1. Biochemistry and Chemical Biology
  2. Cell Biology

The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP

  1. Maria Körner
  2. Susanne R Meyer
  3. Gabriella Marincola
  4. Maximilian J Kern
  5. Clemens Grimm
  6. Christina Schuelein-Voelk
  7. Utz Fischer
  8. Kay Hofmann
  9. Alexander Buchberger  Is a corresponding author
  1. University of Würzburg, Biocenter, Chair of Biochemistry I, Germany
  2. Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Germany
  3. Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Germany
  4. Institute of Genetics, University of Cologne, Germany
https://doi.org/10.7554/eLife.92409
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Cite this article
  1. Maria Körner
  2. Susanne R Meyer
  3. Gabriella Marincola
  4. Maximilian J Kern
  5. Clemens Grimm
  6. Christina Schuelein-Voelk
  7. Utz Fischer
  8. Kay Hofmann
  9. Alexander Buchberger
(2023)
The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP
eLife 12:e92409.
https://doi.org/10.7554/eLife.92409
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  3. Download .RIS
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
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VCF1/2 bind to p97.

(A) Schematic overview of human VCF1 and VCF2 isoforms isolated in a yeast two-hybrid screen. Relevant amino acid residue numbers are shown, and conserved N- and C-terminal sequence motifs are …

Figure 1—source data 1

Related to Figure 1D–F.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig1-data1-v2.zip
Figure 1—figure supplement 1
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Evolutionary conservation of FAM104 proteins.

(A) CLANS diagram of the FAM104 family. The sequence relationship between different FAM104-related proteins is visualized by similarity-based clustering using the CLANS software. Each point …

Figure 2 with 1 supplement
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FAM104 proteins bind to p97 via their C-terminal helix.

(A) Schematic overview of the C-terminal truncations of VCF1 isoform 5 used for yeast two-hybrid analysis. Labeling as in Figure 1A. (B) Yeast two-hybrid analysis of p97 binding to the C-terminally …

Figure 2—source data 1

Related to Figure 2C.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig2-data1-v2.zip
Figure 2—source data 2

Related to Figure 2D, E, and H.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig2-data2-v2.zip
Figure 2—figure supplement 1
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The p97 binding mode is conserved in invertebrate FAM104 proteins.

AlphaFold Multimer model of the C-terminal alpha-helix of Drosophila melanogaster CG14229 (purple) superimposed on the model of VCF1 (turquoise) bound to the N domain of p97 (green) shown in Figure …

Figure 3 with 1 supplement
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VCF1/2 form complexes with p97 and several p97 cofactors in cells.

(A) HEK293T cells ectopically expressing the indicated N-terminally FLAG-epitope-tagged wildtype or C-terminally truncated (Cdel26) VCF1/2 proteins were subjected to anti-FLAG immunoprecipitation …

Figure 3—source data 1

Related to Figure 3A.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data1-v2.zip
Figure 3—source data 2

Related to Figure 3B.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data2-v2.zip
Figure 3—source data 3

Related to Figure 3C.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data3-v2.zip
Figure 3—figure supplement 1
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VCF1/2 associate with several p97-cofactor complexes.

(A) HEK293T cells ectopically expressing the indicated N-terminally FLAG-epitope-tagged VCF1/2 proteins or control cells were subjected to anti-FLAG immunoprecipitation (IP). Input and IP samples …

Figure 3—figure supplement 1—source data 1

Related to Figure 3—figure supplement 1A–C.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-figsupp1-data1-v2.zip
Figure 4 with 1 supplement
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Ectopic expression of VCF1/2 increases nuclear p97 levels.

(A) HeLa cells ectopically expressing full-length, N-terminally FLAG epitope-tagged VCF isoforms 1, 2, and 5, VCF2 isoform 3, and empty vector control, respectively, were analyzed by confocal …

Figure 4—source data 1

Related to Figure 4C and D.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig4-data1-v2.zip
Figure 4—figure supplement 1
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VCF1/2 promote the nuclear localization of p97.

(A) Comparison of the images from Figure 4A and B for full-length VCF1 isoform 1 and its classical nuclear localization signal (cNLS)-deleted variant. The images were processed for higher signal …

Figure 5 with 1 supplement
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Ectopic expression of VCF1 isoforms 1 and 2 promotes the association of p97 with chromatin.

