B ) A 39 bp fragment of ub - HS4 is sufficient to drive ectopic ub - HS formation . Fragments of ub - HS4 were integrated ectopically and ub - K48 ChIP - qPCR was performed for the endogenous ub - HS5 and the ectopic ub - HS4 fragments as depicted in ( A ) . contr . : control , empty YI128 - vector was integrated at LEU2 . Experiments were performed in cdc48 ‑ 6 background . Data represent means ± SD ( n = 2 ) . Experimental mapping and bioinformatic prediction identify a similar ub - HS - motif . Comparison between the experimentally mapped ub - HS4 F7 ( B ) and the consensus motif of all ub - HSs identified by the MEME software .

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B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2). Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software.


E ) Euc1 binds to the ub - HS - motif in a Y1H assay . Gal4 - AD - or Gal4 - AD - Euc1 - encoding plasmids were transformed into a Y1H reporter strain as described in ( D ) . Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3 - amino - triazole ( 3AT ) . Cells were grown at 30 ° C for 3 days .

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E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.


F ) Euc1 is required for formation of ub - HSs . ChIP with ub - K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR . Data represent means ± SD ( n = 4 ) .

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F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of K48 - linked ubiquitin chains were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of Euc1 were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of Euc1 were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


( C ) Euc1 can induce transactivation via its N - terminal 30 amino acids . Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig . 2D and serial dilutions were spotted on control or selective media to test HIS3 - activation . Cells were grown at 30 ° C for 3 days .

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(C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days.


( D ) Quantification of HIS3 mRNA levels from strains used in ( C ) . Cells were grown in liquid media with selection for the transformed plasmids ( SC - Leu ) , harvested in logarithmic growth phase and total mRNA was prepared . After reverse transcription , HIS3 mRNA levels were quantified using qPCR ( RT - qPCR ) , normalized first to ACT1 mRNA and then to the empty vector control strain . Data represent means ± SD ( n = 4 ) . p = 2 . 43 * 10 - 5 ( Student ' s t - test ) .

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(D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).


( A ) Euc1 interacts with SUMO pathway proteins in a yeast two - hybrid ( Y2H ) assay . A Y2H reporter strain ( PJ69 - 7a ) was transformed with Gal4 DNA - binding domain ( BD ) and Gal4 activation domain ( AD ) fusion constructs in the indicated combinations . AD - SUMO - GG can be conjugated to SUMOylation substrates , while AD - SUMO - AA is conjugation - deficient . Cells were spotted on control media or selective media ( - His ) and grown for 3 and 6 days , respectively .

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(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.


( B ) Euc1 is SUMOylated by Ubc9 , Siz1 and Siz2 on K231 . Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO ( top ) and Euc1 ( bottom ) . An up - shifted band corresponding to Euc1SUMO was detected with both antibodies . Asterisks denote non - specific bands .

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(B) Euc1 is SUMOylated by Ubc9, Siz1 and Siz2 on K231. Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up-shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non-specific bands.


( C ) Euc1 is predominantly monoSUMOylated on lysine 231 . Denaturing NiNTA - pulldowns ( NiNTA - PD ) with strains expressing His - tagged SUMO ( HisSUMO ) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter . Covalently SUMO - modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1 . Eluates were probed for SUMO to control for equal pull - down , Pgk1 was probed as input control . Euc1 - KR denotes the K231R mutation here and hereafter .

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(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.


( D ) Euc1 SUMOylation is required for ub - HS formation . Ub - K48 ChIP quantified by qPCR for the indicated strains . The ubx5 ∆ background was used to enhance the ubiquitin signal , similar results were obtained in a WT background . Data represent means ± SD ( n = 3 ) .

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(D) Euc1 SUMOylation is required for ub-HS formation. Ub-K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, similar results were obtained in a WT background. Data represent means ± SD (n = 3).


( E ) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation . Denaturing NiNTA - PDs as described for ( C ) but with strains expressing His - ubiquitin ( HisUbi ) and 3FLAGEuc1 under the control of the ADH promoter . WBs were developed with a FLAG antibody to probe for 3FLAGEuc1 , a ubiquitin blot served as PD - control and Dpm1 as input control . The slx8 - CD allele carries the C206S , C209S - mutations ( Xie et al , 2007 ) .

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(E) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA-PDs as described for (C) but with strains expressing His-ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD-control and Dpm1 as input control. The slx8-CD allele carries the C206S, C209S-mutations (Xie et al, 2007).


SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub - HSs . ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and 3HA - Slx5 in ( F ) and

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SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT


SUMOylated Euc1 is required to recruit Slx8 to ub - HSs . ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and Slx8 - 9myc in ( G )

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SUMOylated Euc1 is required to recruit Slx8 to ub-HSs. ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and Slx8-9myc in (G)


( A ) Euc181 - 183 binds to Slx5 in Y2H assays . Y2H assay performed as in Fig . 4A . Serial dilutions were spotted for each plasmid combination and cells were grown at 30 ° C for 2 days . The Slx5 - RING ∆ construct is deleted for the Slx5 C - terminus from beginning of the RING - domain ( ∆ aa 488 - 619 ) . See also Fig . EV3B .

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(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B.


( B ) Euc1 binds Slx5 in vitro . Recombinant purified GST or GSTEuc1 were used in GST pull - down assays to co - precipitate recombinant 6HisSlx5 .

