Article |
2 Department of Cell Biology, Yale Medical School, New Haven, CT, 06520
Address correspondence to Tom A. Rapoport, Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-0637. Fax: (617) 432-1190. E-mail: tom_rapoport{at}hms.harvard.edu
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Abstract |
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Key Words: p97/Cdc48; ubiquitin; AAA ATPase; protein degradation; ER quality control
* Abbreviations used in this paper: AAA, ATPase associated with diverse cellular activities; NZF, Npl4 zinc finger.
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Introduction |
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p97/Cdc48 belongs to a family of ATPases associated with diverse cellular activities (AAA;* for review see Ogura and Wilkinson, 2001). The characteristic feature of this family is the presence of one or two conserved ATPase domains, referred to as AAA cassettes, which contain Walker A and Walker B motifs that mediate ATP binding and hydrolysis, respectively. p97/Cdc48 is similar to the NSF protein involved in vesicle fusion in the secretory pathway. Like NSF, p97/Cdc48 consists of two ATPase domains (D1 and D2) and an additional NH2-terminal domain (N). Structural studies reveal that the ATPase domains form two hexameric rings stacked on top of each other with a central pore of 15 Å (Rouiller et al., 2000; Zhang et al., 2000). The N domains are located at the side of the D1 ring. Although both p97 and Cdc48 are abundant cytosolic proteins, a significant fraction of them is tightly associated with the ER membrane (Rabouille et al., 1998; Hitchcock et al., 2001).
The ATPase p97/Cdc48 appears to perform different cellular functions depending on its association with cofactors (Meyer et al., 2000). Mammalian p97 can interact in a mutually exclusive manner with either p47 or a dimer consisting of Ufd1 and Npl4. In mammals, the complex of p97p47 appears to play a role in the homotypic fusion of ER and Golgi membranes (Kondo et al., 1997; Rabouille et al., 1998; Uchiyama et al., 2002). The p97p47 complex binds ubiquitinated proteins via a UBA domain in p47, and this domain is essential for its activity in Golgi membrane fusion (Meyer et al., 2002). In Saccharomyces cerevisiae, the complex of Cdc48Ufd1Npl4 has been implicated in the release of polypeptides from the ER membrane (for review see Tsai et al., 2002). This release step is essential for both retrotranslocation of misfolded proteins from the ER into the cytosol, and for the activation of the transcription factor Spt23 from its ER-anchored precursor (Hitchcock et al., 2001; Rape et al., 2001). Mammalian p97 has also been shown to function in retrotranslocation (Ye et al., 2001), but a requirement for the cofactor Ufd1Npl4 has not yet been demonstrated.
Exactly how p97/Cdc48 and its cofactor Ufd1Npl4 extract polypeptides from the ER membrane during retrotranslocation is unclear. It is not known how the ATPase binds to the ER membrane, nor how the two ATPase domains collaborate to "pull" polypeptides out of the membrane. The mechanism of substrate recognition has been particularly difficult to address because its elucidation requires complex systems in which p97/Cdc48 acts on physiological substrates emerging from the ER membrane. Two possibilities of substrate recognition have been proposed. In one model, p97/Cdc48 would interact directly with an unfolded polypeptide segment as it emerges from the ER membrane. Alternatively, the recognition signal could be a polyubiquitin chain attached to a substrate. This model is based on the fact that all polypeptides emerging from the ER undergo polyubiquitination, and that this modification is required for the release of substrates from the ER membrane into the cytosol (for review see Tsai et al., 2002).
Mammalian Ufd1Npl4 has been shown to bind polyubiquitin and has been proposed to mediate substrate binding by p97 (Meyer et al., 2002). A zinc-binding motif in Npl4, the Npl4 zinc finger (NZF) domain, has been identified as the major binding site for ubiquitin (Meyer et al., 2002; Wang et al., 2003). In addition, p97/Cdc48 itself can interact with ubiquitin (Dai and Li, 2001; Rape et al., 2001). The relevance of the reported ubiquitin interactions for retrotranslocation is unclear, particularly because the ubiquitin binding to p97/Cdc48 is weak and the yeast homologue of Npl4 lacks an NZF domain.
