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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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Effect of p97 mutants on MHC class I heavy chain degradation. US11-expressing cells were labeled and permeabilized in the presence of various purified His-p97 proteins. The samples were chase-incubated for different time periods and subjected to immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. Error bars in the bottom graph are derived from three experiments.
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fig4: Effect of p97 mutants on MHC class I heavy chain degradation. US11-expressing cells were labeled and permeabilized in the presence of various purified His-p97 proteins. The samples were chase-incubated for different time periods and subjected to immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. Error bars in the bottom graph are derived from three experiments.

Mentions: Next, we tested whether the mutant p97 proteins have a dominant-negative effect on the retrotranslocation of MHC class I heavy chains (Fig. 3 A–C, top panels). In the absence of added p97 or in the presence of exogenous wild-type p97, a significant fraction of the heavy chains was retrotranslocated into the cytosol during the chase period (Fig. 3 A, top, lane 4; Fig. 3 B, top, lane 16). The cytosolic heavy chains have a slightly faster mobility in the SDS gel because they are deglycosylated by a cytosolic N-glycanase. Likewise, p97 mutants that were defective in substrate interaction (AK, AA, and ΔN) also did not affect retrotranslocation. In contrast, the p97 variants that allowed substrate binding but did not permit ATP hydrolysis (EQ, QQ, KA, and ΔD2; Fig. 1 C) strongly inhibited heavy chain dislocation; the amount of deglycosylated heavy chains in the cytosol fraction (S) was reduced, and the amount of glycosylated heavy chains in the membrane fraction (P) was increased (Fig. 3, top). The D1 hydrolysis mutant QE, which had lower affinity to substrate and had ∼50% residual ATPase activity (Fig. 1 C), had a weak effect on heavy chain retrotranslocation (Fig. 3 A, top, lane 8 vs. lane 4 and lane 7 vs. lane 3; see also Fig. 4)Figure 4.


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)

Effect of p97 mutants on MHC class I heavy chain degradation. US11-expressing cells were labeled and permeabilized in the presence of various purified His-p97 proteins. The samples were chase-incubated for different time periods and subjected to immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. Error bars in the bottom graph are derived from three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172719&req=5

fig4: Effect of p97 mutants on MHC class I heavy chain degradation. US11-expressing cells were labeled and permeabilized in the presence of various purified His-p97 proteins. The samples were chase-incubated for different time periods and subjected to immunoprecipitation with heavy chain (HC) antibodies. The graphs show the quantification of the experiments. Error bars in the bottom graph are derived from three experiments.
Mentions: Next, we tested whether the mutant p97 proteins have a dominant-negative effect on the retrotranslocation of MHC class I heavy chains (Fig. 3 A–C, top panels). In the absence of added p97 or in the presence of exogenous wild-type p97, a significant fraction of the heavy chains was retrotranslocated into the cytosol during the chase period (Fig. 3 A, top, lane 4; Fig. 3 B, top, lane 16). The cytosolic heavy chains have a slightly faster mobility in the SDS gel because they are deglycosylated by a cytosolic N-glycanase. Likewise, p97 mutants that were defective in substrate interaction (AK, AA, and ΔN) also did not affect retrotranslocation. In contrast, the p97 variants that allowed substrate binding but did not permit ATP hydrolysis (EQ, QQ, KA, and ΔD2; Fig. 1 C) strongly inhibited heavy chain dislocation; the amount of deglycosylated heavy chains in the cytosol fraction (S) was reduced, and the amount of glycosylated heavy chains in the membrane fraction (P) was increased (Fig. 3, top). The D1 hydrolysis mutant QE, which had lower affinity to substrate and had ∼50% residual ATPase activity (Fig. 1 C), had a weak effect on heavy chain retrotranslocation (Fig. 3 A, top, lane 8 vs. lane 4 and lane 7 vs. lane 3; see also Fig. 4)Figure 4.

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