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(B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5.


( C ) Euc1 binds to Slx5 in vivo . Cell lysates from an euc1 ∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids ( under EUC1 promoter ) were subjected to immunoprecipitation using anti - FLAG beads . IP - eluates were probed by WB with Slx5 , SUMO and Euc1 antibodies , inputs were probed with Slx5 , FLAG and Dpm1 antibodies ( top to bottom ) . Note that the Euc1 - Slx5 interaction is independent of the Slx8 ligase activity ( slx8 - CD , lanes 5 - 8 ) .

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(C) Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti-FLAG beads. IP-eluates were probed by WB with Slx5, SUMO and Euc1 antibodies, inputs were probed with Slx5, FLAG and Dpm1 antibodies (top to bottom). Note that the Euc1-Slx5 interaction is independent of the Slx8 ligase activity (slx8-CD, lanes 5-8).


( D ) Euc1 Slx5 - binding mutants ( SBM1 , SBM2 ) affect Euc1 - Slx5 interaction in Y2H assays . AD - Euc181 - 183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD - Slx5 constructs as described in Fig . 4A . Mutations : SBM1 : aa 139 - 143 ENQKK > ANAAA , SBM2 : aa 162 - 165 KEVF > AAAA . Serial dilutions were spotted and cells were grown at 30 ° C for 2 days . See Appendix Fig . S6A for expression levels .

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(D) Euc1 Slx5-binding mutants (SBM1, SBM2) affect Euc1-Slx5 interaction in Y2H assays. AD-Euc181-183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD-Slx5 constructs as described in Fig. 4A. Mutations: SBM1: aa 139-143 ENQKK>ANAAA, SBM2: aa 162-165 KEVF>AAAA. Serial dilutions were spotted and cells were grown at 30°C for 2 days. See Appendix Fig. S6A for expression levels.


( E ) Euc1 - SBM constructs show defective Slx5 binding in vivo . Mutations described in ( D ) were introduced into full - length Euc1 constructs ( with or without the K231R mutation ) and FLAG IPs were performed as described in ( C ) . WB analysis showed strong Slx5 - binding defects for the SBM1 / SBM2 and SBM1 + 2 constructs ( top panel , immunoprecipitated Slx5 ) . WBs were probed as in ( C ) .

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(E) Euc1-SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full-length Euc1 constructs (with or without the K231R mutation) and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5-binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C).


( F ) The Slx5 middle domain ( Slx5 - Md ) is required for interaction with Euc1 . Y2H assays with AD - Euc181 - 183 and BD - Slx5 constructs . Slx5 - Md : aa 201 - 335 , Slx5 - Md ∆ : ∆ aa 201 - 338 . Serial dilutions were spotted and cells were grown at 30 ° C for 4 days . See also Fig . EV3C .

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(F) The Slx5 middle domain (Slx5-Md) is required for interaction with Euc1. Y2H assays with AD-Euc181-183 and BD-Slx5 constructs. Slx5-Md: aa 201-335, Slx5-Md∆: ∆aa 201-338. Serial dilutions were spotted and cells were grown at 30°C for 4 days. See also Fig. EV3C.


( G ) Euc1 - SBM constructs show defective binding to the Slx5 - Md . Y2H assay with the indicated constructs as in ( D ) . See Appendix Fig . S6A for expression controls .

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(G) Euc1-SBM constructs show defective binding to the Slx5-Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig. S6A for expression controls.


( A ) Euc1 Slx5 - binding mutations impair Euc1 ubiquitylation . His - ubiquitin - modified proteins were enriched by denaturing NiNTA - PDs as described in Fig . 4E . 3FLAG - tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter . NiNTA - PD eluates were probed with FLAG and ubiquitin ( P4D1 ) antibodies . Whole cell lysates ( input ) were probed with FLAG and Dpm1 antibodies .

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(A) Euc1 Slx5-binding mutations impair Euc1 ubiquitylation. His-ubiquitin-modified proteins were enriched by denaturing NiNTA-PDs as described in Fig. 4E. 3FLAG-tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA-PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies.


( B ) The Slx5 - SIMs and Slx5 - Md are required for Euc1 ubiquitylation . NiNTA - PDs for His - ubiquitin as in ( A ) . All strains expressed wild - type 3FLAGEuc1 from plasmids under the ADH promoter and His - ubiquitin . Slx5 constructs were expressed from plasmids under control of the endogenous promoter . WBs were probed as described in ( A ) , Slx5 - levels were probed using an HA antibody . Asterisk denotes a non - specific band . Slx5 - SIM * : SIMs 1 - 4 were mutated as described ( Xie et al , 2010 ) , for SIM5 , aa 477 - 479 ( IIV ) were mutated to alanines . To avoid possible mislocalization by deletion of the Slx5 - Md , which overlaps with a putative NLS ( Westerbeck et al , 2014 ) , an N - terminal NLS was fused to all constructs . See Appendix Fig . S7C for HU complementation .

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(B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation.


( C ) Euc1 Slx5 - binding mutations ( SBM1 / SBM2 ) reduce / abolish ub - HSs and recruitment of Slx5 to ub - HSs . ChIP - qPCR for ub - K48 ( top ) or Slx5 ( using Slx5 antibody , bottom ) in euc1 ∆ ubx5 ∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter . Data represent means ± SD ( n = 3 ) . See also Appendix Fig . S7E - G .

