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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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The UT3 domain of Ufd1 is required for retrotranslocation. (A) US11-expressing cells were labeled and permeabilized in the presence of different Ufd1–Npl4 (U/N) variants. The added U/N complexes included wild-type U/N or U/N lacking either the NZF domain in Npl4 (U/NΔZF), the UT3 domain in Ufd1 (UΔUT3/N), or both the NZF domain and the UT3 domains (UΔUT3/NΔZF). After incubation for the indicated time periods, the samples were analyzed by immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. (B) Comparison of the dominant-negative effects of the p97 ATPase mutant KA and of the cofactor complex UΔUT3/N. The experiment was performed as in A, but proteasome inhibitors were included during incubation. The samples were analyzed both directly (T) and after fractionation into membrane (P) or cytosol (S) fractions. A portion of the precipitated HC was subjected to a second round of immunoprecipitation with ubiquitin (Ub) antibodies (bottom). Quantification of the polyubiquitin chains is given above the lanes, with the results in lane 2 set to 100%.
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fig8: The UT3 domain of Ufd1 is required for retrotranslocation. (A) US11-expressing cells were labeled and permeabilized in the presence of different Ufd1–Npl4 (U/N) variants. The added U/N complexes included wild-type U/N or U/N lacking either the NZF domain in Npl4 (U/NΔZF), the UT3 domain in Ufd1 (UΔUT3/N), or both the NZF domain and the UT3 domains (UΔUT3/NΔZF). After incubation for the indicated time periods, the samples were analyzed by immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. (B) Comparison of the dominant-negative effects of the p97 ATPase mutant KA and of the cofactor complex UΔUT3/N. The experiment was performed as in A, but proteasome inhibitors were included during incubation. The samples were analyzed both directly (T) and after fractionation into membrane (P) or cytosol (S) fractions. A portion of the precipitated HC was subjected to a second round of immunoprecipitation with ubiquitin (Ub) antibodies (bottom). Quantification of the polyubiquitin chains is given above the lanes, with the results in lane 2 set to 100%.

Mentions: Next, we tested whether the ubiquitin-binding domain in Ufd1 is important for the dislocation and degradation of MHC class I heavy chains. We reasoned that a mutant Ufd1–Npl4 complex lacking the UT3 ubiquitin-binding site would act as a dominant-negative mutant by sequestering endogenous p97 in a defective complex that could no longer efficiently interact with ubiquitin chains on the substrate. Indeed, although addition of either wild-type Ufd1–Npl4 (U/N) or Ufd1–Npl4ΔZF (U/NΔZF) had no effect on heavy chain degradation (Fig. 8Figure 8.


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)

The UT3 domain of Ufd1 is required for retrotranslocation. (A) US11-expressing cells were labeled and permeabilized in the presence of different Ufd1–Npl4 (U/N) variants. The added U/N complexes included wild-type U/N or U/N lacking either the NZF domain in Npl4 (U/NΔZF), the UT3 domain in Ufd1 (UΔUT3/N), or both the NZF domain and the UT3 domains (UΔUT3/NΔZF). After incubation for the indicated time periods, the samples were analyzed by immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. (B) Comparison of the dominant-negative effects of the p97 ATPase mutant KA and of the cofactor complex UΔUT3/N. The experiment was performed as in A, but proteasome inhibitors were included during incubation. The samples were analyzed both directly (T) and after fractionation into membrane (P) or cytosol (S) fractions. A portion of the precipitated HC was subjected to a second round of immunoprecipitation with ubiquitin (Ub) antibodies (bottom). Quantification of the polyubiquitin chains is given above the lanes, with the results in lane 2 set to 100%.
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Related In: Results  -  Collection

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fig8: The UT3 domain of Ufd1 is required for retrotranslocation. (A) US11-expressing cells were labeled and permeabilized in the presence of different Ufd1–Npl4 (U/N) variants. The added U/N complexes included wild-type U/N or U/N lacking either the NZF domain in Npl4 (U/NΔZF), the UT3 domain in Ufd1 (UΔUT3/N), or both the NZF domain and the UT3 domains (UΔUT3/NΔZF). After incubation for the indicated time periods, the samples were analyzed by immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. (B) Comparison of the dominant-negative effects of the p97 ATPase mutant KA and of the cofactor complex UΔUT3/N. The experiment was performed as in A, but proteasome inhibitors were included during incubation. The samples were analyzed both directly (T) and after fractionation into membrane (P) or cytosol (S) fractions. A portion of the precipitated HC was subjected to a second round of immunoprecipitation with ubiquitin (Ub) antibodies (bottom). Quantification of the polyubiquitin chains is given above the lanes, with the results in lane 2 set to 100%.
Mentions: Next, we tested whether the ubiquitin-binding domain in Ufd1 is important for the dislocation and degradation of MHC class I heavy chains. We reasoned that a mutant Ufd1–Npl4 complex lacking the UT3 ubiquitin-binding site would act as a dominant-negative mutant by sequestering endogenous p97 in a defective complex that could no longer efficiently interact with ubiquitin chains on the substrate. Indeed, although addition of either wild-type Ufd1–Npl4 (U/N) or Ufd1–Npl4ΔZF (U/NΔZF) had no effect on heavy chain degradation (Fig. 8Figure 8.

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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