(A) HEK293T cells were transfected with plasmids encoding the indicated N-terminally FLAG-epitope-tagged VCF1 proteins or with empty vector (control). Total protein extracts were prepared by direct …

Figure 5—source data 1

Related to Figure 5A and B.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig5-data1-v2.zip
Figure 5—figure supplement 1
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Control fractionations of cells ectopically expressing Cdel26 and delNLS variants of VCF1 isoforms 1 and 2.

(A) Fractionation into cytoplasmic, nucleoplasmic, and chromatin fractions of lysates from HEK293T cells ectopically expressing the FLAG-tagged, p97 binding-deficient Cdel26 variants of VCF1 …

Figure 5—figure supplement 1—source data 1

Related to Figure 5—figure supplement 1A and B.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
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Deletion of VCF1/2 reduces nuclear p97 levels.

(A) Control, VCF1 single-knockout (KO) and VCF1/2 double-knockout (DKO) HeLa cell pools were analyzed by confocal immunofluorescence microscopy using an antibody against endogenous p97. Scale bars, …

Figure 6—source data 1

Related to Figure 6C, D, and F.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig6-data1-v2.zip
Figure 6—figure supplement 1
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De(p)letion of VCF1/2 reduces nuclear p97 levels.

(A) Z stack images of control- or VCF1-depleted HeLa cells stained for endogenous p97. Shown are maximum intensity projections of stacks consisting of eight z planes with 2.5 µm distance. Scale …

Figure 6—figure supplement 1—source data 1

Related to Figure 6—figure supplement 1C and D.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig6-figsupp1-data1-v2.zip
Figure 7 with 2 supplements
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Deletion of VCF1/2 causes reduced growth and hypersensitivity to p97 inhibition.

(A) HeLa VCF1/2 double-knockout (DKO) and control cells were seeded in 96-well plates, cultivated for 8 d, stained with Hoechst dye and automatically counted using a high-content microscopy …

Figure 7—source data 1

Related to Figure 7A–D and F.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig7-data1-v2.zip
Figure 7—figure supplement 1
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Overexpression of VCF1 isoform 1 causes moderate growth defects.

HEK293T cells ectopically expressing N-terminally FLAG epitope-tagged VCF1 isoform 1 (VCF1 iso1 o/e) or empty vector control, respectively, were seeded in 96-well plates, grown for 24 hr in the …

Figure 7—figure supplement 1—source data 1

Related to Figure 7—figure supplement 1.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig7-figsupp1-data1-v2.zip
Figure 7—figure supplement 2
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VCF1 expression levels in VCF1 and VCF1/2 knockout cell pools.

Steady-state VCF1 mRNA levels of control, VCF1 single-knockout (KO), and VCF1/2 double-knockout (DKO) HeLa cell pools were determined by qRT-PCR using primer pairs specifically amplifying isoforms 1 …

Figure 7—figure supplement 2—source data 1

Related to Figure 7—figure supplement 2.

https://cdn.elifesciences.org/articles/92409/elife-92409-fig7-figsupp2-data1-v2.zip
Author response image 1
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Fractionation of cell lysates after ectopic expression or deletion of VCF1/2.