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(C) Euc1 Slx5-binding mutations (SBM1/SBM2) reduce/abolish ub-HSs and recruitment of Slx5 to ub-HSs. ChIP-qPCR for ub-K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig. S7E-G.


( D ) Slx5 - SIMs and the Slx5 - Md are required for the formation of ub - HSs and recruitment of Slx5 . ChIP - qPCR for ub - K48 ( left ) or Slx5 ( anti - HA ChIP , right ) in ubx5 ∆ cells or slx5 ∆ ubx5 ∆ cells complemented with plasmids expressing the indicated Slx5 - constructs under control of the SLX5 promoter . Data represent means ± SD ( n = 2 ) . See also Appendix Fig . S7H .

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(D) Slx5-SIMs and the Slx5-Md are required for the formation of ub-HSs and recruitment of Slx5. ChIP-qPCR for ub-K48 (left) or Slx5 (anti-HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5-constructs under control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig. S7H.


( A ) Deletion of EUC1 does not lead to ub - HS adjacent gene deregulation , but rather widespread transcriptome changes . Total RNA isolated from WT and euc1 ∆ cells was polyA - enriched and sequenced ( RNAseq , triplicates ) . Significance testing was based on the Wald test , see Materials and Methods for details . See also Appendix Fig . S8A for quantification of selected transcripts by qPCR .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(A) Deletion of EUC1 does not lead to ub-HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA-enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig. S8A for quantification of selected transcripts by qPCR.


( B ) RNAseq transcriptome analysis of EUC1 overexpression as in ( A ) . ∆ euc1 cells with pGAL - EUC1 or pEUC1 - EUC1 ( control ) integrated at the URA3 - locus ( YIplac211 ) were grown to mid - log phase and 2 % galactose was added for 3h . See also Appendix Fig . S8E .

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(B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E.


( C ) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol . For ( C , D , F ) , serial dilutions of the indicated strains were spotted and grown on YPD control ( or selective media ) plates or under conditions as indicated . See also Fig . EV4A - B .

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(C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B.


( D ) EUC1 overexpression toxicity depends on DNA binding and transactivation . The indicated EUC1 alleles with endogenous or galactose - inducible promoters were integrated at the URA3 locus ( YIplac211 , euc1 ∆ background ) and spotted on glucose control or galactose plates to induce EUC1 overexpression . See also Appendix Fig . S9A - C .

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(D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C.


( E ) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding . ChIP - qPCR of Euc1 after 3 hours galactose induction . Note that IP / input ratios of Euc1 signals are shown , also for the control locus ( contr . , TOS1 promoter ) to highlight Euc1 binding at non - ub - HS sites . STI1 - CIN5 : intergenic region . TEC1 - us : upstream ( promoter ) region . Data represent means ± SD ( n = 3 ) . See also Fig . EV4C - D .

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(E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D.


( F ) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity . Experiment as in ( D ) , but with concomitant plasmid - borne overexpression ( ADH promoter ) of SLX5 and SLX8 ( bottom panels ) and on media selecting for SLX5 / SLX8 plasmids . See also Fig . EV4E and Appendix Fig . S9D .

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(F) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid-borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig. EV4E and Appendix Fig. S9D.


EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation ( A ) and in particular with members of the Rpd3L histone deacetylase complex ( B ) . For ( A - D ) , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation (A) and in particular with members of the Rpd3L histone deacetylase complex (B). For (A-D), serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( C ) Plasmid - borne EUC1 complements genetic interactions with Rpd3L subunits . Empty vector ( - ) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media .

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For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (C) Plasmid-borne EUC1 complements genetic interactions with Rpd3L subunits. Empty vector (-) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media.


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( D ) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L . rco1 ∆ and eaf3 ∆ ( Rpd3S ) show similar phenotypes when paired with sds3 ∆ ( Rpd3L ) as euc1 ∆ and are epistatic with euc1 ∆ .

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For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (D) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L. rco1∆ and eaf3∆ (Rpd3S) show similar phenotypes when paired with sds3∆ (Rpd3L) as euc1∆ and are epistatic with euc1∆.


( A ) Euc1 is recruited to ub - HSs upon mild heat stress ( 37 ° C , 90 min ) . ChIP - qPCR experiments with strains and conditions as indicated . Data represent means ± SD ( n = 4 ) , see Appendix Fig . S11B for statistical analysis .

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(A) Euc1 is recruited to ub-HSs upon mild heat stress (37°C, 90 min). ChIP-qPCR experiments with strains and conditions as indicated. Data represent means ± SD (n = 4), see Appendix Fig. S11B for statistical analysis.


The ability to form ub - hotspots is crucial for endogenous EUC1 function . The genetic interaction of EUC1 with SDS3 ( Rpd3L complex ) was rescued with plasmid - borne EUC1 - alleles and serial dilutions were spotted on selective media . ( - ) denotes empty vector .

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The ability to form ub-hotspots is crucial for endogenous EUC1 function. The genetic interaction of EUC1 with SDS3 (Rpd3L complex) was rescued with plasmid-borne EUC1-alleles and serial dilutions were spotted on selective media. (-) denotes empty vector.


