Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4 - PubMed (original) (raw)
Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4
Hemmo H Meyer et al. EMBO J. 2002.
Abstract
The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.
Figures
Fig. 1. The p97 adaptor, Ufd1–Npl4, mediates binding to polyubiquitylated proteins. Recombinant mammalian p97 (lanes 2–4), p47 (lane 5) and Ufd1–Npl4 (UN, lane 6) were biotinylated and immobilized on streptavidin–beads. The p97–beads were either used without further treatment (lane 2) or first saturated with recombinant p47 or UN (lanes 3 and 4, respectively). The immobilized protein complexes were then incubated with a Triton X-100 extract from HeLa cells and bound proteins analyzed by western blotting using a ubiquitin-specific antibody (upper panel). The filter was also stained with Ponceau S to visualize the immobilized proteins (lower panel). Note that only UN efficiently binds polyubiquitylated proteins (pUb) either alone or in combination with p97.
Fig. 2. The binding of p47 and UN to mono-ubiquitin is direct but differentially regulated. (A) The indicated combinations of p97, p47 and UN were incubated with either GST (lanes 1–5) or a ubiquitin–GST fusion protein (Ub–GST, lanes 6–10) immobilized on glutathione– beads. Bound material was analyzed by western blotting using specific antibodies against the proteins indicated on the right; 5% of the input was loaded as a reference (lanes 11 and 12). Note that p97 binds Ub–GST efficiently only as a complex with either p47 (lane 7) or UN (lane 9), and that UN (lane 10), but not p47 (lane 8), can bind Ub–GST independently of p97. (B) Binding and analysis were carried out as in (A) but using sequential incubations. Ub–GST was first incubated with p47 (lane 2) or UN (lane 4), and with p97–47 or p97–UN as a reference (lanes 3 and 5). The beads were then washed and incubated with p97 as indicated. Note that UN (lane 4), but not p47 (lane 2), binds Ub–GST, stays bound during the washes and can then recruit p97 in the second incubation (lane 4).
Fig. 3. The UBA domain of p47 is both necessary and sufficient for mono-ubiquitin binding. (A) Schematic representation of the p47 domain structure with amino acid (aa) numbers indicated. p47 contains a UBX domain at the C-terminus, that has a ubiquitin fold which contributes to p97 binding (Yuan et al., 2001). The N-terminal 45 amino acids constitute the UBA domain. (B) Upper panels: p97 was incubated with immobilized Ub–GST either alone or in the presence of p47(wt) or p47 lacking the UBA domain [p47(ΔUBA)]. Bound proteins were analyzed by western blotting using specific antibodies to the proteins indicated on the right. Note that the UBA domain is needed for binding to Ub–GST. Lower panels: biotinylated p97 was incubated with p47 or p47(ΔUBA) and complexes retrieved using streptavidin–beads. Note that the UBA domain is not required for binding to p97. The panel on the right shows 10% of input material used. (C) p97–p47 was bound to immobilized Ub–GST in the absence (lane 1) or presence of increasing amounts (5, 25 and 125 mol excess over p47) of peptides representing different UBA domains: p47(wt) (lanes 2–4), p47(F41A) mutant (lanes 5–7) and the human homolog of Rad23 (hhRad23, lanes 8–10). Bound material was analyzed by western blotting. Note that the p47(wt) and the hhRad23 peptides bind Ub–GST directly and compete with the p97–p47 complex, whereas the p47(F41A) mutant does so much less efficiently.
Fig. 4. The p47 UBA domain is required for p97–p47-mediated reassembly of cisternal membranes from MGFs. Rat liver Golgi membranes were fragmented by treatment with mitotic HeLa cell lysate, and MGFs were isolated. After incubation with purified p97 alone or in combination with p47(wt), p47(ΔUBA) or p47(F41A), the samples were processed for electron microscopy and the percentage of Golgi membranes in cisternae determined. Results are presented as the mean percentage of cisternal regrowth (± SEM), where 0% represents incubations with buffer alone (38 ± 3% of membrane in cisternae) and 100% represents incubations with p97–p47(wt) (66 ± 2% of membrane in cisternae). Note that both deletion and mutation of the UBA domain reduced the reassembly activity of p97–p47 by 75–80%.
Fig. 5. The zinc finger of mammalian Npl4 (NZF) represents a novel, conserved ubiquitin-binding domain. (A) Domain structure of rat Ufd1 and Npl4: Npl4 contains a single putative zinc finger motif in the C-terminal 29 amino acids (NZF). Ufd1 contains the N-terminal UT3 domain that putatively folds as a double-ψβ-barrel and a UT6 domain that binds both p97 and Npl4. (B) GST–Ufd1 (GST-U) was immobilized on glutathione–beads and saturated with either wild-type Npl4 [N(wt)] or Npl4 with a deletion of the zinc finger [N(ΔZF)]. A fusion protein comprising the Npl4 zinc finger alone (GST–NZF) was also immobilized on glutathione–beads. The beads were then incubated with a detergent lysate from HeLa cells. GST served as a control. Bound proteins were analyzed by western blotting for the presence of either polyubiquitin (upper panel) or p97 (lower panel). Note that the NZF was both necessary and sufficient to mediate binding to polyubiquitylated proteins (pUb). (C) Beads prepared as in (B) were incubated with pure p97 and bound fractions were analyzed by SDS–PAGE followed by Coomassie Blue staining. Ten percent of the input p97 used is shown in the lower panel. Note that the NZF domain does not participate in p97 binding to UN. (D) GST or Ub–GST was immobilized on glutathione–beads and incubated with different combinations of Ufd1–Npl4 [UN(wt)], Ufd1–Npl4ΔZF [UN(ΔZF)] and p97 as indicated. Complexes were isolated and analyzed by western blotting using antibodies specific to the proteins indicated on the right. Note that deletion of the zinc finger abolished binding to Ub–GST. (E) CLUSTAL_W alignment of related zinc finger peptide sequences of different proteins indicated on the left. The rat sequence is identical to that for mouse and humans and is probably representative of all mammals. (F) Peptides indicated with an asterisk in (E) were expressed as fusions with GST and incubated with a HeLa cell lysate. Their ability to bind polyubiquitylated proteins was tested by western blot analysis. See Materials and methods for details.
Fig. 6. The UBA domain of p47, but not the zinc finger domain of Npl4, inhibits polyubiquitylation of Ub–GST in vitro. Ub–GST was expressed in a rabbit reticulocyte lysate in the absence or presence of methylated ubiquitin (me-Ub, 20 µM) or 35 µM of the indicated peptides: p47-UBA(wt), p47-UBA(F41A), Npl4-NZF peptide, a GST–NZF fusion or GST. Samples were fractionated using SDS–PAGE and visualized using a phosphoimager. The radiolabeled Ub–GST fusion protein was polyubiquitylated under control conditions (Ub1–n) by factors present in the lysate. Note that p47-UBA(wt) significantly inhibited polyubiquitylation.
Fig. 7. Hypothetical model for the role played by p97 adaptors in ubiquitin-mediated processes. p47 bound to p97 would bind to substrates both directly and indirectly via mono-ubiquitin (using the UBA domain). Binding would prevent further ubiquitylation. UN would bind mono- or polyubiquitylated substrates via the zinc finger on Npl4 and permit further ubiquitylation. UN would either bind as a complex with p97 or would recruit p97 to the substrate (S).
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