(A) HEK293T cells ectopically expressing the indicated VCF1 isoforms were processed to cytoplasmic, soluble nuclear (nucleoplasmic) and chromatin fractions and analyzed by immunoblotting as …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)XL1 BlueStratageneCat# 200249
Strain, strain background (E. coli)XL10 GoldAgilentCat# 200516-4
Strain, strain background (E. coli)BL21(DE3) pRILAgilentCat# 230245
Strain, strain background (Saccharomyces cerevisiae)PJ69-4AJames et al., 1996N/A
Cell line (Homo sapiens)HeLaATCCCCL-2
Cell line (H. sapiens)HEK293TATCCCRL-3216
Cell line (H. sapiens)HeLa VCF1 knockout cell poolThis studyN/ASee ‘Materials and methods,’ section ‘Mammalian cell culture’
Cell line (H. sapiens)HeLa VCF1/2 double-knockout cell poolThis studyN/A
Cell line (H. sapiens)HeLa non-human-target control cell poolThis studyN/A
Cell line (H. sapiens)HeLa pINDUCER20 control cell poolThis studyN/A
Cell line (H. sapiens)HeLa pINDUCER20 p97 wildtype cell poolThis studyN/A
Cell line (H. sapiens)HeLa pINDUCER20 SV40NLS-p97 cell poolThis studyN/A
Cell line (H. sapiens)HEK293T VCF1 knockout cell poolThis studyN/A
Cell line (H. sapiens)HEK293T VCF1/2 double-knockout cell poolThis studyN/A
Cell line (H. sapiens)HEK293T non-human-target control cell poolThis studyN/A
AntibodyAnti-alpha-tubulin (mouse monoclonal)Sigma-AldrichCat# T5168, RRID:AB_477579WB 1:2500
AntibodyAnti-GAL4-TA (mouse monoclonal)Santa CruzSc-1663WB 1:500
AntibodyAnti-FLAG (rabbit polyclonal)Thermo Fisher ScientificCat# PA1-984B, RRID:AB_347227WB 1:2000
AntibodyAnti-FLAG (mouse monoclonal)Sigma-AldrichCat# F1804WB 1:2000
IF 1:200
AntibodyAnti-VCP (rabbit polyclonal)Bethyl LaboratoriesCat# A300-589A, RRID:AB_495512WB 1:5000
IF 1:300
AntibodyAnti-VCP (mouse monoclonal)Santa CruzCat# sc-57492, RRID:AB_793927IF 1:50
AntibodyAnti-FAF1 (rabbit polyclonal)Max Planck Institute of Biochemistry, animal houseAB65WB 1:10,000
AntibodyAnti-UBXN7 (rabbit polyclonal)Sigma-AldrichCat# HPA049442WB 1:1000
AntibodyAnti-NPL4 (rabbit polyclonal)Sigma-AldrichCat# HPA021560WB 1:1000
AntibodyAnti-UFD1 (rabbit polyclonal)ProteintechCat# 10615WB 1:2000
AntibodyAnti-Ubiquityl-Histone H2B (rabbit monoclonal)Cell SignalingCat# 5546WB 1:1000
AntibodyAnti-UBXN2B (rabbit polyclonal)Lee et al., 2018N/AWB 1:1000
AntibodyAnti-UBXD1 (rabbit polyclonal)NovusNBP2-57653WB 1:1000
AntibodyAnti-MCM7 (rabbit polyclonal)Proteintech11225-1-APWB 1:1000
AntibodyAnti-MYC (rabbit monoclonal)Biocenter, University of Würzburg, made in-houseN/AWB 1:2000
AntibodyAnti-mouse IgG HRP (goat polyclonal)Dianova (Jackson ImmunoResearch)Cat# 115-035-003, RRID:AB_100152891:7500
AntibodyAnti-rabbit IgG HRP (goat polyclonal)Dianova (Jackson ImmunoResearch)Cat# 111-035-045, RRID:AB_23379381:7500
AntibodyAlexa Fluor 488 goat anti-rabbit IgG (H+L)Thermo Fisher ScientificCat# A-11070, RRID:AB_1421341:500
AntibodyAlexa Fluor 488 goat anti-mouse IgG (H+L)Thermo Fisher ScientificCat# A-11017, RRID:AB_1431601:500
AntibodyAlexa Fluor 594 goat anti-rabbit IgG (H+L)Thermo Fisher ScientificCat# A-11072, RRID:AB_1420571:500
AntibodyAlexa Fluor 594 goat anti-mouse IgG (H+L)Thermo Fisher ScientificCat# A-11020, RRID:AB_1419741:500
Recombinant DNA reagentpsPAX2AddgeneCat# 12260
Recombinant DNA reagentpMD2.GAddgeneCat# 12259
Recombinant DNA reagentpCMV-Tag2BAgilentCat# 211172
Recombinant DNA reagentpCMV VCF1 isoform 1This studypAB2225See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentpCMV VCF1 isoform 2This studypAB2208
Recombinant DNA reagentpCMV VCF1 isoform 5This studypAB2223
Recombinant DNA reagentpCMV VCF2 isoform 3This studypAB2277
Recombinant DNA reagentpCMV VCF1 isoform 1 Cdel26This studypAB2280
Recombinant DNA reagentpCMV VCF1 isoform 2 Cdel26This studypAB2275
