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pet15b p97 strep  (Addgene inc)


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    Addgene inc pet15b p97 strep
    Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by <t>p97</t> to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.
    Pet15b P97 Strep, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation."

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.09.020

    Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.
    Figure Legend Snippet: Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Stable Transfection, Isolation, Western Blot, Inhibition, Blocking Assay, Radioactivity, Autoradiography, Control, Expressing

    Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.
    Figure Legend Snippet: Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

    Techniques Used: Ubiquitin Proteomics, Binding Assay, Western Blot, Knock-Out, Immunoprecipitation, Control, Autoradiography, Pulse Chase, Knockdown, Isolation, Mass Spectrometry, Labeling, MTS Assay

    Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.
    Figure Legend Snippet: Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

    Techniques Used: Binding Assay, Mutagenesis, Generated, Incubation, Isolation, Western Blot, Residue, Variant Assay, Irradiation

    Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.
    Figure Legend Snippet: Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

    Techniques Used: Ubiquitin Proteomics, Incubation, Immunoprecipitation, Size-exclusion Chromatography, Migration, Binding Assay

    Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.
    Figure Legend Snippet: Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

    Techniques Used: Western Blot, Incubation, Binding Assay

    Related Articles

    Recombinant:

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DyLight 800 maleimide Thermo Fisher 46621 ATPgS Sigma A1388 Zeba Spin Desalting column Thermo Fisher 89882 Lipofectamine RNAiMAX transfection reagent Thermo Fisher 13778150 Recombinant p37 This study N/A Recombinant p37DSEP (D140-206) This study N/A Recombinant p37SHPmut (F215A/E218A/QKL220-222AAA) This study N/A Recombinant p37DN (D2-81) This study N/A Recombinant p37DUBX (D251-331) This study N/A Recombinant p37 F89pBpA This study N/A Recombinant p37 L182pBpA This study N/A Recombinant His-p97 This study N/A Recombinant p97-Strep-His E314pBpA This study N/A Recombinant p97-His D592pBpA This study N/A Recombinant His-SDS22-PP1g-I3 This study N/A Recombinant His-I3 This study N/A Recombinant His-I3 (V41A/W43A) This study N/A Recombinant SDS22-PP1g This study N/A Recombinant SDS22-PP1g-Eos-I3 This study N/A Recombinant His-NIPP1 Beullens et al., 2000 N/A Recombinant His-diUb-GFP Blythe et al., 2017 N/A Recombinant Ufd1-Npl4 Meyer et al., 2002 N/A Critical Commercial Assays CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat. no. G3580 Deposited Data Original unprocessed gels, blots, and microscopy images used in this manuscript This study; Mendeley data https://doi.org/10.17632/ hfsb92kdh5.1 Mass spectrometry label-free quantification data: PP1g-specific interactors in wild-type HeLa cells versus p47 knockout cells with additional depletion of p37 and UBXN2A This study; PRIDE repository dataset identifier PXD010750 https://www.ebi.ac.uk/pride/ archive/ Experimental Models: Cell Lines HEK293 FlpIn T-REx p97 (wt)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p97 (E578Q)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p47-strep-HA This study N/A HEK293 FlpIn T-REx p37-strep-HA This study N/A HEK293 FlpIn T-REx UBXN2A-strep-HA This study N/A HEK293 FlpIn T-REx UBXN11-strep-HA This study N/A HEK293 FlpIn T-REx Ufd1-strep-HA This study N/A HeLa Kyoto RRID CVCL_1922 HeLa FlpIn T-REx GFP-strep-HA This study N/A HeLa FlpIn T-REx PP1g-strep This study N/A HeLa p47 knockout This study N/A Sf9 Invitrogen 11496015 Oligonucleotides siInhibitor-3 s1: CUGCUGTAUUUAUGAGAAATT Microsynth N/A siInhibitor-3 s2: GUAGAAUGGACAAGUGACATT Microsynth N/A sip97 s1: GGAGUUCAAAGUGGUGGAAACAGAUTT Microsynth N/A sip97 s2: AAGUAGGGUAUGAUGACAUUGTT Microsynth N/A (Continued on next page) e2 Molecular Cell 72, 1–12.e1–e6, November 15, 2018 .. REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/ .. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hemmo Meyer (hemmo.meyer@uni-due.de).