In this paper, we have addressed the mechanism by which the p97 ATPase and its cofactor Ufd1Npl4 function in retrotranslocation. Our results provide insight into the ATPase cycle, the mode of interaction of the complex with the membrane, and particularly the mechanism of substrate recognition. We propose a dual recognition mechanism in which a nonubiquitinated segment of a substrate is initially recognized by p97 itself, and subsequently, the polyubiquitin chain is bound by both p97 and the cofactor Ufd1Npl4.
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Results |
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To test the role of the ATPase domains in D1 and D2 in vivo, we made the QE and EQ mutations in Cdc48, the yeast homologue of p97. The mutants were tested for complementation of a cdc48 temperature-sensitive strain at the nonpermissive temperature (Fig. 1 D). When the mutants were expressed under a gal promoter in the presence of galactose, no growth was seen at 37°C, in contrast to the results with the wild-type protein (Fig. 1 D, top right), although the transgenes were expressed at similar levels (Fig. S2). At permissive temperature (30°C), the EQ mutant exerted a strong dominant-negative effect on cell growth, whereas the effect of the QE mutant was much weaker (Fig. 1 D, top left). As expected, when the proteins were not expressed in the presence of glucose, the cells grew at 30°C, but not 37°C (Fig. 1 D, bottom). These data show that ATP hydrolysis in both ATPase domains is required for the function of the ATPase in vivo.
Cofactor and membrane interaction domains of p97
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 N protein, all p97 variants were able to interact with both Ufd1Npl4 and p47 (Fig. 2 A). The results show that the binding of both cofactors requires the N domain of p97.
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Substrate interaction of p97
Next, we investigated the association of the p97 mutants with a retrotranslocation substrate. The substrate chosen was MHC class I heavy chain, which is targeted for retrotranslocation and subsequent proteasomal degradation in cells expressing the human cytomegalovirus protein US11 (Wiertz et al., 1996). The process can be recapitulated in permeabilized cells (Shamu et al., 1999). To determine binding of p97 to MHC class I heavy chains, US11-expressing cells were treated with proteasome inhibitors, labeled with [35S]methionine, and permeabilized with the detergent digitonin. His-tagged p97 variants were added, the cells were incubated for a chase period, and then they were fractionated into membrane (P) and cytosol (S) fractions. A portion of the samples was subjected to immunoprecipitation with heavy chain (HC) antibodies to monitor the amount of substrate in each sample (Fig. 3, AC, top panels). The remainder of the samples was subjected to sequential immunoprecipitation with His and HC antibodies to detect p97-associated heavy chains (Fig. 3, AC, bottom panels). Previous experiments provided evidence that association of the exogenous p97 with heavy chains occurs during retrotranslocation because (1) binding to p97 was seen preferentially with heavy chains on the membrane; (2) the interaction was reduced when the membranes were solubilized before addition of p97; and (3) no binding was seen with heavy chains of cells that did not express US11 (Ye et al., 2001).
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Next, we tested whether the mutant p97 proteins have a dominant-negative effect on the retrotranslocation of MHC class I heavy chains (Fig. 3 AC, top panels). In the absence of added p97 or in the presence of exogenous wild-type p97, a significant fraction of the heavy chains was retrotranslocated into the cytosol during the chase period (Fig. 3 A, top, lane 4; Fig. 3 B, top, lane 16). The cytosolic heavy chains have a slightly faster mobility in the SDS gel because they are deglycosylated by a cytosolic N-glycanase. Likewise, p97 mutants that were defective in substrate interaction (AK, AA, and N) also did not affect retrotranslocation. In contrast, the p97 variants that allowed substrate binding but did not permit ATP hydrolysis (EQ, QQ, KA, and
D2; Fig. 1 C) strongly inhibited heavy chain dislocation; the amount of deglycosylated heavy chains in the cytosol fraction (S) was reduced, and the amount of glycosylated heavy chains in the membrane fraction (P) was increased (Fig. 3, top). The D1 hydrolysis mutant QE, which had lower affinity to substrate and had
50% residual ATPase activity (Fig. 1 C), had a weak effect on heavy chain retrotranslocation (Fig. 3 A, top, lane 8 vs. lane 4 and lane 7 vs. lane 3; see also Fig. 4). These data indicate that the dominant-negative effect of the p97 mutants is a consequence of sequestering heavy chains in a defective ATPase complex.
