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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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p97 binds to heavy chains that were not ubiquitinated. (A) US11-expressing cells were labeled and permeabilized. The cytosol was removed by high speed centrifugation and replaced with cow liver cytosol that was ubiquitin depleted (−Ub). Where indicated, purified ubiquitin was added back to the depleted cytosol (+Ub). After chase incubation, the samples were either analyzed directly by immunoprecipitation with heavy chain (HC) antibodies (T), or first fractionated into membrane (P) and cytosol (S) fractions before immunoprecipitation with HC antibodies. (B) Purified His-p97 was added during the permeabilization of labeled cells. A portion of the membrane and cytosol fractions was subjected to immunoprecipitation with HC antibodies (top), and the rest was analyzed by sequential immunoprecipitation with His and HC antibodies (bottom). (C) Astrocytoma cells stably expressing hemagglutinin (HA)-tagged MHC class I heavy chains were permeabilized and incubated with ubiquitin-depleted cytosol in the presence of purified wild-type His-p97 or the indicated His-tagged mutant proteins. The membrane fraction was subjected to immunoprecipitation with HA antibodies (top) or His and HA antibodies (bottom). Note that the increase in the amount of p97-associated, HA-tagged heavy chains is less apparent than that in B, which may result from heavy chain overexpression.
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fig5: p97 binds to heavy chains that were not ubiquitinated. (A) US11-expressing cells were labeled and permeabilized. The cytosol was removed by high speed centrifugation and replaced with cow liver cytosol that was ubiquitin depleted (−Ub). Where indicated, purified ubiquitin was added back to the depleted cytosol (+Ub). After chase incubation, the samples were either analyzed directly by immunoprecipitation with heavy chain (HC) antibodies (T), or first fractionated into membrane (P) and cytosol (S) fractions before immunoprecipitation with HC antibodies. (B) Purified His-p97 was added during the permeabilization of labeled cells. A portion of the membrane and cytosol fractions was subjected to immunoprecipitation with HC antibodies (top), and the rest was analyzed by sequential immunoprecipitation with His and HC antibodies (bottom). (C) Astrocytoma cells stably expressing hemagglutinin (HA)-tagged MHC class I heavy chains were permeabilized and incubated with ubiquitin-depleted cytosol in the presence of purified wild-type His-p97 or the indicated His-tagged mutant proteins. The membrane fraction was subjected to immunoprecipitation with HA antibodies (top) or His and HA antibodies (bottom). Note that the increase in the amount of p97-associated, HA-tagged heavy chains is less apparent than that in B, which may result from heavy chain overexpression.

Mentions: Although the majority of the heavy chains bound to p97 were unmodified (Fig. 3, bottom), the results did not exclude that they were originally polyubiquitinated when they bound to p97 and were subsequently de-ubiquitinated by an isopeptidase. De-ubiquitination must, in fact, be a very efficient process because the heavy chains accumulating in the cytosol in the presence of proteasome inhibitor are mostly unmodified (Fig. 3 A, top, lane 4), despite the fact that they must have been polyubiquitinated on the membrane (Shamu et al., 2001). To test whether p97 can interact with heavy chains that were never modified, the cytosol in permeabilized US11 cells was replaced with ubiquitin-depleted cow liver cytosol (Shamu et al., 2001). Pulse-chase experiments showed that in the absence of ubiquitin, the heavy chains were stable and remained in the ER membrane (Fig. 5Figure 5.


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 binds to heavy chains that were not ubiquitinated. (A) US11-expressing cells were labeled and permeabilized. The cytosol was removed by high speed centrifugation and replaced with cow liver cytosol that was ubiquitin depleted (−Ub). Where indicated, purified ubiquitin was added back to the depleted cytosol (+Ub). After chase incubation, the samples were either analyzed directly by immunoprecipitation with heavy chain (HC) antibodies (T), or first fractionated into membrane (P) and cytosol (S) fractions before immunoprecipitation with HC antibodies. (B) Purified His-p97 was added during the permeabilization of labeled cells. A portion of the membrane and cytosol fractions was subjected to immunoprecipitation with HC antibodies (top), and the rest was analyzed by sequential immunoprecipitation with His and HC antibodies (bottom). (C) Astrocytoma cells stably expressing hemagglutinin (HA)-tagged MHC class I heavy chains were permeabilized and incubated with ubiquitin-depleted cytosol in the presence of purified wild-type His-p97 or the indicated His-tagged mutant proteins. The membrane fraction was subjected to immunoprecipitation with HA antibodies (top) or His and HA antibodies (bottom). Note that the increase in the amount of p97-associated, HA-tagged heavy chains is less apparent than that in B, which may result from heavy chain overexpression.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: p97 binds to heavy chains that were not ubiquitinated. (A) US11-expressing cells were labeled and permeabilized. The cytosol was removed by high speed centrifugation and replaced with cow liver cytosol that was ubiquitin depleted (−Ub). Where indicated, purified ubiquitin was added back to the depleted cytosol (+Ub). After chase incubation, the samples were either analyzed directly by immunoprecipitation with heavy chain (HC) antibodies (T), or first fractionated into membrane (P) and cytosol (S) fractions before immunoprecipitation with HC antibodies. (B) Purified His-p97 was added during the permeabilization of labeled cells. A portion of the membrane and cytosol fractions was subjected to immunoprecipitation with HC antibodies (top), and the rest was analyzed by sequential immunoprecipitation with His and HC antibodies (bottom). (C) Astrocytoma cells stably expressing hemagglutinin (HA)-tagged MHC class I heavy chains were permeabilized and incubated with ubiquitin-depleted cytosol in the presence of purified wild-type His-p97 or the indicated His-tagged mutant proteins. The membrane fraction was subjected to immunoprecipitation with HA antibodies (top) or His and HA antibodies (bottom). Note that the increase in the amount of p97-associated, HA-tagged heavy chains is less apparent than that in B, which may result from heavy chain overexpression.
Mentions: Although the majority of the heavy chains bound to p97 were unmodified (Fig. 3, bottom), the results did not exclude that they were originally polyubiquitinated when they bound to p97 and were subsequently de-ubiquitinated by an isopeptidase. De-ubiquitination must, in fact, be a very efficient process because the heavy chains accumulating in the cytosol in the presence of proteasome inhibitor are mostly unmodified (Fig. 3 A, top, lane 4), despite the fact that they must have been polyubiquitinated on the membrane (Shamu et al., 2001). To test whether p97 can interact with heavy chains that were never modified, the cytosol in permeabilized US11 cells was replaced with ubiquitin-depleted cow liver cytosol (Shamu et al., 2001). Pulse-chase experiments showed that in the absence of ubiquitin, the heavy chains were stable and remained in the ER membrane (Fig. 5Figure 5.

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