SLX5 also shows negative genetic interactions with SDS3 upon heat stress . Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated . See also Appendix Fig . S12C - D . Note that all strains used in ( A - C ) contained an extra copy of MED11 ,

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SLX5 also shows negative genetic interactions with SDS3 upon heat stress. Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated. See also Appendix Fig. S12C-D. Note that all strains used in (A-C) contained an extra copy of MED11,


All ORFs for the sense non - AUG - dependent translation of the ( G4C2 ) n ~ 188 , DPR coding for ( GA ) , ( GP ) , ( GR ) can be detected and quantified using a filter - trap binding assay probing for the c - terminal HA tag of the C9 DPR reporter construct . The ( GA ) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct . n = 5 .

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All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.


The C9orf72 NRE non - AUG - dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models . The Dendra2 ( green ) c - terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES - mIFP - mIFP - NES ( NES - mIFP ) fusion protein cytoplasmic marker .

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The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.


C9 DPR reporter constructs show cell - type variability in the number of cells that are DPR positive and the overall DPR levels . Transient cotransfections of HEK293T cells robustly undergo G4C2 - Dendra2 non - AUG - dependent translation compared to NSC34 or rat primary cortical neurons . DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post - transfection only in cells expressing the co - transfected NES - mIFP construct . DPR fluorescent intensity was normalized to NES - mIFP fluorescent intensity to adjust for cell specific AUG - dependent translation levels ; represented as fold - change relative to HEK293T . HEK293T : n = 5 with m > 500 cells analyzed per n . NSC34 : n = 5 with m > 500 cells per n . Rat primary cortical neuron : n = 5 with m > 40 cells per n . ( * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 ) See also Figure EV1 - 2 .

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C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.


Quantitative fluorescent microscopy imaging shows a dose - dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator , staurosporine ( SSP ) . The C9 DPR reporter in the GP frame was utilized for these experiments . n = 4 .

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Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.


Filter trap binding assays using DPR - specific antibodies show all DPR levels are increased for the sense - coding C9 DPR reporter when NSC34 cells are challenged with stress - inducing compounds . These stress - inducing compounds broadly cover distinct or overlapping cellular pathways ( see Table EV2 ) Antibodies specific for the C9orf72 NRE DPRs , poly - GA , - GP , or - GR , were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs . n = 4 .

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Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.


Quantitative fluorescent microscopy imaging shows that NSC34 , rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress . In NES - mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress - inducing compounds . Fluorescent intensity was not normalized to NES - mIFP fluorescent levels . NSC34 : n = 10 with m > 500 cells analyzed per n . Cortical neuron : n = 6 with m > 40 cells per n . Endogenously expression of DPRs in iPS spinal motor neurons ( iPS sMN ) derived from C9orf72 NRE patients increases following induction of cellular stress . DPR fluorescent intensity in iPS sMN was measured using the DPR - specific antibodies described above . IPS sMN : n = 4 with m > 20 cells per n .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.


Fluorescent microscopy images shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients . Glutamate - induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801 , respectively . Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors : glutamate , AMPA , NMDA , and HC . Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for quantification . DPR fluorescent intensity in iPS sMN was measured using DPR - specific antibodies . Changes in DPR levels are relative to non - stressed cells ( CTRL ) Cortical neuron : n = 6 with m > 40 cells per n . IPSC sMN : n = 4 with m > 25 cells per n .

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Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.


Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter .

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Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.


Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons . No stimulation and low stimulation frequencies did not increase levels of DPR . The C9 DPR reporter , monitoring the GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 cells per n . ( * * * * P < 0 . 0001 ) .

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Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).


Filter trap binding assays show that ISR proteins are enhanced by all stressors . Significant increases following cellular stress treatments are seen in the ratio of phospho - eif2α to total eif2α as well as phospho - PERK and ATF4 levels compared to untreated controls . Changes in ISR proteins are normalized to GAPDH and shown relative to non - stressed or DMSO - only treated cells ( CTRL ) depending on the vehicle compounds were dissolved in . n = 4 .

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Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.


Fluorescent intensity measurements show the levels of two ISR - related proteins , PERK and relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative images indicate fluorescent intensity differences for phospho - eif2α and phospho - PERK levels 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR - related protein levels , which parallels reduced DPR levels ( Figure 3B ) . Changes in ISR proteins are relative to non - stressed cells ( CTRL ) .

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Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had the most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR levels as the PERK inhibitor , GSK2606414 ( 500 nM ) .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to the treatments in NSC34 , 1 , 3DBM shows the most significant decrease in DPR production .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.


Analysis of ER architecture and dynamics as a function of RNF26 in U2OS cells ectopically expressing mCherry - KDEL and transfected with either control siRNA ( siC ) or siRNAs targeting RNF26 ( siRNF26 - 1 or siRNF26 - 2 ) . B . Time - lapse imaging . Top panels : representative confocal fluorescence images of Cherry - KDEL ( white ) at the start of time - lapse t = 0 ( top ) . Middle panels : time color coded overlays of 60x1s frames , each assigned a different color LUT ( white - same ER location across all frames ; color - ER movement between frames ) . Bottom panels : overlays of twice eroded ER sheet masks ( magenta ) with mCherry - KDEL signal ( green ) . Masks were generated using ImageJ software . Zoom - ins show select PN and PP regions . Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively .