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Ubiquitination is not required for substrate recognition by p97
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. 5 A, lanes 6 and 7 vs. lanes 2 and 3). To test substrate binding of p97, we added His-tagged p97 to permeabilized cells incubated for different time periods in cytosol containing or lacking ubiquitin, and performed sequential immunoprecipitations with antibodies to the His tag and to the heavy chains. In the absence of ubiquitin, a significant fraction of the heavy chains was bound to p97 (Fig. 5 B, bottom, lanes 712). The fraction increased during the chase incubation, indicating that the movement of heavy chains from the ER lumen to the cytoplasmic side of the ER membrane is independent of ubiquitination. When purified ubiquitin was added back to ubiquitin-depleted cytosol, an interaction of substrate with p97 was only seen at early time points during the chase incubation (Fig. 5 B, bottom, lanes 1318); because ubiquitin restored degradation of the heavy chains (Fig. 5 B, top), there was little material left for interaction with p97 at later time points. As expected, no material was immunoprecipitated when His-tagged p97 was omitted from the incubation (Fig. 5 B, bottom, lanes 16). As expected from the previous results (Fig. 3), the QQ, but not the N and AA mutants of p97, interacted with nonubiquitinated substrate (Fig. 5 C). These data suggest that p97 can specifically interact with substrate that has not been modified by a polyubiquitin chain, and that binding of p97 is an early step after emergence of the polypeptide from the ER.
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To test the specificity of the ubiquitin interaction with the cofactor Ufd1Npl4, we used in vitrosynthesized polyubiquitin chains with linkages through lysine 63 (K63). These chains were synthesized with the yeast ubiquitinconjugating enzyme (E2) consisting of Ubc13 and Mms2 (Hofmann and Pickart, 1999; the purity of the dimer is shown in Fig. 6 A, lane 3). These polyubiquitin chains were also bound to both the U/N and U/N/p97 complexes (Fig. 6 E, top, lane 8 and lane 10). However, the lysine 63 chains were not bound when the NZF domain was missing in Npl4 (Fig. 6 E, top, lane 10; <5% of the input material was bound). Thus, in the absence of the NZF domain, the interaction of the Ufd1Npl4 complex with polyubiquitin chains is specific for the lysine 48 linkage. The NZF domain, on the other hand, appears to be promiscuous, and interacts with both lysine 48 and lysine 63linked ubiquitin chains.
Because the yeast homologue of Npl4 lacks the NZF domain, one might expect that the yeast Cdc48Ufd1Npl4 complex interacts with lysine 48 but not lysine 63linked polyubiquitin chains. To test this possibility, we replaced the wild-type Ufd1 protein in S. cerevisiae with a fusion of Ufd1 to an IgG-binding domain (Ufd1PrA). The cells were homogenized and the Cdc48Ufd1Npl4 complex isolated by binding to IgG beads. As expected, Cdc48 was bound to the beads together with the tagged Ufd1 protein (Fig. 6 F, bottom, lane 3 and lane 6). When incubated with in vitrosynthesized polyubiquitin chains, the complex indeed bound chains with lysine 48 linkage (Fig. 6 F, top, lane 3), but not those with lysine 63 linkage (Fig. 6 F, lane 6). Because both the yeast Cdc48Ufd1Npl4 complex and the mammalian Ufd1Npl4ZF lacking the NZF domain discriminate between lysine 48 and lysine 63 linkages, these data suggest that the unidentified ubiquitin-binding site in the Ufd1Npl4 complex may be the one relevant for the conserved function in ER protein degradation.
