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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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Ubiquitin recognition by the p97–Ufd1–Npl4 complex. (A) A GST fusion to the cytosolic fragment of the ubiquitin ligase gp78, the ubiquitin-conjugating enzyme Ubc7, and the ubiquitin-conjugating complex consisting of the Ubc13 and Mms2 were all expressed in E. coli and purified. Shown is a Coomassie-stained gel after SDS-PAGE with mol wt markers on the right side. (B) In vitro ubiquitination reactions were performed with the indicated components in the presence of ubiquitin activation enzyme (E1) and ATP. The samples were analyzed by immunoblotting with ubiquitin antibodies. Ub, Ub2, and Ub(n) indicate the positions of ubiquitin, di-ubiquitin, and polyubiquitin chains, respectively. (C) Shown is a comparison of wild-type ubiquitin (Ub) with methylated ubiquitin (Ub-m) and a mutant ubiquitin carrying a substitution of lysine 48 by arginine (UbK48R). The reactions were performed as in B. (D) Ubiquitin mutants with single lysines at the indicated position were tested in reactions similar to those in B. (E) Polyubiquitin chains were synthesized with either Ubc7/GST-gp78c (lanes 1–5) or Ubc13/Mms2 (lanes 6–10), generating chains with lysine 48 and lysine 63 linkages, respectively. These chains were incubated with the indicated proteins, and the samples were subjected to immunoprecipitation with Ufd1 antibodies (lanes 2–5 and lanes 7–10). Bound ubiquitin chains were detected by immunoblotting with ubiquitin antibodies (Ub; top). A portion of the immunoprecipitates was analyzed by immunoblotting with His (middle) or Ufd1 and Npl4 antibodies (bottom). UbK48 and UbK63 indicate chains with lysine 48 and lysine 63 linkages, respectively. (F) Yeast cytosol from a control strain (UFD1) or a strain expressing protein A–tagged UFD1 (UFD1PrA) was incubated with IgG beads. The beads were incubated with in vitro–synthesized polyubiquitin chains, and the bound material was analyzed by immunoblotting with ubiquitin antibodies (top) and Cdc48 antibodies (bottom).
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fig6: Ubiquitin recognition by the p97–Ufd1–Npl4 complex. (A) A GST fusion to the cytosolic fragment of the ubiquitin ligase gp78, the ubiquitin-conjugating enzyme Ubc7, and the ubiquitin-conjugating complex consisting of the Ubc13 and Mms2 were all expressed in E. coli and purified. Shown is a Coomassie-stained gel after SDS-PAGE with mol wt markers on the right side. (B) In vitro ubiquitination reactions were performed with the indicated components in the presence of ubiquitin activation enzyme (E1) and ATP. The samples were analyzed by immunoblotting with ubiquitin antibodies. Ub, Ub2, and Ub(n) indicate the positions of ubiquitin, di-ubiquitin, and polyubiquitin chains, respectively. (C) Shown is a comparison of wild-type ubiquitin (Ub) with methylated ubiquitin (Ub-m) and a mutant ubiquitin carrying a substitution of lysine 48 by arginine (UbK48R). The reactions were performed as in B. (D) Ubiquitin mutants with single lysines at the indicated position were tested in reactions similar to those in B. (E) Polyubiquitin chains were synthesized with either Ubc7/GST-gp78c (lanes 1–5) or Ubc13/Mms2 (lanes 6–10), generating chains with lysine 48 and lysine 63 linkages, respectively. These chains were incubated with the indicated proteins, and the samples were subjected to immunoprecipitation with Ufd1 antibodies (lanes 2–5 and lanes 7–10). Bound ubiquitin chains were detected by immunoblotting with ubiquitin antibodies (Ub; top). A portion of the immunoprecipitates was analyzed by immunoblotting with His (middle) or Ufd1 and Npl4 antibodies (bottom). UbK48 and UbK63 indicate chains with lysine 48 and lysine 63 linkages, respectively. (F) Yeast cytosol from a control strain (UFD1) or a strain expressing protein A–tagged UFD1 (UFD1PrA) was incubated with IgG beads. The beads were incubated with in vitro–synthesized polyubiquitin chains, and the bound material was analyzed by immunoblotting with ubiquitin antibodies (top) and Cdc48 antibodies (bottom).

Mentions: Because the p97-dependent release of substrate from the membrane requires polyubiquitination, we tested how the polyubiquitin chain is recognized by p97 and its cofactor Ufd1–Npl4. To address interactions between polyubiquitin and the ATPase complex, we used polyubiquitin chains synthesized in vitro using purified recombinant proteins. The polyubiquitination reaction contained ubiquitin, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme Ubc7 (E2), and a GST fusion to a cytosolic fragment of the ubiquitin ligase gp78 (E3; the purity of the recombinant E2 and E3 proteins is shown in Fig. 6Figure 6.