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Analysis of ER architecture and dynamics as a function of RNF26 in U2OS cells ectopically expressing mCherry-KDEL and transfected with either control siRNA (siC) or siRNAs targeting RNF26 (siRNF26-1 or siRNF26-2). B. Time-lapse imaging. Top panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded overlays of 60x1s frames, each assigned a different color LUT (white - same ER location across all frames; color - ER movement between frames). Bottom panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively.


I . CLIMP63 abundance as a function of RNF26 . Top : representative immunoblot of U2OS cells transfected with the indicated siRNAs stained against endogenous CLIMP63 and Vinculin ( loading control ) . Bottom : quantification of CLIMP63 abundance , normalized to Vinculin and expressed relative to control ( siC = 1 . 0 ) , n = 4 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test with error bars reflecting + / - SD )

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I. CLIMP63 abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with the indicated siRNAs stained against endogenous CLIMP63 and Vinculin (loading control). Bottom: quantification of CLIMP63 abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test with error bars reflecting +/- SD)


J , K . Effect of RNF26 depletion on basal ER stress using IRE1 - mediated XBP1 splicing as a readout . J . Relative abundance of RNF26 transcript normalized to GAPDH ; n = 3 independent experiments . All transcripts were detected by qPCR . Plots report values relative to control ( siC = 1 . 0 ) . K . Quantification of IRE1 - dependent XBP1 splicing . XBP - s and XBP1 transcripts isolated from U2OS cells transfected with the indicated siRNAs were detected by qPCR , normalized to GAPDH , and expressed as ratio , n = 3 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test J and K with error bars reflecting + / - SD ) p < 0 . 05 ; * * * p < 0 . 001 , ns : not significant .

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J, K. Effect of RNF26 depletion on basal ER stress using IRE1-mediated XBP1 splicing as a readout. J. Relative abundance of RNF26 transcript normalized to GAPDH; n=3 independent experiments. All transcripts were detected by qPCR. Plots report values relative to control (siC=1.0). K. Quantification of IRE1-dependent XBP1 splicing. XBP-s and XBP1 transcripts isolated from U2OS cells transfected with the indicated siRNAs were detected by qPCR, normalized to GAPDH, and expressed as ratio, n=3 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test J and K with error bars reflecting +/- SD) p<0.05; *** p<0.001, ns: not significant.


C , D . Proximity biotinylation of endogenous vimentin by 2HA - TurboID - RNF26 , catalytically inactive point mutant ( I382R ) , or RING domain truncation mutant ( ∆ RING ) in HeLa cells following 30 min incubation with biotin prior to lysis . C . Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin . D . Quantifications of vimentin biotinylation by 2HA - TurboID - RNF26 and mutants normalized to auto biotinylated HA - tagged RNF26 species and expressed relative to RNF26 WT , n = 6 independent experiments performed in U2OS and HeLa cells . Data information : WCL : whole cell lysate . Graphs in D report mean ( red line ) and SEM ( error bars ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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C, D. Proximity biotinylation of endogenous vimentin by 2HA-TurboID-RNF26, catalytically inactive point mutant (I382R), or RING domain truncation mutant (∆RING) in HeLa cells following 30 min incubation with biotin prior to lysis. C. Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin. D. Quantifications of vimentin biotinylation by 2HA-TurboID-RNF26 and mutants normalized to auto biotinylated HA-tagged RNF26 species and expressed relative to RNF26 WT, n=6 independent experiments performed in U2OS and HeLa cells. Data information: WCL: whole cell lysate. Graphs in D report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.


E . In vitro co - precipitation of purified recombinant Rhodamine - labeled RING - I382R ( 304 - 433 ) with purified recombinant His - tagged vimentin on Ni - NTA beads . Rhodamine in - gel fluorescent scan and immunoblot against vimentin are shown , representative of three independent experiments .

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E. In vitro co-precipitation of purified recombinant Rhodamine-labeled RING-I382R (304-433) with purified recombinant His-tagged vimentin on Ni-NTA beads. Rhodamine in-gel fluorescent scan and immunoblot against vimentin are shown, representative of three independent experiments.


G . Proximity - based biotinylation of endogenous vimentin by 2HA - TurboID - I382R , - ∆ RING , and - I382R - ∆ C lacking amino acids 423 - 433 . Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments . Data information : WCL : whole cell lysate .

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G. Proximity-based biotinylation of endogenous vimentin by 2HA-TurboID-I382R, -∆RING, and -I382R-∆C lacking amino acids 423-433. Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments. Data information: WCL: whole cell lysate.


H . In vitro peptide binding assay . Rhodamine - linked C - terminal peptide of RNF26 ( Rho - RGILQTLNVYL ) or free Rhodamine were incubated at 10nM with increasing concentrations of His - vimentin and binding was measured using fluorescence polarization ; n = 3 independent experiments . Data information H report mean ( red line ) and SEM ( error bars ) of sample values ( open circles ) . Significance was assessed using Student ' s t - test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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H. In vitro peptide binding assay. Rhodamine-linked C-terminal peptide of RNF26 (Rho-RGILQTLNVYL) or free Rhodamine were incubated at 10nM with increasing concentrations of His-vimentin and binding was measured using fluorescence polarization; n=3 independent experiments. Data information H report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.


