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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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The majority of ubiquitinated heavy chain is not glycosylated. (A) US11 cells were labeled with 3H-mannose in the presence (lane 4) or absence (lane 3) of ZL3VS, denaturing SDS lysates were made, and heavy chains were immunoprecipitated with αHC serum. [35S]methionine-labeled heavy chain immunoprecipitates from another experiment (US11 cells treated with ZL3VS) were run alongside the 3H-mannose–labeled samples (lanes 1 and 2). The asterisk indicates high molecular mass species, most likely ubiquitinated heavy chains, that are immunoprecipitated with αHC serum. The exposure time of this gel is 24 d. (B) US11 cells treated with ZL3VS were labeled with [35S]methionine and chased intact for 0 or 15 min. Denaturing SDS lysates were made and subjected to sequential immunoprecipitation with αHC and αUb serum, as described for Fig. 6 B. One-half of each αUb precipitate was analyzed directly by SDS PAGE (lanes 1 and 2) and the other half was precipitated with Con A–Sepharose, either in the presence (+) or absence (−) of 0.5 M methyl α-d-mannopyranoside. Note that the two panels are from a single exposure (4 wk) of the same gel; the lanes were separated to help clarify the experimental procedure. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. (C) US11 cells from the experiment shown in B but chased intact for 7 min. Con A precipitations were done as in B after immunoprecipitation with αHC serum. The two panels are from a single exposure (3 d) of the same gel.
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Figure 8: The majority of ubiquitinated heavy chain is not glycosylated. (A) US11 cells were labeled with 3H-mannose in the presence (lane 4) or absence (lane 3) of ZL3VS, denaturing SDS lysates were made, and heavy chains were immunoprecipitated with αHC serum. [35S]methionine-labeled heavy chain immunoprecipitates from another experiment (US11 cells treated with ZL3VS) were run alongside the 3H-mannose–labeled samples (lanes 1 and 2). The asterisk indicates high molecular mass species, most likely ubiquitinated heavy chains, that are immunoprecipitated with αHC serum. The exposure time of this gel is 24 d. (B) US11 cells treated with ZL3VS were labeled with [35S]methionine and chased intact for 0 or 15 min. Denaturing SDS lysates were made and subjected to sequential immunoprecipitation with αHC and αUb serum, as described for Fig. 6 B. One-half of each αUb precipitate was analyzed directly by SDS PAGE (lanes 1 and 2) and the other half was precipitated with Con A–Sepharose, either in the presence (+) or absence (−) of 0.5 M methyl α-d-mannopyranoside. Note that the two panels are from a single exposure (4 wk) of the same gel; the lanes were separated to help clarify the experimental procedure. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. (C) US11 cells from the experiment shown in B but chased intact for 7 min. Con A precipitations were done as in B after immunoprecipitation with αHC serum. The two panels are from a single exposure (3 d) of the same gel.

Mentions: Having demonstrated the occurrence of ubiquitinated heavy chains, we wished to know whether ubiquitination precedes deglycosylation or vice versa. Two independent approaches were taken to determine the fraction of ubiquitinated heavy chains that are glycosylated. First, intact US11 cells were treated with proteasome inhibitors and labeled with 3H-mannose. Very little 3H-mannose-labeled, ubiquitinated heavy chain was detected. On very long (2 mo) exposures of gels from three independent experiments, the amount of 3H-mannose–labeled heavy chain precipitated with αUb serum was barely above background levels (data not shown). Furthermore, in αHC immunoprecipitations, 3H-mannose–labeled, nonubiquitinated heavy chains were efficiently recovered, but very little mannose label was detectable in the area of the high molecular mass, ubiquitinated HC species (Fig. 8 A, lanes 3 and 4). In contrast, such species were readily detectable in 35S-labeled cells (Fig. 8 A, lanes 1 and 2). Ubiquitinated glycosylated heavy chains account, at most, for only 1–2% of all glycosylated heavy chains in these experiments. Thus, it appears that the majority of the ubiquitinated heavy chains in US11 cells are deglycosylated.