Next, we determined that the novel binding site is contained in Ufd1. A purified mammalian Npl4 protein lacking the NZF motif (Npl4ZF) 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. 7 B, lane 3). A GST fusion to the NH2-terminal 215 residues of Ufd1 (UT3 domain; Fig. 7 A), which was predicted to contain a double
barrel fold (Coles et al., 1999; Golbik et al., 1999), also bound ubiquitin chains (Fig. 7 B, lane 4). On the other hand, a GST fusion to the COOH-terminal UT6 domain (Fig. 7 A), containing binding sites for both p97 and Npl4 (Hetzer et al., 2001), was inactive (Fig. 7 B, lane 5).
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The interactions of p97 and Ufd1 with polyubiquitin appear to be synergistic. When both components were immobilized together, polyubiquitin binding was significantly stronger than with either component alone (Fig. 7 D, lane 6 and 7 vs. lanes 35). A similarly strong binding was seen when all three components (p97, Ufd1, and Npl4ZF) were together in a ternary complex (Fig. 7 E, lane 7). These interactions are again specific for lysine 48linked polyubiquitin chains (Fig. 7 E, lanes 17 vs. lanes 814). Together, our data suggest that the Ufd1Npl4 complex has an evolutionarily conserved function in mediating ubiquitin binding of the ATPase complex. The Ufd1Npl4p97 complex contains two conserved and cooperating polyubiquitin-binding sites, one in p97 itself, and one in the UT3 domain of Ufd1; the mammalian complex also contains the NZF domain that may further increase the binding of polyubiquitin.
The ubiquitin-binding domain of Ufd1 is required for retrotranslocation
Next, we tested whether the ubiquitin-binding domain in Ufd1 is important for the dislocation and degradation of MHC class I heavy chains. We reasoned that a mutant Ufd1Npl4 complex lacking the UT3 ubiquitin-binding site would act as a dominant-negative mutant by sequestering endogenous p97 in a defective complex that could no longer efficiently interact with ubiquitin chains on the substrate. Indeed, although addition of either wild-type Ufd1Npl4 (U/N) or Ufd1Npl4ZF (U/N
ZF) had no effect on heavy chain degradation (Fig. 8 A, top; for quantification, see top right panel), the corresponding complexes lacking the UT3 domain (U
UT3/N and U
UT3/N
ZF) were inhibitory (Fig. 8 A, bottom). Because all complexes tested bind p97 with similar affinity (unpublished data), the results suggest that lack of ubiquitin interaction may cause the dominant-negative effect. With the mutant lacking UT3 (Ufd1
UT3/Npl4), polyubiquitinated heavy chains accumulated on the membrane (Fig. 8 B, bottom, lane 11 vs. lane 3), as they did when the ATPase mutant KA was added (Fig. 8 B, lane 7; quantification given above the lanes). In controls, the ubiquitinated heavy chains were released into the cytosol (Fig. 8 B, lane 4). Thus, both ubiquitin binding and ATP hydrolysis by p97Ufd1Npl4 complex are required for the release of polyubiquitinated substrates into the cytosol. Ubiquitin binding to UT3, and not to the NZF domain of Npl4, appears to be important for retrotranslocation.
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Discussion |
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We propose that the polypeptide substrate is transported through the central pore in the double barrel structure of p97 in the direction of the D1 to the D2 ring. Our data indeed suggest that the D1 ring is proximal to the membrane because the N domain, located at its side, is required for membrane interaction of p97. However, because the N domain is also involved in cofactor binding, it is unclear which component makes direct contact with the membrane. Because a mutant of p97 defective in substrate binding could still bind to the membrane, substrate binding cannot be the only linkage of the ATPase complex with the membrane. An interaction with an unidentified membrane receptor may explain why a large fraction of p97 and of its yeast homologue Cdc48 is firmly bound to the ER.
