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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

IEF demonstrates that deglycosylated heavy chains accumulate in permeabilized US11 cells. Samples from the experiment shown in Fig. 1 D were treated or mock-treated with PNGase F as indicated, and analyzed by one-dimensional IEF as described (Ploegh 1995). Arrows point to bands corresponding to deglycosylated MHC class I heavy chains. The different HLA gene products migrate with disparate, though characteristic, isoelectric points.
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Figure 2: IEF demonstrates that deglycosylated heavy chains accumulate in permeabilized US11 cells. Samples from the experiment shown in Fig. 1 D were treated or mock-treated with PNGase F as indicated, and analyzed by one-dimensional IEF as described (Ploegh 1995). Arrows point to bands corresponding to deglycosylated MHC class I heavy chains. The different HLA gene products migrate with disparate, though characteristic, isoelectric points.

Mentions: As in intact cells (Fig. 1 A, lanes 1–3 and 7–9), in the permeabilized cell system, heavy chains were degraded only when US11 was present (Fig. 1 B compare lanes 1–6 with lanes 7–12). The half-life of MHC class I heavy chain in permeabilized US11 cells was ∼10 min (Fig. 1 C), somewhat longer than its 2–3-min half-life in intact US11 cells (Wiertz et al. 1996a). In the presence of the proteasome inhibitors ZL3VS (Fig. 1 D) or lactacystin (data not shown), degradation was largely prevented and a lower molecular mass heavy chain species accumulated in US11 cells. This species is endoglycosidase H (Endo H) resistant (data not shown) and its molecular mass by SDS-PAGE corresponds to that of the deglycosylated heavy chain species that accumulates in the cytosol of intact US11 cells treated with proteasome inhibitors (Fig. 1 A lanes 4–6). As was observed in intact cells, the change in molecular mass of the heavy chain in the permeabilized cells is accompanied by a change in isoelectric point upon hydrolysis of the glycoamide bond. This is best seen by comparison with bacterial N-glycanase (PNGase F) treated samples (Fig. 2 compare lane 3 with lanes 2 and 6). Thus, deglycosylated heavy chains also accumulate in permeabilized US11 cells in the presence of proteasome inhibitor.


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)

IEF demonstrates that deglycosylated heavy chains accumulate in permeabilized US11 cells. Samples from the experiment shown in Fig. 1 D were treated or mock-treated with PNGase F as indicated, and analyzed by one-dimensional IEF as described (Ploegh 1995). Arrows point to bands corresponding to deglycosylated MHC class I heavy chains. The different HLA gene products migrate with disparate, though characteristic, isoelectric points.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164983&req=5

Figure 2: IEF demonstrates that deglycosylated heavy chains accumulate in permeabilized US11 cells. Samples from the experiment shown in Fig. 1 D were treated or mock-treated with PNGase F as indicated, and analyzed by one-dimensional IEF as described (Ploegh 1995). Arrows point to bands corresponding to deglycosylated MHC class I heavy chains. The different HLA gene products migrate with disparate, though characteristic, isoelectric points.
Mentions: As in intact cells (Fig. 1 A, lanes 1–3 and 7–9), in the permeabilized cell system, heavy chains were degraded only when US11 was present (Fig. 1 B compare lanes 1–6 with lanes 7–12). The half-life of MHC class I heavy chain in permeabilized US11 cells was ∼10 min (Fig. 1 C), somewhat longer than its 2–3-min half-life in intact US11 cells (Wiertz et al. 1996a). In the presence of the proteasome inhibitors ZL3VS (Fig. 1 D) or lactacystin (data not shown), degradation was largely prevented and a lower molecular mass heavy chain species accumulated in US11 cells. This species is endoglycosidase H (Endo H) resistant (data not shown) and its molecular mass by SDS-PAGE corresponds to that of the deglycosylated heavy chain species that accumulates in the cytosol of intact US11 cells treated with proteasome inhibitors (Fig. 1 A lanes 4–6). As was observed in intact cells, the change in molecular mass of the heavy chain in the permeabilized cells is accompanied by a change in isoelectric point upon hydrolysis of the glycoamide bond. This is best seen by comparison with bacterial N-glycanase (PNGase F) treated samples (Fig. 2 compare lane 3 with lanes 2 and 6). Thus, deglycosylated heavy chains also accumulate in permeabilized US11 cells in the presence of proteasome inhibitor.

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