A - E . Analysis of RNF26 sequence determinants with respect to the intracellular distribution of RNF26 and its colocalization with vimentin filaments . A . Representative confocal fluorescence images of U2OS cells ectopically expressing RFP - RNF26 ( green ) wild - type ( WT ) or mutants ( I382R , ∆ RING , ∆ C ) , fixed and immunostained against endogenous vimentin ( magenta ) and VAP - A ( magenta ) . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions . B . Colocalization ( Manders ' ) analysis of RFP - RNF26 and mutants overlap with vimentin . Plot reports n WT = 38 , n I382R = 31 , n ΔRING = 34 , n ΔC = 22 cells analysed from three independent experiments . C . Colocalization ( Manders ' ) analysis of RFP - RNF26 and mutants with VAP - A . Plot reports n WT = 29 , n I382R = 21 , n ΔRING = 17 , n ΔC = 16 cells analysed from three independent experiments . D . Perinuclearity of RFP - RNF26 as a function of its RING domain determinants . Plot reports n WT = 72 , n I382R = 48 , n ΔRING = 37 , n ΔC = 28 cells analysed from three independent experiments . E . Intracellular distribution of vimentin as a function of RNF26 ( mutant ) overexpression . Plot reports n WT59 , n I382R = 58 , n ΔRING = 36 , n ΔC = 32 technical replicates from three independent experiments . Values capped at 10 . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm . Statistical analyses were performed using Students ' T - test ( B , D error bars indicate mean - / + SD ) or Mann - Whitney U test ( C , E error bars indicate median and 95 % confidence interval ) ; * * * p < 0 . 001 , ns : not significant .

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A-E. Analysis of RNF26 sequence determinants with respect to the intracellular distribution of RNF26 and its colocalization with vimentin filaments. A. Representative confocal fluorescence images of U2OS cells ectopically expressing RFP-RNF26 (green) wild-type (WT) or mutants (I382R, ∆RING, ∆C), fixed and immunostained against endogenous vimentin (magenta) and VAP-A (magenta). Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. B. Colocalization (Manders') analysis of RFP-RNF26 and mutants overlap with vimentin. Plot reports n WT=38, n I382R=31, n ΔRING=34, n ΔC=22 cells analysed from three independent experiments. C. Colocalization (Manders') analysis of RFP-RNF26 and mutants with VAP-A. Plot reports n WT=29, n I382R=21, n ΔRING=17, n ΔC=16 cells analysed from three independent experiments. D. Perinuclearity of RFP-RNF26 as a function of its RING domain determinants. Plot reports n WT=72, n I382R=48, n ΔRING=37, n ΔC=28 cells analysed from three independent experiments. E. Intracellular distribution of vimentin as a function of RNF26 (mutant) overexpression. Plot reports n WT59, n I382R=58, n ΔRING=36, n ΔC=32 technical replicates from three independent experiments. Values capped at 10. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test (B, D error bars indicate mean -/+ SD) or Mann-Whitney U test (C, E error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.


F , G . Analysis of vimentin cytoskeleton distribution as a function of RNF26 . F . Representative confocal fluorescence images of control cells , cells silenced for RNF26 ( si # 1 ) , or RNF26 KO cells . Zoom - ins highlight PN regions . G . Perinuclearity of vimentin in the presence or absence of RNF26 . Plot reports n control = 98 , n siRNF26 = 132 , n RNF26 KO = 41 technical replicates from three independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm . Statistical analyses were performed using Students ' T - test Mann - Whitney U test error bars indicate median and 95 % confidence interval ) ; * * * p < 0 . 001 , ns : not significant .

O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G. Analysis of vimentin cytoskeleton distribution as a function of RNF26. F. Representative confocal fluorescence images of control cells, cells silenced for RNF26 (si#1), or RNF26 KO cells. Zoom-ins highlight PN regions. G. Perinuclearity of vimentin in the presence or absence of RNF26. Plot reports n control=98, n siRNF26=132, n RNF26 KO=41 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test Mann-Whitney U test error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.


H . Vimentin abundance as a function of RNF26 . Top : representative immunoblot of U2OS cells transfected with siRNF26 # 1 stained against endogenous vimentin and Vinculin ( loading control ) . Bottom : quantification of vimentin abundance , normalized to Vinculin and expressed relative to control ( siC = 1 . 0 ) , n = 4 independent experiments . Statistical analyses were performed using Students ' T - test error bars indicate mean - / + SD ) ; * * * p < 0 . 001 , ns : not significant .

O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Vimentin abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with siRNF26#1 stained against endogenous vimentin and Vinculin (loading control). Bottom: quantification of vimentin abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Statistical analyses were performed using Students' T-test error bars indicate mean -/+ SD) ; *** p<0.001, ns: not significant.