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)

Ubiquitin recognition by the p97–Ufd1–Npl4 complex. (A) A GST fusion to the cytosolic fragment of the ubiquitin ligase gp78, the ubiquitin-conjugating enzyme Ubc7, and the ubiquitin-conjugating complex consisting of the Ubc13 and Mms2 were all expressed in E. coli and purified. Shown is a Coomassie-stained gel after SDS-PAGE with mol wt markers on the right side. (B) In vitro ubiquitination reactions were performed with the indicated components in the presence of ubiquitin activation enzyme (E1) and ATP. The samples were analyzed by immunoblotting with ubiquitin antibodies. Ub, Ub2, and Ub(n) indicate the positions of ubiquitin, di-ubiquitin, and polyubiquitin chains, respectively. (C) Shown is a comparison of wild-type ubiquitin (Ub) with methylated ubiquitin (Ub-m) and a mutant ubiquitin carrying a substitution of lysine 48 by arginine (UbK48R). The reactions were performed as in B. (D) Ubiquitin mutants with single lysines at the indicated position were tested in reactions similar to those in B. (E) Polyubiquitin chains were synthesized with either Ubc7/GST-gp78c (lanes 1–5) or Ubc13/Mms2 (lanes 6–10), generating chains with lysine 48 and lysine 63 linkages, respectively. These chains were incubated with the indicated proteins, and the samples were subjected to immunoprecipitation with Ufd1 antibodies (lanes 2–5 and lanes 7–10). Bound ubiquitin chains were detected by immunoblotting with ubiquitin antibodies (Ub; top). A portion of the immunoprecipitates was analyzed by immunoblotting with His (middle) or Ufd1 and Npl4 antibodies (bottom). UbK48 and UbK63 indicate chains with lysine 48 and lysine 63 linkages, respectively. (F) Yeast cytosol from a control strain (UFD1) or a strain expressing protein A–tagged UFD1 (UFD1PrA) was incubated with IgG beads. The beads were incubated with in vitro–synthesized polyubiquitin chains, and the bound material was analyzed by immunoblotting with ubiquitin antibodies (top) and Cdc48 antibodies (bottom).
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Related In: Results  -  Collection

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fig6: Ubiquitin recognition by the p97–Ufd1–Npl4 complex. (A) A GST fusion to the cytosolic fragment of the ubiquitin ligase gp78, the ubiquitin-conjugating enzyme Ubc7, and the ubiquitin-conjugating complex consisting of the Ubc13 and Mms2 were all expressed in E. coli and purified. Shown is a Coomassie-stained gel after SDS-PAGE with mol wt markers on the right side. (B) In vitro ubiquitination reactions were performed with the indicated components in the presence of ubiquitin activation enzyme (E1) and ATP. The samples were analyzed by immunoblotting with ubiquitin antibodies. Ub, Ub2, and Ub(n) indicate the positions of ubiquitin, di-ubiquitin, and polyubiquitin chains, respectively. (C) Shown is a comparison of wild-type ubiquitin (Ub) with methylated ubiquitin (Ub-m) and a mutant ubiquitin carrying a substitution of lysine 48 by arginine (UbK48R). The reactions were performed as in B. (D) Ubiquitin mutants with single lysines at the indicated position were tested in reactions similar to those in B. (E) Polyubiquitin chains were synthesized with either Ubc7/GST-gp78c (lanes 1–5) or Ubc13/Mms2 (lanes 6–10), generating chains with lysine 48 and lysine 63 linkages, respectively. These chains were incubated with the indicated proteins, and the samples were subjected to immunoprecipitation with Ufd1 antibodies (lanes 2–5 and lanes 7–10). Bound ubiquitin chains were detected by immunoblotting with ubiquitin antibodies (Ub; top). A portion of the immunoprecipitates was analyzed by immunoblotting with His (middle) or Ufd1 and Npl4 antibodies (bottom). UbK48 and UbK63 indicate chains with lysine 48 and lysine 63 linkages, respectively. (F) Yeast cytosol from a control strain (UFD1) or a strain expressing protein A–tagged UFD1 (UFD1PrA) was incubated with IgG beads. The beads were incubated with in vitro–synthesized polyubiquitin chains, and the bound material was analyzed by immunoblotting with ubiquitin antibodies (top) and Cdc48 antibodies (bottom).
Mentions: Because the p97-dependent release of substrate from the membrane requires polyubiquitination, we tested how the polyubiquitin chain is recognized by p97 and its cofactor Ufd1–Npl4. To address interactions between polyubiquitin and the ATPase complex, we used polyubiquitin chains synthesized in vitro using purified recombinant proteins. The polyubiquitination reaction contained ubiquitin, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme Ubc7 (E2), and a GST fusion to a cytosolic fragment of the ubiquitin ligase gp78 (E3; the purity of the recombinant E2 and E3 proteins is shown in Fig. 6Figure 6.

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