    CRISPR:

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DyLight 800 maleimide Thermo Fisher 46621 ATPgS Sigma A1388 Zeba Spin Desalting column Thermo Fisher 89882 Lipofectamine RNAiMAX transfection reagent Thermo Fisher 13778150 Recombinant p37 This study N/A Recombinant p37DSEP (D140-206) This study N/A Recombinant p37SHPmut (F215A/E218A/QKL220-222AAA) This study N/A Recombinant p37DN (D2-81) This study N/A Recombinant p37DUBX (D251-331) This study N/A Recombinant p37 F89pBpA This study N/A Recombinant p37 L182pBpA This study N/A Recombinant His-p97 This study N/A Recombinant p97-Strep-His E314pBpA This study N/A Recombinant p97-His D592pBpA This study N/A Recombinant His-SDS22-PP1g-I3 This study N/A Recombinant His-I3 This study N/A Recombinant His-I3 (V41A/W43A) This study N/A Recombinant SDS22-PP1g This study N/A Recombinant SDS22-PP1g-Eos-I3 This study N/A Recombinant His-NIPP1 Beullens et al., 2000 N/A Recombinant His-diUb-GFP Blythe et al., 2017 N/A Recombinant Ufd1-Npl4 Meyer et al., 2002 N/A Critical Commercial Assays CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat. no. G3580 Deposited Data Original unprocessed gels, blots, and microscopy images used in this manuscript This study; Mendeley data https://doi.org/10.17632/ hfsb92kdh5.1 Mass spectrometry label-free quantification data: PP1g-specific interactors in wild-type HeLa cells versus p47 knockout cells with additional depletion of p37 and UBXN2A This study; PRIDE repository dataset identifier PXD010750 https://www.ebi.ac.uk/pride/ archive/ Experimental Models: Cell Lines HEK293 FlpIn T-REx p97 (wt)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p97 (E578Q)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p47-strep-HA This study N/A HEK293 FlpIn T-REx p37-strep-HA This study N/A HEK293 FlpIn T-REx UBXN2A-strep-HA This study N/A HEK293 FlpIn T-REx UBXN11-strep-HA This study N/A HEK293 FlpIn T-REx Ufd1-strep-HA This study N/A HeLa Kyoto RRID CVCL_1922 HeLa FlpIn T-REx GFP-strep-HA This study N/A HeLa FlpIn T-REx PP1g-strep This study N/A HeLa p47 knockout This study N/A Sf9 Invitrogen 11496015 Oligonucleotides siInhibitor-3 s1: CUGCUGTAUUUAUGAGAAATT Microsynth N/A siInhibitor-3 s2: GUAGAAUGGACAAGUGACATT Microsynth N/A sip97 s1: GGAGUUCAAAGUGGUGGAAACAGAUTT Microsynth N/A sip97 s2: AAGUAGGGUAUGAUGACAUUGTT Microsynth N/A (Continued on next page) e2 Molecular Cell 72, 1–12.e1–e6, November 15, 2018 .. REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/ .. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hemmo Meyer (hemmo.meyer@uni-due.de).

    Plasmid Preparation:

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DyLight 800 maleimide Thermo Fisher 46621 ATPgS Sigma A1388 Zeba Spin Desalting column Thermo Fisher 89882 Lipofectamine RNAiMAX transfection reagent Thermo Fisher 13778150 Recombinant p37 This study N/A Recombinant p37DSEP (D140-206) This study N/A Recombinant p37SHPmut (F215A/E218A/QKL220-222AAA) This study N/A Recombinant p37DN (D2-81) This study N/A Recombinant p37DUBX (D251-331) This study N/A Recombinant p37 F89pBpA This study N/A Recombinant p37 L182pBpA This study N/A Recombinant His-p97 This study N/A Recombinant p97-Strep-His E314pBpA This study N/A Recombinant p97-His D592pBpA This study N/A Recombinant His-SDS22-PP1g-I3 This study N/A Recombinant His-I3 This study N/A Recombinant His-I3 (V41A/W43A) This study N/A Recombinant SDS22-PP1g This study N/A Recombinant SDS22-PP1g-Eos-I3 This study N/A Recombinant His-NIPP1 Beullens et al., 2000 N/A Recombinant His-diUb-GFP Blythe et al., 2017 N/A Recombinant Ufd1-Npl4 Meyer et al., 2002 N/A Critical Commercial Assays CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat. no. G3580 Deposited Data Original unprocessed gels, blots, and microscopy images used in this manuscript This study; Mendeley data https://doi.org/10.17632/ hfsb92kdh5.1 Mass spectrometry label-free quantification data: PP1g-specific interactors in wild-type HeLa cells versus p47 knockout cells with additional depletion of p37 and UBXN2A This study; PRIDE repository dataset identifier PXD010750 https://www.ebi.ac.uk/pride/ archive/ Experimental Models: Cell Lines HEK293 FlpIn T-REx p97 (wt)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p97 (E578Q)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p47-strep-HA This study N/A HEK293 FlpIn T-REx p37-strep-HA This study N/A HEK293 FlpIn T-REx UBXN2A-strep-HA This study N/A HEK293 FlpIn T-REx UBXN11-strep-HA This study N/A HEK293 FlpIn T-REx Ufd1-strep-HA This study N/A HeLa Kyoto RRID CVCL_1922 HeLa FlpIn T-REx GFP-strep-HA This study N/A HeLa FlpIn T-REx PP1g-strep This study N/A HeLa p47 knockout This study N/A Sf9 Invitrogen 11496015 Oligonucleotides siInhibitor-3 s1: CUGCUGTAUUUAUGAGAAATT Microsynth N/A siInhibitor-3 s2: GUAGAAUGGACAAGUGACATT Microsynth N/A sip97 s1: GGAGUUCAAAGUGGUGGAAACAGAUTT Microsynth N/A sip97 s2: AAGUAGGGUAUGAUGACAUUGTT Microsynth N/A (Continued on next page) e2 Molecular Cell 72, 1–12.e1–e6, November 15, 2018 .. REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/ .. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hemmo Meyer (hemmo.meyer@uni-due.de).