A - D . Effect of vimentin ablation on intracellular distribution of RNF26 . A . Representative confocal images of parental and vimentin knockout ( Vim KO ) U2OS cells ectopically expressing GFP - RNF26 ( green ) , fixed and immunostained against endogenous VAP - A ( magenta ) . B . Validation of vimentin knockout . Immunoblots against endogenous vimentin and Vinculin ( loading control ) of a Vim KO clonal cell line and their parental U2OS cells are shown , representative of three independent experiments . Molecular weight markers are as indicated . C . Quantification of GFP - RNF26 signal distribution expressed as perinuclearity ratio . Graph reports n Parental = 73 , n Vim KO = 43 technical replicates from three independent experiments . D . Colocalization ( Manders ' ) analysis of VAP - A overlapping with GFP - RNF26 ( left plot ) and vice versa ( right plot ) . Plots report on subset of cells from data set in ( B ) , same as in ( C ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test ( D Mann - Whitney U test ( C error bars indicate median and 95 % confidence interval ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D. Effect of vimentin ablation on intracellular distribution of RNF26. A. Representative confocal images of parental and vimentin knockout (Vim KO) U2OS cells ectopically expressing GFP-RNF26 (green), fixed and immunostained against endogenous VAP-A (magenta). B. Validation of vimentin knockout. Immunoblots against endogenous vimentin and Vinculin (loading control) of a Vim KO clonal cell line and their parental U2OS cells are shown, representative of three independent experiments. Molecular weight markers are as indicated. C. Quantification of GFP-RNF26 signal distribution expressed as perinuclearity ratio. Graph reports n Parental = 73, n Vim KO = 43 technical replicates from three independent experiments. D. Colocalization (Manders') analysis of VAP-A overlapping with GFP-RNF26 (left plot) and vice versa (right plot). Plots report on subset of cells from data set in (B), same as in (C). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test (D Mann-Whitney U test (C error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.


E , F . Effect of vimentin or RNF26 ablation on the intracellular distribution of endolysosomes . E . Representative confocal images of parental , RNF26 KO , vimentin KO # 1 U2OS cells , vimentin KO # 1 cells ectopically complemented with untagged wild type vimentin ( Vim KO + Rescue ) , fixed and immunostained against endogenous LAMP1 . F . Quantification of LAMP1 signal distribution expressed as perinuclearity ratio . Graph reports on n Parental = 145 , n R26 KO = 64 , n Vim KO # 1 = 119 , n Vim KO # 1 / Rescue = 32 technical replicates from three independent experiments . Values capped at 6 . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Mann - Whitney U test F error bars indicate median and 95 % confidence interval ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. Effect of vimentin or RNF26 ablation on the intracellular distribution of endolysosomes. E. Representative confocal images of parental, RNF26 KO, vimentin KO #1 U2OS cells, vimentin KO#1 cells ectopically complemented with untagged wild type vimentin (Vim KO + Rescue), fixed and immunostained against endogenous LAMP1. F. Quantification of LAMP1 signal distribution expressed as perinuclearity ratio. Graph reports on n Parental = 145, n R26 KO = 64, n Vim KO#1 = 119, n Vim KO#1/Rescue= 32 technical replicates from three independent experiments.Values capped at 6. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Mann-Whitney U test F error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.


G , H . Effects of vimentin ablation on trafficking of extracellular materials to lysosomes . G . Time - lapse imaging . Representative confocal overlays of parental and vimentin KO # 2 U2OS cells , stained with SiR - Lysosome ( magenta ) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 ( green ) , taken soon after the start ( t = 5 min ) and at the end ( t = 120 min ) of time - lapse are shown . Zoom - ins highlight select PN regions in overlay and single channels . H . Colocalization ( Manders ' ) of SiR - Lysosome overlapping SR101 at t = 120 minutes following SR101 addition . Graph reports on n Parental = 71 , n Vim KO = 69 technical replicates from 3 independent experiments . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Zoom - ins designate regions encompassing perinuclear and peripheral areas . All scale bars = 10µm . Graphs report mean ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test H error bars indicate mean - / + SD ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_LINE O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H. Effects of vimentin ablation on trafficking of extracellular materials to lysosomes. G. Time-lapse imaging. Representative confocal overlays of parental and vimentin KO#2 U2OS cells, stained with SiR-Lysosome (magenta) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 (green), taken soon after the start (t=5 min) and at the end (t=120 min) of time-lapse are shown. Zoom-ins highlight select PN regions in overlay and single channels. H. Colocalization (Manders') of SiR-Lysosome overlapping SR101 at t=120 minutes following SR101 addition. Graph reports on n Parental = 71, n Vim KO = 69 technical replicates from 3 independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test H error bars indicate mean -/+ SD) ; * p<0.05, ** p<0.01, *** p<0.001.


Effect of vimentin ablation on ER organization and dynamics . Analysis of parental versus vimentin KO # 1 U2OS cells ectopically expressing mCherry - KDEL . D . Time - lapse imaging . Left panels : representative confocal fluorescence images of Cherry - KDEL ( white ) at the start of time - lapse t = 0 ( top ) . Middle panels : time color coded ( TCC ) overlays of 60x1s frames . Right panels : overlays of twice eroded ER sheet masks ( magenta ) with mCherry - KDEL signal ( green ) . Masks were generated using ImageJ software . Zoom - ins show select PN and PP regions . Scale bar = 10µm .

O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O

Effect of vimentin ablation on ER organization and dynamics. Analysis of parental versus vimentin KO#1 U2OS cells ectopically expressing mCherry-KDEL. D. Time-lapse imaging. Left panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded (TCC) overlays of 60x1s frames. Right panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Scale bar = 10µm.


D , Consequences of RNF26 , UBE2J1 , and vimentin loss on ERQC formation . D . Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated , treated in the absence ( - ) or presence ( + ) of tunicamycin ( 5ug / mL , O / N ) , fixed and immunostained against endogenous calnexin ( white ) .

