Cooperative assembly of p97 complexes involved in replication termination

Abstract

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97^Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97^Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97^{Ufd1-Npl4-Ubxn7} and p97^{Ufd1-Npl4-Faf1} also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.

Fig. 1. Ubxn7 promotes unloading of terminated CMG in interphase.

a Analysis of chromatin-bound proteins in mock- or Ubxn7-depleted extracts in the absence or presence of p97i. Plasmid was incubated in total egg lysate to carry out licensing, and then exposed to nucleoplasmic extract (NPE) to initiate replication. Extracts were supplemented with rUbxn7 variants as indicated. At 20 min after NPE addition, plasmids were recovered with LacR-coated beads and immunoblotted for the indicated proteins. Ub-Mcm7, ubiquitylated Mcm7; Ubxn7^ΔUBA, deletion of the 1-74 N-terminal amino acids of Ubxn7; Ubxn7^UBA*, I46A/L65A/L73A amino acid substitutions in the UBA domain; Ubxn7^UIM*, S321A amino acid substitution in the UIM domain; Ubxn7^UBA*/UIM*, combined I46A/L65A/L73A and S321A amino acid substitutions; Ubxn7^UBX*, P486G amino acid substitution in the UBX domain. See panel (c) for Ubxn7 domain composition and Supplementary Fig. 1d for immunodepletion efficiency. Lanes 1-8 and 9-16 are parts of the same experiment and were analyzed on separate Western blots processed and imaged in parallel under identical conditions. b Coomassie-stained SDS PAGE gel of recombinant wild-type (WT) and mutant variants of Ubxn7. Abbreviations as in a. c Domain composition and known protein-protein interactions of Xenopus Ubxn7. UBX ubiquitin regulatory X domain, UIM Ubiquitin Interacting Motif, UBA ubiquitin-associated domain, UAS ubiquitin associating domain. Other abbreviations as in a. d A model for the Ubxn7-mediated p97 recruitment to ubiquitylated CMG during interphase termination. e A model for p97 recruitment to ubiquitylated CMG by UBA mutants of Ubxn7. f A model for p97 recruitment to ubiquitylated CMG by the UIM mutant of Ubxn7. g A model for p97 recruitment to ubiquitylated CMG the double UBA and UIM mutant of Ubxn7. h A model for p97 recruitment to ubiquitylated CMG by the UBX mutant of Ubxn7. In d–h, Ub ubiquitin, ZF NZF domain of Npl4, N8 Nedd8, red dotted lines indicate weak protein–protein interactions. Source data are provided as a Source Data file.

Fig. 2. The p97^Ufd1-Npl4 complex initiates CMG unloading in interphase.

a Analysis of chromatin-bound proteins in mock- or Npl4-depleted egg extracts in the absence p97i. Extracts were supplemented with recombinant wild-type or Ufd1-Npl4 mutants as indicated. At 25 and 45 min after replication initiation, plasmid DNA was recovered with LacR-coated beads and immunoblotted for the indicated proteins. ΔUT3, deletion of 2-215 amino acids of Ufd1. Ub-Mcm7, ubiquitylated Mcm7. Other abbreviations as in d. See also Supplementary Fig. 2a for mock and Ufd1-Npl4 immunodepletion efficiency. b An image of polyacrylamide gel of recombinant wild-type and mutant variants of Ufd1-Npl4. Abbreviations as in d. c As in a, but extracts were supplemented with p97i. See also Supplementary Fig. 2c for mock and Ufd1-Npl4 immunodepletion efficiency. “Input, 1%”, “No DNA” lanes, and lanes 1–10 are parts of the same experiment that were analyzed on separate Western blots processed and imaged in parallel under identical conditions. For Sld5, the contrast of the whole image was adjusted using ImageJ to enhance visibility of the bands. d Domain composition of Xenopus Npl4 and Ufd1. UBXL, ubiquitin regulatory X-like domain; MPN, Mpr1, Pad1 N-terminal domain; NZF, NPL4 Zinc Finger domain; UT3, Ufd1 truncation 3 domain; UT6, Ufd1 truncation 6 domain. LV, T592L/F593V amino acid substitutions in NZF; AAE, L240A/W243A/R244E amino acid substitutions in MPN. Source data are provided as a Source Data file.