Our results provide insight into how the p97Ufd1Npl4 complex recognizes retrotranslocation substrates. p97 itself recognizes both polyubiquitinated and nonubiquitinated polypeptides. The strongest evidence that p97 can interact with nonmodified substrate comes from our experiments in which polyubiquitination was prevented. Because p97 binding to nonmodified substrate was similarly observed in a crude permeabilized cell system in the presence of ubiquitin, the interaction is not caused by ubiquitin depletion and is of physiological relevance. Although direct contact of p97 with substrate remains to be demonstrated, our data show that the interaction is nucleotide dependent, as observed for other ATPases (for review see Bukau and Horwich, 1998; Hoskins et al., 2000). An appealing possibility is that p97 recognizes unfolded hydrophobic polypeptide segments as demonstrated with model substrates (Thoms, 2002). The VAT protein, a homologue of p97 in archaebacteria, must also recognize nonubiquitinated substrates, because no ubiquitination exists in these organisms.
In a pulse-chase experiment, the amount of p97-associated substrate increased with time when ubiquitination was abolished, suggesting that p97 may interact with nonmodified polypeptides as soon as they emerge from the ER membrane, preventing their backsliding into the ER lumen (Fig. 9 B). Although it has been proposed that ubiquitin mediates the initial interaction of the transcription factor Spt23 with the Cdc48Ufd1Npl4 complex (Rape et al., 2001), binding to an unfolded segment of the polypeptide has not been excluded. At least for retrotranslocation substrates, it is likely that polyubiquitination succeeds substrate binding to p97.
Our data suggest that the polyubiquitin chain is recognized by both p97 and the cofactor Ufd1Npl4. The finding that p97 can weakly interact with polyubiquitin chains is in agreement with previous results (Dai and Li, 2001; Rape et al., 2001). The interaction of p97 with ubiquitin appears to be distinct from that with nonmodified substrate because it can be observed with the AA mutant of p97 that is defective in substrate binding. Polyubiquitin binding to the Ufd1Npl4 complex was previously ascribed to the NZF domain of Npl4 (Meyer et al., 2002). With a more sensitive binding assay, we now demonstrate that the UT3 domain of Ufd1 contains an additional ubiquitin-binding site that has a lower affinity than the NZF domain, but appears to be more important for retrotranslocation. This domain is similar in sequence among all eukaryotic Ufd1 proteins, consistent with a conserved function in retrotranslocation, and a cofactor complex lacking this domain acts as a dominant-negative mutant in retrotranslocation. These data also demonstrate that the mammalian cofactor Ufd1Npl4, like its homologue in yeast, plays a role in ER protein degradation. The UT3 domain recognizes polyubiquitin chains in a lysine 48 (but not lysine 63) linkage, as might be expected from the similar preference by the proteasome acting downstream of p97 in the ER degradation pathway. Longer polyubiquitin chains are the preferred substrate for Ufd1, in agreement with our previous finding that a certain chain length is required for substrate release from the membrane (Shamu et al., 2001). Whether the ubiquitin-binding NZF domain of Npl4 plays a role in retrotranslocation is unclear because it is missing in the yeast protein, does not show specificity for the ubiquitin linkage, and because a mammalian cofactor complex lacking this domain is not dominant negative. It could play a supporting role in ER protein degradation in mammalian cells, or could be involved in p97Ufd1Npl4 dependent processes other than retrotranslocation.
Our data suggest that p97 and Ufd1 act synergistically in polyubiquitin binding. One possibility is that the proteins together form a binding site with higher affinity. Specifically, double barrel folds are present in both the UT3 domain of Ufd1 and the N domain of p97 (Coles et al., 1999; Golbik et al., 1999), and these could both bind weakly to polyubiquitin chains containing lysine 48 linkages. When present together in a complex, the simultaneous interaction of the double
barrel folds with polyubiquitin chains could increase the affinity.
