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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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ATPase activity of p97 mutants. (A) Scheme of p97 constructs used in this work (N, NH2-terminal domain; D1, D2, first and second ATPase domain, respectively). The name of each construct is shown on the left. For the wild-type protein (WT), the conserved lysine (K) in the Walker A motif and glutamate (E) in the Walker B motif are indicated. For the mutants, the alanine (A) or glutamine (Q) altered residue is shown. (B) The p97 proteins were expressed in E. coli, purified, and subjected to SDS-PAGE. Shown is the gel after staining with Coomassie blue. (C) The ATPase activity of the different p97 proteins was measured. Shown is the mean of three experiments. (D) Wild-type Cdc48, the yeast homologue of p97, or ATP hydrolysis mutants (QE or EQ) were expressed under the gal promoter in the presence of galactose in the temperature-sensitive cdc48–3 yeast strain at the permissive (30°C) or nonpermissive (37°C) temperature (top). As control, the cells were grown on glucose to repress the genes (bottom).
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fig1: ATPase activity of p97 mutants. (A) Scheme of p97 constructs used in this work (N, NH2-terminal domain; D1, D2, first and second ATPase domain, respectively). The name of each construct is shown on the left. For the wild-type protein (WT), the conserved lysine (K) in the Walker A motif and glutamate (E) in the Walker B motif are indicated. For the mutants, the alanine (A) or glutamine (Q) altered residue is shown. (B) The p97 proteins were expressed in E. coli, purified, and subjected to SDS-PAGE. Shown is the gel after staining with Coomassie blue. (C) The ATPase activity of the different p97 proteins was measured. Shown is the mean of three experiments. (D) Wild-type Cdc48, the yeast homologue of p97, or ATP hydrolysis mutants (QE or EQ) were expressed under the gal promoter in the presence of galactose in the temperature-sensitive cdc48–3 yeast strain at the permissive (30°C) or nonpermissive (37°C) temperature (top). As control, the cells were grown on glucose to repress the genes (bottom).

Mentions: p97 contains an NH2-terminal domain (N) and two AAA cassettes (D1 and D2; Fig. 1Figure 1.


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)

ATPase activity of p97 mutants. (A) Scheme of p97 constructs used in this work (N, NH2-terminal domain; D1, D2, first and second ATPase domain, respectively). The name of each construct is shown on the left. For the wild-type protein (WT), the conserved lysine (K) in the Walker A motif and glutamate (E) in the Walker B motif are indicated. For the mutants, the alanine (A) or glutamine (Q) altered residue is shown. (B) The p97 proteins were expressed in E. coli, purified, and subjected to SDS-PAGE. Shown is the gel after staining with Coomassie blue. (C) The ATPase activity of the different p97 proteins was measured. Shown is the mean of three experiments. (D) Wild-type Cdc48, the yeast homologue of p97, or ATP hydrolysis mutants (QE or EQ) were expressed under the gal promoter in the presence of galactose in the temperature-sensitive cdc48–3 yeast strain at the permissive (30°C) or nonpermissive (37°C) temperature (top). As control, the cells were grown on glucose to repress the genes (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172719&req=5

fig1: ATPase activity of p97 mutants. (A) Scheme of p97 constructs used in this work (N, NH2-terminal domain; D1, D2, first and second ATPase domain, respectively). The name of each construct is shown on the left. For the wild-type protein (WT), the conserved lysine (K) in the Walker A motif and glutamate (E) in the Walker B motif are indicated. For the mutants, the alanine (A) or glutamine (Q) altered residue is shown. (B) The p97 proteins were expressed in E. coli, purified, and subjected to SDS-PAGE. Shown is the gel after staining with Coomassie blue. (C) The ATPase activity of the different p97 proteins was measured. Shown is the mean of three experiments. (D) Wild-type Cdc48, the yeast homologue of p97, or ATP hydrolysis mutants (QE or EQ) were expressed under the gal promoter in the presence of galactose in the temperature-sensitive cdc48–3 yeast strain at the permissive (30°C) or nonpermissive (37°C) temperature (top). As control, the cells were grown on glucose to repress the genes (bottom).
Mentions: p97 contains an NH2-terminal domain (N) and two AAA cassettes (D1 and D2; Fig. 1Figure 1.

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
Related in: MedlinePlus