Fig. 3. Ubiquitin chains mediate interaction between UBA-UBX proteins and p97^Ufd1-Npl4.

a A model for ubiquitin chain-mediated complex assembly between p97^Ufd1-Npl4 and UBA-UBX proteins. b Co-immunoprecipitates of FLAG-rUbxn7 from Ubxn7- or Ubxn7/Npl4-depleted egg extracts in the presence or absence of p97i or Culi were analyzed with the indicated antibodies. FLAG co-immunoprecipitates were washed with 50 mM or 100 mM KCl in IP buffers. See Supplementary Fig. 3a for verification that Ubxn7 and Npl4 were depleted. Note: Cul2 undergoes de-neddylation during the FLAG immunoprecipitation procedure, and the degree of de-neddylation varies between extracts. De-neddylation is partially prevented by p97i (lanes 14 vs. 18), and further inhibited by 100 mM KCl (lane 30). Recovery of both neddylated and de-neddylated Cul2 is inhibited by Culi (lane 16), confirming that neddylation is required for its association with Ubxn7. Black arrowheads, neddylated Cul2; blue arrowheads, un-neddylated Cul2. Red asterisks, non-specific bands. N8-Cul2, neddylated Cul2. Lanes 1–12 and 13–36 are parts of the same experiment that were analyzed on separate Western blots processed and imaged in parallel under identical conditions. For Cul2 and Ufd1, the contrast of the whole image was adjusted using ImageJ to enhance visibility of the bands. c Co-immunoprecipitates of GST or wild type GST-Ufd1-Npl4 heterodimer from non-replicating egg extracts in the presence of the indicated additives were analyzed with indicated antibodies. Black arrowheads indicate neddylated Cul2 (E1i also blocks neddylating enzymes). Red arrowheads most likely indicate a cross-reacting band or non-specific recovery of unneddylated Cul2. For Cul2, the contrast of the whole image was adjusted using ImageJ to enhance visibility of the bands. Red asterisks, non-specific bands. See Supplementary Fig. 3c for verification of Npl4 depletion. Source data are provided as a Source Data file.

Fig. 4. Faf1’s back-up function in CMG unloading can be enhanced by Ubxn7’s UIM motif.

a Analysis of chromatin-bound proteins in mock-, Ubxn7-, Faf1-, or Ubxn7/Faf1-depleted extracts. At indicated time points after replication initiation, plasmid DNA was recovered with LacR-coated beads and immunoblotted for the indicated proteins. Lanes 1–9 and 10–21 are parts of the same experiment that were analyzed on separate Western blots processed and imaged in parallel under identical conditions. Red asterisks indicate non-specific band that is likely contamination with non-reduced heavy chains of rabbit IgG. See Supplementary Fig. 6a for immunodepletion efficiency and 6b for independent replicate. Ub-Mcm7, ubiquitylated Mcm7. b An image of polyacrylamide gel of recombinant wild-type Faf1 and Faf1^UIM chimera. Black arrowheads indicate Faf1-specific bands. c Analysis of co-immunoprecipitates of FLAG-rUbxn7 or FLAG-rFaf1 with K48-linked ubiquitin chains. Recombinant Ubxn7 and Faf1 were detected by using anti-FLAG antibodies. Red asterisk indicates C-terminal truncation of recombinant Faf1. IP immunoprecipitation. d Domain composition of Xenopus Faf1 and the Faf1^UIM chimera. In Faf1^UIM, the UIM motif of Ubxn7 was inserted between amino acid residues 293 and 294 of Faf1. UBX ubiquitin regulatory X domain, UIM Ubiquitin Interacting Motif, UBA ubiquitin-associated domain, UBL1, 2 ubiquitin like domain 1, 2, UAS ubiquitin associating domain. e Analysis of chromatin-bound proteins in mock-, Ubxn7-, Ubxn7-Npl4-, or Ubxn7/Faf1-depleted extracts in the presence of p97i and wild-type Ubxn7, wild-type Faf1, or the Faf1^UIM chimera. See also Supplementary Fig. 7a for immunodepletion efficiency. Source data are provided as a Source Data file.

Fig. 5. hUBXD7 is required for CMG unloading in mammalian cells.

a Quantification of MCM7 levels on chromatin in the G2/M phase of cell cycle in wild-type HFT cells in the presence or absence of the p97 inhibitor CB-5083 and in hUBXD7 KO cells. Bars represent mean, and error bars represent SD from three independent repeats. Asterisks indicate statistical significance compared with the control, as determined by one way ANOVA and Tukey test for multiple comparisons (***P < 0.0001). See also Supplementary Fig. 7. b Wild-type or hUBXD7 KO HFT cells were allowed to grow asynchronously or they were synchronized with double thymidine and then optionally released into RO3306 for 10 h, as indicated. Soluble and chromatin bound proteins were blotted for the indicated proteins. The images are all part of the same Western blot, which was cropped to remove irrelevant information between lanes 3–4. A, asynchronous. Source data are provided as a Source Data file.

Fig. 6. A model for p97^{Ufd1-Npl4-Ubxn7} assembly on ubiquitylated CMG.

a Ubxn7 binds weakly and transiently to ubiquitylated CMG-CRL2^Lrr1 prior to p97^Ufd1-Npl4 binding. b Multi-valent and cooperative interactions stabilize p97^{Ufd1-Npl4-Ubxn7} binding to ubiquitylated CMG-CRL2^Lrr1, allowing initiation of ubiquitin chain unfolding. c Ubiquitylated Mcm7 is extracted from CMG and threaded through the central pore of p97.