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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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Related in: MedlinePlus

p97 binding to cofactors and membrane requires its N domain. (A) p97 binding to cofactors. Purified His-tagged p97 proteins (His-p97) were added to rat liver cytosol in the presence of 1 M NaCl. After dilution of the salt to 150 mM, p97 was immunoprecipitated with His antibodies. Bound proteins were detected by immunoblotting with antibodies to the indicated proteins. (B) Association of p97 with the membrane. Purified His-p97 proteins were added to permeabilized astrocytoma cells. After incubation, the cells were fractionated into membrane (P) and cytosol (S) fractions. The amount of His-p97 in each fraction was determined by immunoblotting with His antibodies (top). Immunoblotting for calnexin served as a loading control for the membrane fraction (bottom).
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fig2: p97 binding to cofactors and membrane requires its N domain. (A) p97 binding to cofactors. Purified His-tagged p97 proteins (His-p97) were added to rat liver cytosol in the presence of 1 M NaCl. After dilution of the salt to 150 mM, p97 was immunoprecipitated with His antibodies. Bound proteins were detected by immunoblotting with antibodies to the indicated proteins. (B) Association of p97 with the membrane. Purified His-p97 proteins were added to permeabilized astrocytoma cells. After incubation, the cells were fractionated into membrane (P) and cytosol (S) fractions. The amount of His-p97 in each fraction was determined by immunoblotting with His antibodies (top). Immunoblotting for calnexin served as a loading control for the membrane fraction (bottom).

Mentions: To test the interaction of p97 with its cofactors, purified p97 variants were incubated with rat liver cytosol at high salt to break endogenous complexes. After lowering the salt concentration, complexes were immunoprecipitated with antibodies directed against the NH2-terminal His tag of p97. The precipitates were analyzed by immunoblotting with antibodies to Ufd1 and p47. With the exception of the ΔN protein, all p97 variants were able to interact with both Ufd1–Npl4 and p47 (Fig. 2Figure 2.


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)

p97 binding to cofactors and membrane requires its N domain. (A) p97 binding to cofactors. Purified His-tagged p97 proteins (His-p97) were added to rat liver cytosol in the presence of 1 M NaCl. After dilution of the salt to 150 mM, p97 was immunoprecipitated with His antibodies. Bound proteins were detected by immunoblotting with antibodies to the indicated proteins. (B) Association of p97 with the membrane. Purified His-p97 proteins were added to permeabilized astrocytoma cells. After incubation, the cells were fractionated into membrane (P) and cytosol (S) fractions. The amount of His-p97 in each fraction was determined by immunoblotting with His antibodies (top). Immunoblotting for calnexin served as a loading control for the membrane fraction (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: p97 binding to cofactors and membrane requires its N domain. (A) p97 binding to cofactors. Purified His-tagged p97 proteins (His-p97) were added to rat liver cytosol in the presence of 1 M NaCl. After dilution of the salt to 150 mM, p97 was immunoprecipitated with His antibodies. Bound proteins were detected by immunoblotting with antibodies to the indicated proteins. (B) Association of p97 with the membrane. Purified His-p97 proteins were added to permeabilized astrocytoma cells. After incubation, the cells were fractionated into membrane (P) and cytosol (S) fractions. The amount of His-p97 in each fraction was determined by immunoblotting with His antibodies (top). Immunoblotting for calnexin served as a loading control for the membrane fraction (bottom).
Mentions: To test the interaction of p97 with its cofactors, purified p97 variants were incubated with rat liver cytosol at high salt to break endogenous complexes. After lowering the salt concentration, complexes were immunoprecipitated with antibodies directed against the NH2-terminal His tag of p97. The precipitates were analyzed by immunoblotting with antibodies to Ufd1 and p47. With the exception of the ΔN protein, all p97 variants were able to interact with both Ufd1–Npl4 and p47 (Fig. 2Figure 2.

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