Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, United Kingdom
Figures
Figure 1 with 1 supplement
Reconstituted disassembly of ubiquitylated CMG helicase by purified human p97-UFD1-NPL4.
(A) Budding yeast CMG helicase (with an internal FLAG-tag on the Cdc45 subunit) was ubiquitylated in vitro, bound to beads coated with anti-FLAG antibody and then incubated with recombinant human …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig1-data1-v2.pdf
Figure 1—figure supplement 1
Characterisation of ubiquitin chain formation under the conditions used in this study.
(A) Reconstituted CMG ubiquitylation reactions were performed as described in Materials and methods, in the presence of 25 nM E2, 10 nM E3, and 6 µM of the indicated form of ubiquitin (wt [wild …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig1-figsupp1-data1-v2.pdf
Figure 2 with 3 supplements
Human p97-UFD1-NPL4 has a much higher ubiquitin threshold than yeast Cdc48-Ufd1-Npl4.
(A) Reconstituted CMG ubiquitylation reactions were performed under conditions that produced a single chain of up to ~12 ubiquitins on CMG-Mcm7 (2.5 nM E2, 1 nM E3; see Figure 1—figure supplement 1B)…
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Time course analysis of CMG disassembly reactions by human p97-UFD1-NPL4.
CMG ubiquitylation reactions were performed as in Figure 2, with the indicated concentrations of E2 and E3 enzymes. Subsequently, ubiquitylated CMG was bound to anti-FLAG beads before incubation …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig2-figsupp1-data1-v2.pdf
Figure 2—figure supplement 2
Human p97-UFD1-NPL4 preferentially disassembles extensively ubiquitylated CMG helicase.
CMG ubiquitylation and disassembly reactions were performed as above, except that the disassembly reactions contained the indicated concentrations of p97-UFD1-NPL4, in addition to 15 nM …
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig2-figsupp2-data1-v2.pdf
Figure 2—figure supplement 3
Disassembly of CMG helicase by human p97-UFD1-NPL4 does not require multiple ubiquitin chains, or other ubiquitin chain linkages apart from K48.
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Figure 2—figure supplement 3—source data 1
Source data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig2-figsupp3-data1-v2.pdf
Figure 3 with 2 supplements
The Ufd1-Npl4/UFD1-NPL4 adaptor complex determines the ubiquitin threshold of Cdc48/p97.
(A) CMG helicase was ubiquitylated as in Figure 1—figure supplement 1A, then bound to anti-FLAG beads. Disassembly reactions were then performed as indicated in the presence of yeast Cdc48 or human …
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Sequence alignment of human and yeast orthologues of p97 and UFD1-NPL4.
(A) Human p97 and yeast Cdc48 were aligned with Clustal Omega software and the output viewed with MView (Madeira et al., 2019). (B, C) Similar analysis for NPL4/Npl4 and UFD1/Ufd1.
Figure 3—figure supplement 2
The NPL4 groove is essential for disassembly of ubiquitylated CMG by p97-UFD1-NPL4, whereas the NZF domain is largely dispensable.
(A) Domain analysis of human NPL4 UBXL = UBX like domain that interacts with a p97 N-terminal domain; ZF1-2 are Zn fingers that anchor the MPN domain to p97; MPN is the ‘Mpr1/Pad1 N-terminal domain’ …
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Figure 3—figure supplement 2—source data 1
Source data for Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig3-figsupp2-data1-v2.pdf
Figure 4 with 3 supplements
UBXN7, FAF1, and FAF2 reduce the ubiquitin threshold of p97-UFD1-NPL4.
(A) Domain organisation of the indicated UBX proteins. UBA = ‘UBiquitin-Associated’ domain that binds ubiquitin; UBX = ‘UBiquitin regulatory X’ domain that binds p97; UBL = ‘UBiquitin-Like’ domain …
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig4-data1-v2.pdf
Figure 4—figure supplement 1
Purified human UBX proteins bind to p97-UFD1-NPL4.
(A) Purified UBX proteins. (B) Proteins were mixed as indicated and the factors associated with 6HisNPL4 were isolated on Ni-NTA beads as described in Materials and methods. The bound proteins were …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig4-figsupp1-data1-v2.pdf
Figure 4—figure supplement 2
Stimulation of p97-UFD1-NPL4 activity by UBX proteins requires the NPL4 groove and suppresses loss of the ubiquitin-binding NPL4-NZF.
CMG ubiquitylation is performed as in Figure 1—figure supplement 1A. Disassembly reactions were then carried out as in Figure 1, in the presence of the indicated factors. Variants of NPL4 correspond …
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig4-figsupp2-data1-v2.pdf
Figure 4—figure supplement 3
Human UBX proteins stimulate CMG helicase disassembly by yeast Cdc48 in the presence of human UFD1-NPL4.
CMG was ubiquitylated as in Figure 1—figure supplement 1D, before binding to anti-FLAG beads. Disassembly reactions were then performed in the presence of the indicated factors.