Recombinant DNA reagentpCMV VCF1 isoform 5 Cdel26This studypAB2276
Recombinant DNA reagentpCMV VCF1 isoform 1 delNLSThis studypAB2230
Recombinant DNA reagentpCMV VCF1 isoform 2 delNLSThis studypAB2231
Recombinant DNA reagentpCMV VCF1 isoform 5 delNLSThis studypAB2227
Recombinant DNA reagentpGEX-4T1CytivaCat# 28954549
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 1This studypAB2212See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 2This studypAB2195
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 5This studypAB2213
Recombinant DNA reagentpGEX-4T1 VCF2 isoform 3This studypAB2298
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 1 Cdel26This studypAB2295
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 2 Cdel26This studypAB2296
Recombinant DNA reagentpGEX-4T1 VCF1 isoform 5 Cdel26This studypAB2297
Recombinant DNA reagentpGEX-4T1 VCF2 isoform 3 Cdel26This studypAB2299
Recombinant DNA reagentpGEX-4T1 VCF1 isof. 5 NL->AAThis studypAB3163
Recombinant DNA reagentpGEX-4T1 VCF1 isof. 5 NL->RRThis studypAB3162
Recombinant DNA reagentmini-pRSETAPerrett et al., 1999N/A
Recombinant DNA reagentmini-pRSETA UBXN7This studypAB2008See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentmini-pRSETA p47Allen et al., 2006pAB356
Recombinant DNA reagentpQE30 FAF1Jensen et al., 2001N/A
Recombinant DNA reagentpET28a(+) UFD1Fernández-Sáiz and Buchberger, 2010pAB425
Recombinant DNA reagentpET21d NPL4Fernández-Sáiz and Buchberger, 2010pAB1340
Recombinant DNA reagentpProExHT p97Fernández-Sáiz and Buchberger, 2010pAB1312
Recombinant DNA reagentpProExHT p97 N domainFernández-Sáiz and Buchberger, 2010pAB1342
Recombinant DNA reagentpProExHT p97 ND1Fernández-Sáiz and Buchberger, 2010pAB1343
Recombinant DNA reagentpProExHT p97 ΔNRothballer et al., 2007pAB749
Recombinant DNA reagentpGAD-C1James et al., 1996N/A
Recombinant DNA reagentpGAD-C1 VCF1 isoform 1This studypAB2217See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentpGAD-C1 VCF1 isoform 2This studypAB2205
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5This studypAB2215
Recombinant DNA reagentpGAD-C1 VCF2 isoform 3This studypAB2233
Recombinant DNA reagentpGBDUJames et al., 1996N/A
Recombinant DNA reagentpGBDU p97This studypAB1184See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5 Cdel4This studypAB2312
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5 Cdel7This studypAB2313
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5 Cdel13This studypAB2314
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5 Cdel18This studypAB2315
Recombinant DNA reagentpGAD-C1 VCF1 isoform 5 Cdel26This studypAB2235
Recombinant DNA reagentpINDUCER20Meerbrey et al., 2011pAB3012
Recombinant DNA reagentpINDUCER20 p97 wildtypeThis studypAB3041See ‘Materials and methods,’ section ‘Plasmids’
Recombinant DNA reagentpINDUCER20 SV40NLS-p97This studypAB3261
Recombinant DNA reagentpLentiCRISPRv2AddgeneCat# 52961
Recombinant DNA reagentNon-human control sgRNAs in pLentiCRISPRv2Manuel Kaulich, University of FrankfurtN/A
Recombinant DNA reagentVCF1 sgRNAs in pLentiCRISPRv2Manuel Kaulich, University of FrankfurtN/A
Recombinant DNA reagentVCF2 sgRNAs in pLentiCRISPRv2Manuel Kaulich, University of FrankfurtN/A
Sequence-based reagentON-TARGETplus human FAM104A siRNA-SMARTpoolDharmaconCat# L-015015-02-0005
Sequence-based reagentON-TARGETplus human UBXN2B siRNA-SMARTpoolDharmaconCat# L-025945-01-0005
Sequence-based reagentON-TARGETplus non-targeting poolDharmaconCat# D-001810-10-05
Sequence-based reagentFAM104A_1_fwdThis paperqPCR primerCTCCGTCCCAGGAAAAGGAG
Sequence-based reagentFAM104A_1_revThis paperqPCR primerAGGGTTTCTGCTACTTCTTTTGG
Sequence-based reagentFAM104A_2_fwdThis paperqPCR primerTGGCAACGAAGAAGACAACC
Sequence-based reagentFAM104A_2_revThis paperqPCR primerTCACTGCCTGAAGACTCTGTG
