenerating system from the permeabilization reactions significantly reduced the amount of deglycosylated heavy chain that appeared (Fig. 5, lanes 5–8). When remaining ATP was depleted by the addition of hexokinase and glucose, no deglycosylated heavy chain was detectable (Fig. 5, lanes 9–12). Moreover, ATP could not be substituted with the nonhydrolyzable ATP analogue AMPPNP (Fig. 5, lanes 13–16). Thus, as in intact cells, the US11-dependent accumulation of deglycosylated heavy chain in permeabilized cells requires ATP. Taken together, these data demonstrate that the permeabilized cell system faithfully reproduces the US11-dependent degradation of MHC class I heavy chain seen in intact cells.

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