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Figure 4—figure supplement 3—source data 1
Source data for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig4-figsupp3-data1-v2.pdf
Figure 5 with 3 supplements
Mapping domains of UBXN7, FAF1, and FAF2 that stimulate the unfoldase activity of p97-UFD1-NPL4.
(A) Truncations of UBXN7. (B) Purified proteins. (C) CMG was ubiquitylated as in Figure 1—figure supplement 1D and then bound to anti-FLAG beads. Disassembly reactions were performed as above, in …
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig5-data1-v2.pdf
Figure 5—figure supplement 1
Association of UBXN7 and FAF1 truncations with p97-UFD1-NPL4.
(A) Purified truncated versions of UBXN7, as in Figure 5A. (B) Association of UBXN7 truncations with p97-UFD1-NPL4 was monitored by pull downs of 6HisNPL4 on Ni-NTA beads, as in Figure 4—figure …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig5-figsupp1-data1-v2.pdf
Figure 5—figure supplement 2
The coiled-coil and UBX domains of C.elegans UBXN-3 are required to stimulate the unfoldase activity of CDC-48_UFD-1_NPL-4.
(A) The indicated truncations of C. elegans UBXN-3 (upper panel) were purified together with C. elegans CDC-48 and UFD-1_NPL-4 (lower panel; UN = UFD-1_NPL-4). (B) C. elegans CMG was ubiquitylated …
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Figure 5—figure supplement 2—source data 1
Source data for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig5-figsupp2-data1-v2.pdf
Figure 5—figure supplement 3
Ubiquitin binding of human FAF1.
(A) Domain structure of human FAF1 and associated truncations, as in Figure 5D. (B) As in Figure 3—figure supplement 2, the indicated His-tagged bait proteins (14His-Smt3FAF1 or 14His-Smt3UFD1-NPL4) …
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Figure 5—figure supplement 3—source data 1
Source data for Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig5-figsupp3-data1-v2.pdf
Figure 6 with 5 supplements
Partial redundancy between UBXN7 and FAF1 for CMG helicase disassembly during S-phase and mitosis in mouse ES cells.
(A) CMG was isolated from extracts of mouse embryonic stem cells (mES) with the indicated genotypes, by immunoprecipitation of GFP-tagged SLD5 subunit of the helicase. (B) Equivalent experiment in …
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig6-data1-v2.pdf
Figure 6—figure supplement 1
Sequence alignment of human and mouse orthologues of p97, NPL4, and UFD1.
(A) Human and mouse orthologues of p97 were aligned with Clustal Omega software and the output viewed with MView (Madeira et al., 2019). (B, C) Similar analysis for NPL4 and UFD1.
Figure 6—figure supplement 2
Sequence alignment of human and mouse orthologues of FAF1, FAF2, and UBXN7.
(A) Human and mouse orthologues of FAF1 were aligned with Clustal Omega software and the output viewed with MView (Madeira et al., 2019). (B, C) Similar analysis for FAF2 and UBXN7.
Figure 6—figure supplement 3
The deletion of the Faf1, Ubxn7, and Faf2 genes by CRISPR-Cas9.
(A–C) Loci and corresponding guide RNAs (gRNAs) that were used to target the D10A ‘nickase’ mutant of Cas9 to the Faf1, Ubxn7, and Faf2 loci in mouse ES cells. See Materials and methods for further …
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Figure 6—figure supplement 3—source data 1
Source data for Figure 6—figure supplement 3.
- https://cdn.elifesciences.org/articles/76763/elife-76763-fig6-figsupp3-data1-v2.pdf
Figure 6—figure supplement 4
Rescue of FAF1∆ UBXN7∆ mouse ES cells by expression of human FAF1 or UBXN7.
(A) Scheme for expressing human FAF1 or UBXN7 from the CAG promoter at the Rosa26 locus in mouse ES cells. A donor vector was integrated as shown, in GFP-SLD5 UBXN7∆ FAF1∆ cells. (B) Three …
Figure 6—figure supplement 5
Mitotic CMG disassembly is blocked in mES cells that lack TRAIP.
TRAIP∆ cells expressing GFP-SLD5 from the endogenous Gins4 (Sld5) locus were grown as in Figure 6C and monitored by time-lapse video analysis. Three examples are shown of mitotic entry following …
Figure 7
Cells lacking FAF1 and UBXN7 are sensitive to global inhibition of cullin ligase activity.
(A) Cells of the indicated genotypes were treated as shown and then grown on 10 cm plates for 6 days. Surviving colonies were fixed and stained with crystal violet. (B) Viability of cells treated …
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/76763/elife-76763-transrepform1-v2.pdf
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Supplementary file 1
Reagents and resources used in this study.
- https://cdn.elifesciences.org/articles/76763/elife-76763-supp1-v2.docx
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Supplementary file 2
Plasmids generated in this study.
- https://cdn.elifesciences.org/articles/76763/elife-76763-supp2-v2.docx