Sequence-based reagentHPRT_fwdThis paperqPCR primerTGGACAGGACTGAACGTCTTG
Sequence-based reagentHPRT_revThis paperqPCR primerCAGTCATAGGAATGGATCTATCAC
Sequence-based reagentPBGD_fwdThis paperqPCR primerCCCTGGAGAAGAATGAAGTGG
Sequence-based reagentPBGD_revThis paperqPCR primerTTCTCTGGCAGGGTTTCTAGG
Sequence-based reagentFam104A1-KO-2-R_79This papergRNACGTAGCTTCCATCCGCCAGC
Sequence-based reagentFam104A1-KO-3-R_38This papergRNACCTCGGGCCTTGGCTCTCGC
Sequence-based reagentFam104A2-KO-1-R_190This papergRNATGTCCGGGCTATTGATGCTG
Sequence-based reagentFam104A2-KO-2-R_170This papergRNAACCGCGCAGAACGCTTTGTT
Sequence-based reagentFam104A3-KO-1-R_172This papergRNACTCCGCGAAGAGAGGGAACA
Sequence-based reagentFam104A3-KO-3-R_70This papergRNACCGAAACACAACCCCCTCTG
Sequence-based reagentFam104B1-KO-1-R_187This papergRNACTGTATCTTGAGAATCCTGA
Sequence-based reagentFam104B1-KO-2-R_18This papergRNACTTGCTCTCTCTGGGATATT
Sequence-based reagentFam104B1-KO-3-R_139This papergRNACATTAATATCCCAGAGAGAG
Sequence-based reagentFam104B2-KO-1-R_19This papergRNAGATTGTTACTGAACCCGATG
Sequence-based reagentFam104B2-KO-2-R_85This papergRNAGTTTTCATGGAGTGATAATG
Sequence-based reagentNon-human-target-309-KO-1-R_156This papergRNAAACATGACGTTCAAGATTGG
Sequence-based reagentNon-human-target-365-KO-5-R_5This papergRNAACCACTGTTCTACGCGCAGG
Sequence-based reagentNon-human-target-415-KO-2-R_24This papergRNATTGAACGGGCCGCGGAAGCG
Sequence-based reagentNon-human-target-42-KO-15-R_115This papergRNACCCGCATGACACCGTCACTT
Peptide, recombinant proteinBiotin- CQGLYFHINQTLREAHFHSLQHRG-COOHPANATecs GmbHN/ASee ‘Materials and methods,’ section ‘In vitro binding assays’
Commercial assay or kitPierce BCA Protein Assay KitThermo Fisher ScientificCat# 23225
Commercial assay or kitQuikChange XLII Mutagenesis KitAgilentCat# 200521
Commercial assay or kitTranscriptor High Fidelity cDNA synthesis kitRocheCat# 5081963001
Commercial assay or kitNucleoSpin RNA kitMacherey-NagelCat# REF 740955
Chemical compound, drugCamptothecinSelleckchemNSC-100880
Chemical compound, drugCB-5083SelleckchemCat# S8101
Commercial assay or kitClarity Western ECL SubstrateBio-RadCat# 1705061
Chemical compound, drugcOmplete, EDTA-free Protease Inhibitor CocktailRocheCat# 04693132001
Chemical compound, drugGlutathione Sepharose 4 Fast FlowCytivaCat# 17513202
Chemical compound, drugNi-NTA AgaroseQIAGENCat# 30230
Chemical compound, drugOpti-MEMThermo Fisher ScientificCat# 31985602
Chemical compound, drugPolybreneSanta CruzCat# sc-134220
Chemical compound, drugPolyethylenimine (PEI)PolysciencesCat# 23966-1
Chemical compound, drugProLong Glass Antifade MountantThermo Fisher ScientificCat# P36980
Chemical compound, drugAnti FLAG-M2 affinity agarose beadsSigma-AldrichCat# A2220
Chemical compound, drugOligofectamineThermo Fisher ScientificCat# 12252011
Chemical compound, drugBenzonase NucleaseSigma-AldrichCat# 70664
Chemical compound, drugPowerUp SYBR Green Master MixThermo FisherCat# A25741
Chemical compound, drugProtein G Sepharose 4 fast flowCytivaCat# 17-5132-01
Software, algorithmFijihttp://fiji.scRRID:SCR_002285
Software, algorithmImage Lab SoftwareBio-RadRRID:SCR_014210
Software, algorithmCellProfilerhttps://cellprofiler.org/RRID:SCR_007358
Software, algorithmGraphPad Prismhttps://www.graphpad.com/scientific-software/prism/RRID:SCR_002798
Software, algorithmQuantStudio Design & Analysis Softwarehttps://www.thermofisher.com/de/de/home/global/forms/life-science/quantstudio-3-5-software.html

Additional files

Supplementary file 1

Summary of VCF1/2 yeast two-hybrid hits.

https://cdn.elifesciences.org/articles/92409/elife-92409-supp1-v2.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/92409/elife-92409-mdarchecklist1-v2.pdf

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