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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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Polyubiquitin binding to p97 and Ufd1. (A) Scheme of Ufd1 and mutant constructs used in the binding assays. The black box indicates the double Ψ barrel domain. The shaded box shows the p97- and Npl4-binding domain UT6. (B) Polyubiquitin chains synthesized with Ubc7/GST-gp78c were added to glutathione beads containing bound GST fusions to full-length Ufd1 or fragments of it. The bound fraction was analyzed by immunoblotting with ubiquitin and GST antibodies (top and bottom panels, respectively). The stars in the bottom panel indicate degradation products of GST-UT6. (C) A GST fusion to the UT6 domain was immobilized on glutathione beads in the absence (no p97) or presence of His-tagged wild-type p97 or AA mutant. Binding of polyubiquitin chains with lysine 48 linkages was tested as in B. Bound proteins were also analyzed by immunoblotting with p97 (middle) or GST (bottom) antibodies. As a control, binding of polyubiquitin chains with lysine 63 linkages was tested (lanes 5–8). (D) GST fusions to the UT6 domain or Ufd1 were immobilized on glutathione beads in the absence (no p97) or presence of excess untagged or His-tagged p97. Polyubiquitin binding was analyzed as in C. (E) GST fusions to the UT6 fragment and Ufd1 were immobilized in the presence of p97 or p97 plus Npl4 lacking the NZF domain (Npl4ΔZF). Polyubiquitin binding was tested as in D. Lanes 8–14 show controls with lysine 63–linked polyubiquitin chains.
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fig7: Polyubiquitin binding to p97 and Ufd1. (A) Scheme of Ufd1 and mutant constructs used in the binding assays. The black box indicates the double Ψ barrel domain. The shaded box shows the p97- and Npl4-binding domain UT6. (B) Polyubiquitin chains synthesized with Ubc7/GST-gp78c were added to glutathione beads containing bound GST fusions to full-length Ufd1 or fragments of it. The bound fraction was analyzed by immunoblotting with ubiquitin and GST antibodies (top and bottom panels, respectively). The stars in the bottom panel indicate degradation products of GST-UT6. (C) A GST fusion to the UT6 domain was immobilized on glutathione beads in the absence (no p97) or presence of His-tagged wild-type p97 or AA mutant. Binding of polyubiquitin chains with lysine 48 linkages was tested as in B. Bound proteins were also analyzed by immunoblotting with p97 (middle) or GST (bottom) antibodies. As a control, binding of polyubiquitin chains with lysine 63 linkages was tested (lanes 5–8). (D) GST fusions to the UT6 domain or Ufd1 were immobilized on glutathione beads in the absence (no p97) or presence of excess untagged or His-tagged p97. Polyubiquitin binding was analyzed as in C. (E) GST fusions to the UT6 fragment and Ufd1 were immobilized in the presence of p97 or p97 plus Npl4 lacking the NZF domain (Npl4ΔZF). Polyubiquitin binding was tested as in D. Lanes 8–14 show controls with lysine 63–linked polyubiquitin chains.

Mentions: Next, we determined that the novel binding site is contained in Ufd1. A purified mammalian Npl4 protein lacking the NZF motif (Npl4ΔZF) did not bind ubiquitin chains in the absence of Ufd1 (unpublished data), whereas a GST fusion to full-length Ufd1 interacted with polyubiquitin chains containing lysine 48 linkages (Fig. 7Figure 7.


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)

Polyubiquitin binding to p97 and Ufd1. (A) Scheme of Ufd1 and mutant constructs used in the binding assays. The black box indicates the double Ψ barrel domain. The shaded box shows the p97- and Npl4-binding domain UT6. (B) Polyubiquitin chains synthesized with Ubc7/GST-gp78c were added to glutathione beads containing bound GST fusions to full-length Ufd1 or fragments of it. The bound fraction was analyzed by immunoblotting with ubiquitin and GST antibodies (top and bottom panels, respectively). The stars in the bottom panel indicate degradation products of GST-UT6. (C) A GST fusion to the UT6 domain was immobilized on glutathione beads in the absence (no p97) or presence of His-tagged wild-type p97 or AA mutant. Binding of polyubiquitin chains with lysine 48 linkages was tested as in B. Bound proteins were also analyzed by immunoblotting with p97 (middle) or GST (bottom) antibodies. As a control, binding of polyubiquitin chains with lysine 63 linkages was tested (lanes 5–8). (D) GST fusions to the UT6 domain or Ufd1 were immobilized on glutathione beads in the absence (no p97) or presence of excess untagged or His-tagged p97. Polyubiquitin binding was analyzed as in C. (E) GST fusions to the UT6 fragment and Ufd1 were immobilized in the presence of p97 or p97 plus Npl4 lacking the NZF domain (Npl4ΔZF). Polyubiquitin binding was tested as in D. Lanes 8–14 show controls with lysine 63–linked polyubiquitin chains.
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fig7: Polyubiquitin binding to p97 and Ufd1. (A) Scheme of Ufd1 and mutant constructs used in the binding assays. The black box indicates the double Ψ barrel domain. The shaded box shows the p97- and Npl4-binding domain UT6. (B) Polyubiquitin chains synthesized with Ubc7/GST-gp78c were added to glutathione beads containing bound GST fusions to full-length Ufd1 or fragments of it. The bound fraction was analyzed by immunoblotting with ubiquitin and GST antibodies (top and bottom panels, respectively). The stars in the bottom panel indicate degradation products of GST-UT6. (C) A GST fusion to the UT6 domain was immobilized on glutathione beads in the absence (no p97) or presence of His-tagged wild-type p97 or AA mutant. Binding of polyubiquitin chains with lysine 48 linkages was tested as in B. Bound proteins were also analyzed by immunoblotting with p97 (middle) or GST (bottom) antibodies. As a control, binding of polyubiquitin chains with lysine 63 linkages was tested (lanes 5–8). (D) GST fusions to the UT6 domain or Ufd1 were immobilized on glutathione beads in the absence (no p97) or presence of excess untagged or His-tagged p97. Polyubiquitin binding was analyzed as in C. (E) GST fusions to the UT6 fragment and Ufd1 were immobilized in the presence of p97 or p97 plus Npl4 lacking the NZF domain (Npl4ΔZF). Polyubiquitin binding was tested as in D. Lanes 8–14 show controls with lysine 63–linked polyubiquitin chains.
Mentions: Next, we determined that the novel binding site is contained in Ufd1. A purified mammalian Npl4 protein lacking the NZF motif (Npl4ΔZF) did not bind ubiquitin chains in the absence of Ufd1 (unpublished data), whereas a GST fusion to full-length Ufd1 interacted with polyubiquitin chains containing lysine 48 linkages (Fig. 7Figure 7.

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