On the basis of our results, we propose a model of dual substrate recognition by the p97Ufd1Npl4 complex; a nonubiquitinated, unfolded segment of a retrotranslocation substrate would initially be recognized by p97 itself (Fig. 9 B). Once a polyubiquitin chain has been attached to the substrate, it would be recognized by both p97 and the cofactor Ufd1 in the complex. This interaction may activate the ATPase to pull the polypeptide chains out of the membrane. Purified p97 has a relatively high intrinsic ATPase activity, and therefore stimulation of the ATPase activity may require that the basal activity in the cell be inhibited, perhaps by the membrane receptor of p97. In the cytosol, the basal ATPase activity of p97 is inhibited by the cofactor p47 (Meyer et al., 1998). Consistent with our model, mutants in the p97Ufd1Npl4 complex that can bind the substrate, but either lack ubiquitin interaction or are defective in the ATPase cycle, are dominant negative for retrotranslocation. The dual recognition model explains why p97 interacts with nonubiquitinated substrates and why polyubiquitination is nevertheless required for its function. The unmodified segments of the polypeptide chain may be inserted as a loop into the central cavity of the p97 double barrel, whereas the bulky polyubiquitin chains may stay bound at the side of the barrel. Loop insertion of a substrate polypeptide is also discussed for the proteasome (Liu et al., 2003).
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Materials and methods |
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Antibodies, chemicals, and proteins
Antibodies to MHC class I heavy chain, ubiquitin, and the His tag have been described previously (Ye et al., 2001). Antibodies to mammalian Ufd1, Npl4, and p47 were described by Meyer et al. (2000). The Cdc48 antibodies were a gift from M. Latterich (McGill University, Montreal, Canada). Calnexin and GST antibodies were purchased from StressGen Biotechnologies and Amersham Biosciences, respectively; proteasome inhibitor MG132 was purchased from Calbiochem; bovine ubiquitin was purchased from Sigma-Aldrich; and ubiquitin K48R, methylated ubiquitin, and ubiquitin activating enzyme (E1) was purchased from Affinity BioReagents, Inc.
Protein purification
His-tagged proteins were all purified using Ni-NTA beads (QIAGEN). With the exception of p97 variants, all proteins eluted from the Ni column were further fractionated on a Mono-Q column using a 0500-mM potassium chloride gradient. The p97 variants were further purified by size-exclusion chromatography on a SuperdexTM 200 HR (10/30) column in 50 mM Tris/HCl, pH 8.0, 150 mM potassium chloride, 5% glycerol, and 2 mM magnesium chloride. The complex of His-Ubc13/His-Mms2 was assembled on the same size-exclusion column using the same conditions. GST-gp78c and GST-Ubc7 were purified with glutathione beads. GST-gp78c was eluted with reduced glutathione, whereas Ubc7 was cleaved off from the beads using thrombin. The eluted proteins were further purified on a Mono-Q column. The purification of GST-Ufd1, GST-UT3, GST-UT6, Npl4, Npl4ZF, Ufd1-His, UT3-His, and His-UT6, as well as the assembly of various Ufd1Npl4 complexes was described previously (Meyer et al., 2002). His-UT6 and Npl4 as well as His-UT6 and Npl4
ZF were coexpressed in E. coli and purified on Ni-NTA. All purified proteins were stored at -80°C in 50 mM Tris/HCl, pH 8.0, 150 mM potassium chloride, 2 mM magnesium chloride, and 5% glycerol.
ATPase measurements, nucleotide and cofactor binding, and membrane association
The ATPase activity of p97 variants was measured as described previously (Meyer et al., 1998). To test nucleotide binding by the p97 variants, 100 µg proteins were incubated with 0.2 mM ATP plus 2 µCi [32P]ATP in 50 mM Tris/HCl, pH 8.0, 150 mM KCl, 1 mM magnesium chloride, and 15% glycerol at 4°C for 20 min, and fractionated by a gel filtration column (NAP5; Amersham Biosciences). Nucleotides in each fraction were analyzed by TLC. The cofactor-binding assay was performed as before with slight modification (Meyer et al., 2000). 2-µg purified His-tagged p97 proteins were added to 500 µg rat liver cytosol in 1 M NaCl, 50 mM Hepes, pH 7.6, 250 mM sucrose, 50 mM potassium acetate, 6 mM magnesium chloride, 1 mM EDTA, and 1 mM DTT plus protease inhibitor. The salt concentration was adjusted to 150 mM with 20 mM Hepes, pH 7.6 and 1 mM DTT. p97 proteins were immunoprecipitated with His antibodies, and the bound proteins were analyzed by immunoblotting with p47, Ufd1, and His antibodies. For membrane-binding experiments, US11 cells were permeabilized with 0.028% digitonin in the presence of various p97 proteins at a concentration of 1 µM. After incubation, the membranes were sedimented and washed with permeabilization buffer without detergent (25 mM Hepes, pH 7.3, 115 mM potassium acetate, 5 mM sodium acetate, 2.5 mM magnesium chloride, and 0.5 mM EGTA). Proteins in the membrane and cytosol fractions were solubilized in sample buffer and analyzed by immunoblotting.
