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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 cycle and substrate recognition of p97. (A) Proposed communication between the two ATPase rings (D1 and D2) of p97. The D2 domain hydrolyzes ATP (T) and releases an orthophosphate (Pi), first converting the ring into its ADP-bound state (D). ATP hydrolysis in D1 follows. (B) A dual recognition model for p97–Ufd1–Npl4 function in retrotranslocation. Protein retrotranslocation is proposed to proceed in four steps. (I) A polypeptide chain emerging from the ER may be able to slide back and forth through the membrane (arrows), probably through a protein-conducting channel. The p97–Ufd1–Npl4 complex is recruited to the membrane by an unknown receptor (green). (II) A nonubiquitinated substrate is initially recognized by p97 (the ATPase domains D1 and D2 are shown in yellow, the N domain in blue). This interaction may prevent the substrate from backsliding into the ER lumen. The substrate also undergoes ubiquitination (red) at the membrane. (III) Once the ubiquitin chain (red) reaches a certain length, it interacts with p97 and the cofactor complex Ufd1–Npl4 (black). (IV) p97 uses ATP hydrolysis to move the polypeptide into the cytosol.
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fig9: ATPase cycle and substrate recognition of p97. (A) Proposed communication between the two ATPase rings (D1 and D2) of p97. The D2 domain hydrolyzes ATP (T) and releases an orthophosphate (Pi), first converting the ring into its ADP-bound state (D). ATP hydrolysis in D1 follows. (B) A dual recognition model for p97–Ufd1–Npl4 function in retrotranslocation. Protein retrotranslocation is proposed to proceed in four steps. (I) A polypeptide chain emerging from the ER may be able to slide back and forth through the membrane (arrows), probably through a protein-conducting channel. The p97–Ufd1–Npl4 complex is recruited to the membrane by an unknown receptor (green). (II) A nonubiquitinated substrate is initially recognized by p97 (the ATPase domains D1 and D2 are shown in yellow, the N domain in blue). This interaction may prevent the substrate from backsliding into the ER lumen. The substrate also undergoes ubiquitination (red) at the membrane. (III) Once the ubiquitin chain (red) reaches a certain length, it interacts with p97 and the cofactor complex Ufd1–Npl4 (black). (IV) p97 uses ATP hydrolysis to move the polypeptide into the cytosol.

Mentions: Our results shed light on how the AAA ATPase p97 functions together with its cofactor Ufd1–Npl4 in protein retrotranslocation. One conclusion from the study of different mutants is that the two ATPase domains of p97 are functionally coupled. The activity of the second ATPase domain (D2) requires nucleotide binding, but not hydrolysis by the first ATPase domain (D1), indicating that the D2 domain may hydrolyze nucleotides when D1 is in its ATP-bound state. It appears that D2 can only bind ATP when D1 is in its nucleotide-bound state. In contrast, the ATPase activity of D1 depends on both ATP binding and hydrolysis by the D2 domain, suggesting that the D1 domain likely hydrolyzes ATP when D2 is in its ADP-bound form. Therefore, we propose an ATPase cycle in which there is alternate ATP hydrolysis in the two ATPase rings (Fig. 9Figure 9.


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 cycle and substrate recognition of p97. (A) Proposed communication between the two ATPase rings (D1 and D2) of p97. The D2 domain hydrolyzes ATP (T) and releases an orthophosphate (Pi), first converting the ring into its ADP-bound state (D). ATP hydrolysis in D1 follows. (B) A dual recognition model for p97–Ufd1–Npl4 function in retrotranslocation. Protein retrotranslocation is proposed to proceed in four steps. (I) A polypeptide chain emerging from the ER may be able to slide back and forth through the membrane (arrows), probably through a protein-conducting channel. The p97–Ufd1–Npl4 complex is recruited to the membrane by an unknown receptor (green). (II) A nonubiquitinated substrate is initially recognized by p97 (the ATPase domains D1 and D2 are shown in yellow, the N domain in blue). This interaction may prevent the substrate from backsliding into the ER lumen. The substrate also undergoes ubiquitination (red) at the membrane. (III) Once the ubiquitin chain (red) reaches a certain length, it interacts with p97 and the cofactor complex Ufd1–Npl4 (black). (IV) p97 uses ATP hydrolysis to move the polypeptide into the cytosol.
© Copyright Policy
Related In: Results  -  Collection

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fig9: ATPase cycle and substrate recognition of p97. (A) Proposed communication between the two ATPase rings (D1 and D2) of p97. The D2 domain hydrolyzes ATP (T) and releases an orthophosphate (Pi), first converting the ring into its ADP-bound state (D). ATP hydrolysis in D1 follows. (B) A dual recognition model for p97–Ufd1–Npl4 function in retrotranslocation. Protein retrotranslocation is proposed to proceed in four steps. (I) A polypeptide chain emerging from the ER may be able to slide back and forth through the membrane (arrows), probably through a protein-conducting channel. The p97–Ufd1–Npl4 complex is recruited to the membrane by an unknown receptor (green). (II) A nonubiquitinated substrate is initially recognized by p97 (the ATPase domains D1 and D2 are shown in yellow, the N domain in blue). This interaction may prevent the substrate from backsliding into the ER lumen. The substrate also undergoes ubiquitination (red) at the membrane. (III) Once the ubiquitin chain (red) reaches a certain length, it interacts with p97 and the cofactor complex Ufd1–Npl4 (black). (IV) p97 uses ATP hydrolysis to move the polypeptide into the cytosol.
Mentions: Our results shed light on how the AAA ATPase p97 functions together with its cofactor Ufd1–Npl4 in protein retrotranslocation. One conclusion from the study of different mutants is that the two ATPase domains of p97 are functionally coupled. The activity of the second ATPase domain (D2) requires nucleotide binding, but not hydrolysis by the first ATPase domain (D1), indicating that the D2 domain may hydrolyze nucleotides when D1 is in its ATP-bound state. It appears that D2 can only bind ATP when D1 is in its nucleotide-bound state. In contrast, the ATPase activity of D1 depends on both ATP binding and hydrolysis by the D2 domain, suggesting that the D1 domain likely hydrolyzes ATP when D2 is in its ADP-bound form. Therefore, we propose an ATPase cycle in which there is alternate ATP hydrolysis in the two ATPase rings (Fig. 9Figure 9.

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