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Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.

Ye Y, Meyer HH, Rapoport TA - J. Cell Biol. (2003)

Bottom Line: A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome.Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1.We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

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Association of p97 with MHC class I heavy chains. Cells expressing the cytomegalovirus protein US11 were incubated with proteasome inhibitors and [35S]methionine, and were then permeabilized in the presence of various purified His-p97 proteins. After incubation, samples were taken at different time points of the chase period and fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (A–C, top). The remainder of the samples was subjected to sequential immunoprecipitation with His and HC antibodies to detect heavy chains bound to p97 (A–C, bottom). HC+CHO and HC−CHO indicate the glycosylated and deglycosylated forms of the heavy chains, respectively.
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fig3: Association of p97 with MHC class I heavy chains. Cells expressing the cytomegalovirus protein US11 were incubated with proteasome inhibitors and [35S]methionine, and were then permeabilized in the presence of various purified His-p97 proteins. After incubation, samples were taken at different time points of the chase period and fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (A–C, top). The remainder of the samples was subjected to sequential immunoprecipitation with His and HC antibodies to detect heavy chains bound to p97 (A–C, bottom). HC+CHO and HC−CHO indicate the glycosylated and deglycosylated forms of the heavy chains, respectively.

Mentions: Next, we investigated the association of the p97 mutants with a retrotranslocation substrate. The substrate chosen was MHC class I heavy chain, which is targeted for retrotranslocation and subsequent proteasomal degradation in cells expressing the human cytomegalovirus protein US11 (Wiertz et al., 1996). The process can be recapitulated in permeabilized cells (Shamu et al., 1999). To determine binding of p97 to MHC class I heavy chains, US11-expressing cells were treated with proteasome inhibitors, labeled with [35S]methionine, and permeabilized with the detergent digitonin. His-tagged p97 variants were added, the cells were incubated for a chase period, and then they were fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (Fig. 3Figure 3.


Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.

Ye Y, Meyer HH, Rapoport TA - J. Cell Biol. (2003)

Association of p97 with MHC class I heavy chains. Cells expressing the cytomegalovirus protein US11 were incubated with proteasome inhibitors and [35S]methionine, and were then permeabilized in the presence of various purified His-p97 proteins. After incubation, samples were taken at different time points of the chase period and fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (A–C, top). The remainder of the samples was subjected to sequential immunoprecipitation with His and HC antibodies to detect heavy chains bound to p97 (A–C, bottom). HC+CHO and HC−CHO indicate the glycosylated and deglycosylated forms of the heavy chains, respectively.
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Related In: Results  -  Collection

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fig3: Association of p97 with MHC class I heavy chains. Cells expressing the cytomegalovirus protein US11 were incubated with proteasome inhibitors and [35S]methionine, and were then permeabilized in the presence of various purified His-p97 proteins. After incubation, samples were taken at different time points of the chase period and fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (A–C, top). The remainder of the samples was subjected to sequential immunoprecipitation with His and HC antibodies to detect heavy chains bound to p97 (A–C, bottom). HC+CHO and HC−CHO indicate the glycosylated and deglycosylated forms of the heavy chains, respectively.
Mentions: Next, we investigated the association of the p97 mutants with a retrotranslocation substrate. The substrate chosen was MHC class I heavy chain, which is targeted for retrotranslocation and subsequent proteasomal degradation in cells expressing the human cytomegalovirus protein US11 (Wiertz et al., 1996). The process can be recapitulated in permeabilized cells (Shamu et al., 1999). To determine binding of p97 to MHC class I heavy chains, US11-expressing cells were treated with proteasome inhibitors, labeled with [35S]methionine, and permeabilized with the detergent digitonin. His-tagged p97 variants were added, the cells were incubated for a chase period, and then they were fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (Fig. 3Figure 3.

Bottom Line: A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome.Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1.We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

Show MeSH