Dislocation and degradation of heavy chains and substrate binding
The experiments were performed as described previously (Ye et al., 2001). Recombinant proteins were added at a 1-µM concentration. Immunoprecipitation with heavy chain antibodies and reprecipitation with ubiquitin antibodies were done as described previously (Shamu et al., 1999). The interaction of p97 with MHC heavy chains was analyzed as described previously (Ye et al., 2001). Ubiquitin depletion experiments were performed as described previously by incubating cow liver cytosol with a GST fusion to a mutant of the ubiquitin-conjugating enzyme Ubc2B, which carries a Ser instead of a Cys in the active site (Shamu et al., 2001).
In vitro ubiquitination and ubiquitin binding
Polyubiquitin chains were synthesized at 37°C with either 4 µM Ubc7, 1µM GST-gp78c, or 2 µM His-Ubc13/His-Mms2 in buffers containing 25 mM Tris/HCl, pH 7.4, 2 mM magnesium/ATP, 0.1 mM DTT, 110 nM E1 enzyme, and 20 µM ubiquitin. The binding experiments were performed at 4°C in 0.3 ml of 50 mM Hepes, pH 7.3, 150 mM potassium chloride, 2.5 mM magnesium chloride, 5% glycerol, 2 mM ß-mercaptoethanol, 0.1% Triton X-100, and 1 mg/ml BSA. 20-µl polyubiquitin chains were incubated with 1 µg of the various purified recombinant proteins. The proteins were then precipitated with specific antibodies, and bound ubiquitin chains were detected by immunoblotting with ubiquitin antibodies. To test ubiquitin chain binding to GST fusion proteins, the latter were first immobilized on glutathione beads. The ubiquitin chains were preabsorbed with glutathione beads to remove the E3 and ubiquitin chains associated with it, before addition to the immobilized proteins. To test ubiquitin binding of the yeast Cdc48Ufd1Npl4 complex, the endogenous gene for Ufd1 was replaced by a gene coding for a fusion of Ufd1 with the IgG-binding domain of protein A. Cytosol from the mutant yeast cells was incubated with IgG beads to isolate the Cdc48Ufd1Npl4 complex, and was then incubated with polyubiquitin chains.
Experiments with yeast
The yeast cdc483 strain was transformed with vector or plasmids coding for either wild-type Cdc48 or mutant Cdc48 proteins. The cells were serially diluted and plated on galactose- or glucose-containing agar. They were incubated at 30 or 37°C for 3 d.
Online supplemental material
Fig. S1 shows nucleotide binding to various His-tagged p97 proteins, and Fig. S2 shows that mutant Cdc48 defective in ATP hydrolysis is expressed at similar levels as the wild-type protein in S. cerevisiae. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200302169/DC1.
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Acknowledgments |
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Y. Ye is supported by the Helen Hay Whitney postdoctoral fellowship. H.H. Meyer is supported by grants to Graham Warren from the National Institutes of Health (NIH) and the Human Frontier Science Program, and T.A. Rapoport by NIH grants (R01GM52586 and R01GM54238). T.A. Rapoport is a Howard Hughes Medical Institute Investigator.
Submitted: 27 February 2003
Revised: 16 May 2003
Accepted: 20 May 2003
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