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)

The majority of ubiquitinated heavy chain is not glycosylated. (A) US11 cells were labeled with 3H-mannose in the presence (lane 4) or absence (lane 3) of ZL3VS, denaturing SDS lysates were made, and heavy chains were immunoprecipitated with αHC serum. [35S]methionine-labeled heavy chain immunoprecipitates from another experiment (US11 cells treated with ZL3VS) were run alongside the 3H-mannose–labeled samples (lanes 1 and 2). The asterisk indicates high molecular mass species, most likely ubiquitinated heavy chains, that are immunoprecipitated with αHC serum. The exposure time of this gel is 24 d. (B) US11 cells treated with ZL3VS were labeled with [35S]methionine and chased intact for 0 or 15 min. Denaturing SDS lysates were made and subjected to sequential immunoprecipitation with αHC and αUb serum, as described for Fig. 6 B. One-half of each αUb precipitate was analyzed directly by SDS PAGE (lanes 1 and 2) and the other half was precipitated with Con A–Sepharose, either in the presence (+) or absence (−) of 0.5 M methyl α-d-mannopyranoside. Note that the two panels are from a single exposure (4 wk) of the same gel; the lanes were separated to help clarify the experimental procedure. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. (C) US11 cells from the experiment shown in B but chased intact for 7 min. Con A precipitations were done as in B after immunoprecipitation with αHC serum. The two panels are from a single exposure (3 d) of the same gel.
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Figure 8: The majority of ubiquitinated heavy chain is not glycosylated. (A) US11 cells were labeled with 3H-mannose in the presence (lane 4) or absence (lane 3) of ZL3VS, denaturing SDS lysates were made, and heavy chains were immunoprecipitated with αHC serum. [35S]methionine-labeled heavy chain immunoprecipitates from another experiment (US11 cells treated with ZL3VS) were run alongside the 3H-mannose–labeled samples (lanes 1 and 2). The asterisk indicates high molecular mass species, most likely ubiquitinated heavy chains, that are immunoprecipitated with αHC serum. The exposure time of this gel is 24 d. (B) US11 cells treated with ZL3VS were labeled with [35S]methionine and chased intact for 0 or 15 min. Denaturing SDS lysates were made and subjected to sequential immunoprecipitation with αHC and αUb serum, as described for Fig. 6 B. One-half of each αUb precipitate was analyzed directly by SDS PAGE (lanes 1 and 2) and the other half was precipitated with Con A–Sepharose, either in the presence (+) or absence (−) of 0.5 M methyl α-d-mannopyranoside. Note that the two panels are from a single exposure (4 wk) of the same gel; the lanes were separated to help clarify the experimental procedure. Nonspecific bands that precipitate with Staph A alone are identified by the asterisk. (C) US11 cells from the experiment shown in B but chased intact for 7 min. Con A precipitations were done as in B after immunoprecipitation with αHC serum. The two panels are from a single exposure (3 d) of the same gel.
Mentions: Having demonstrated the occurrence of ubiquitinated heavy chains, we wished to know whether ubiquitination precedes deglycosylation or vice versa. Two independent approaches were taken to determine the fraction of ubiquitinated heavy chains that are glycosylated. First, intact US11 cells were treated with proteasome inhibitors and labeled with 3H-mannose. Very little 3H-mannose-labeled, ubiquitinated heavy chain was detected. On very long (2 mo) exposures of gels from three independent experiments, the amount of 3H-mannose–labeled heavy chain precipitated with αUb serum was barely above background levels (data not shown). Furthermore, in αHC immunoprecipitations, 3H-mannose–labeled, nonubiquitinated heavy chains were efficiently recovered, but very little mannose label was detectable in the area of the high molecular mass, ubiquitinated HC species (Fig. 8 A, lanes 3 and 4). In contrast, such species were readily detectable in 35S-labeled cells (Fig. 8 A, lanes 1 and 2). Ubiquitinated glycosylated heavy chains account, at most, for only 1–2% of all glycosylated heavy chains in these experiments. Thus, it appears that the majority of the ubiquitinated heavy chains in US11 cells are deglycosylated.

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