    Software:

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DyLight 800 maleimide Thermo Fisher 46621 ATPgS Sigma A1388 Zeba Spin Desalting column Thermo Fisher 89882 Lipofectamine RNAiMAX transfection reagent Thermo Fisher 13778150 Recombinant p37 This study N/A Recombinant p37DSEP (D140-206) This study N/A Recombinant p37SHPmut (F215A/E218A/QKL220-222AAA) This study N/A Recombinant p37DN (D2-81) This study N/A Recombinant p37DUBX (D251-331) This study N/A Recombinant p37 F89pBpA This study N/A Recombinant p37 L182pBpA This study N/A Recombinant His-p97 This study N/A Recombinant p97-Strep-His E314pBpA This study N/A Recombinant p97-His D592pBpA This study N/A Recombinant His-SDS22-PP1g-I3 This study N/A Recombinant His-I3 This study N/A Recombinant His-I3 (V41A/W43A) This study N/A Recombinant SDS22-PP1g This study N/A Recombinant SDS22-PP1g-Eos-I3 This study N/A Recombinant His-NIPP1 Beullens et al., 2000 N/A Recombinant His-diUb-GFP Blythe et al., 2017 N/A Recombinant Ufd1-Npl4 Meyer et al., 2002 N/A Critical Commercial Assays CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Promega Cat. no. G3580 Deposited Data Original unprocessed gels, blots, and microscopy images used in this manuscript This study; Mendeley data https://doi.org/10.17632/ hfsb92kdh5.1 Mass spectrometry label-free quantification data: PP1g-specific interactors in wild-type HeLa cells versus p47 knockout cells with additional depletion of p37 and UBXN2A This study; PRIDE repository dataset identifier PXD010750 https://www.ebi.ac.uk/pride/ archive/ Experimental Models: Cell Lines HEK293 FlpIn T-REx p97 (wt)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p97 (E578Q)-myc/strep Ritz et al., 2011 N/A HEK293 FlpIn T-REx p47-strep-HA This study N/A HEK293 FlpIn T-REx p37-strep-HA This study N/A HEK293 FlpIn T-REx UBXN2A-strep-HA This study N/A HEK293 FlpIn T-REx UBXN11-strep-HA This study N/A HEK293 FlpIn T-REx Ufd1-strep-HA This study N/A HeLa Kyoto RRID CVCL_1922 HeLa FlpIn T-REx GFP-strep-HA This study N/A HeLa FlpIn T-REx PP1g-strep This study N/A HeLa p47 knockout This study N/A Sf9 Invitrogen 11496015 Oligonucleotides siInhibitor-3 s1: CUGCUGTAUUUAUGAGAAATT Microsynth N/A siInhibitor-3 s2: GUAGAAUGGACAAGUGACATT Microsynth N/A sip97 s1: GGAGUUCAAAGUGGUGGAAACAGAUTT Microsynth N/A sip97 s2: AAGUAGGGUAUGAUGACAUUGTT Microsynth N/A (Continued on next page) e2 Molecular Cell 72, 1–12.e1–e6, November 15, 2018 .. REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/ .. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hemmo Meyer (hemmo.meyer@uni-due.de).



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    Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by <t>p97</t> to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.
    E314amb, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Activity Assay, Binding Assay, Mutagenesis, Stable Transfection, Isolation, Western Blot, Inhibition, Blocking Assay, Radioactivity, Autoradiography, Control, Expressing

    Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Knock-Out, Immunoprecipitation, Control, Autoradiography, Pulse Chase, Knockdown, Isolation, Mass Spectrometry, Labeling, MTS Assay

    Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Isolation, Western Blot, Residue, Variant Assay, Irradiation

    Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Ubiquitin Proteomics, Incubation, Immunoprecipitation, Size-exclusion Chromatography, Migration, Binding Assay

    Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Western Blot, Incubation, Binding Assay

    Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Activity Assay, Binding Assay, Mutagenesis, Stable Transfection, Isolation, Western Blot, Inhibition, Blocking Assay, Radioactivity, Autoradiography, Control, Expressing

    Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Knock-Out, Immunoprecipitation, Control, Autoradiography, Pulse Chase, Knockdown, Isolation, Mass Spectrometry, Labeling, MTS Assay

    Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Isolation, Western Blot, Residue, Variant Assay, Irradiation

    Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Ubiquitin Proteomics, Incubation, Immunoprecipitation, Size-exclusion Chromatography, Migration, Binding Assay

    Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

    Journal: Molecular cell

    Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

    doi: 10.1016/j.molcel.2018.09.020

    Figure Lengend Snippet: Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

    Techniques: Western Blot, Incubation, Binding Assay