O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O

D, Consequences of RNF26, UBE2J1, and vimentin loss on ERQC formation. D. Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated, treated in the absence (-) or presence (+) of tunicamycin (5ug/mL, O/N), fixed and immunostained against endogenous calnexin (white).


F , G . Consequences of RNF26 , UBE2J1 , and vimentin loss on induction of PERK - mediated UPR signaling by tunicamycin . F . U2OS cells , transfected or genetically modified as indicated , were treated in the absence ( - ) or presence ( + ) of tunicamycin ( 2 . 5µg / mL , 5hrs ) . Immunoblots of whole cell lysates against endogenous PERK , phospho - eIF2a , ATF4 , HERP1 , and Vinculin ( loading control ) are shown , representative of three independent experiments . G . CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence ( - ) or presence ( + ) of tunicamycin ( 2 . 5ug / mL , O / N ) were detected by qPCR and normalized to GAPDH ; n siC ( - ) = 4 , n siC ( + ) = 4 , n siRNF26 # 1 ( + ) = 3 , n siRNF26 # 2 ( + ) = 4 , n PARENTAL ( + ) = 4 , n Vim KO ( + ) = 4 independent experiments . Data information : Graphs report mean or median ( red line ) of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test G error bars indicate mean - / + SEM ) p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G. Consequences of RNF26, UBE2J1, and vimentin loss on induction of PERK-mediated UPR signaling by tunicamycin. F. U2OS cells, transfected or genetically modified as indicated, were treated in the absence (-) or presence (+) of tunicamycin (2.5µg/mL, 5hrs). Immunoblots of whole cell lysates against endogenous PERK, phospho-eIF2a, ATF4, HERP1, and Vinculin (loading control) are shown, representative of three independent experiments. G. CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence (-) or presence (+) of tunicamycin (2.5ug/mL, O/N) were detected by qPCR and normalized to GAPDH; n siC (-)=4, n siC (+)=4, n siRNF26#1 (+)=3, n siRNF26#2 (+)=4, n PARENTAL (+)=4, n Vim KO (+)=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test G error bars indicate mean -/+ SEM) p<0.05, ** p<0.01, *** p<0.001.


A , Analysis of localization of RNF26 sequence variants and HERP1 or HERP2 in the ER . A . Representative confocal images of U2OS cells transfected with GFP - RNF26 WT , RFP - RNF26 I382R , GFP - RNF26 ΔRING and HERP1 or GFP - RNF26 WT with HA - HERP2 that were stained for HA and endogenous VAP - A . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

A, Analysis of localization of RNF26 sequence variants and HERP1 or HERP2 in the ER. A. Representative confocal images of U2OS cells transfected with GFP-RNF26 WT, RFP-RNF26 I382R, GFP-RNF26 ΔRING and HERP1 or GFP-RNF26 WT with HA-HERP2 that were stained for HA and endogenous VAP-A. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


C . Analysis of determinants for RNF26 complex formation with ERQC proteins HERP1 and HRD1 . U2OS cells were transfected with RFP - RNF26 variants ( WT , I382R , ΔRING ) or EV with either HA - HERP1 or HA - HRD1 before lysis in 1 % DMNG detergent , pulldown with RFP - TRAP beads and analysis by SDS - PAGE and WB . Representative images of three independent replicates .

O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

C. Analysis of determinants for RNF26 complex formation with ERQC proteins HERP1 and HRD1. U2OS cells were transfected with RFP-RNF26 variants (WT, I382R, ΔRING) or EV with either HA-HERP1 or HA-HRD1 before lysis in 1% DMNG detergent, pulldown with RFP-TRAP beads and analysis by SDS-PAGE and WB. Representative images of three independent replicates.


D , E . Analysis of localization of ERQC domains with vimentin . D . Representative confocal images of U2OS cells transfected with HA - HERP1 that were stained for endogenous vimentin . Indicated single channels and their corresponding color overlays are shown . Zoom - ins highlight PN regions where vimentin surrounds HERP1 - labeled ERQC domains . E . Line graph analysis of HA - HERP1 ( magenta ) and vimentin ( green ) signals from E ( bottom zoom image ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E. Analysis of localization of ERQC domains with vimentin. D. Representative confocal images of U2OS cells transfected with HA-HERP1 that were stained for endogenous vimentin. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions where vimentin surrounds HERP1-labeled ERQC domains. E. Line graph analysis of HA-HERP1 (magenta) and vimentin (green) signals from E (bottom zoom image). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


F , G . Analysis of HERP1 - induced ERQC domain localization as a function of vimentin and RNF26 . F . Representative confocal fluorescence images of control cells , RNF26 KO cells , or vimentin KO # 2 cells , transfected with HA - HERP1 and immunostained for HA . Zoom - ins highlight PN regions . G . Perinuclearity analysis of HA - HERP1 in the presence or absence of RNF26 or vimentin . Plot reports median ( red line ) and 95 % confidence interval ( error bar ) of sample values ( open circle ) , n Parental = 79 , n R26 KO = 51 , n Vim KO = 45 technical replicates from three independent experiments ( Mann - Whitney U test ; * * p < 0 . 01 , * * * p < 0 . 001 ) . Graphs report median ( red line ) and 95 % confidence interval ( error bars ) of sample values ( open circles ) . Data information : Cell and nuclear boundaries are demarcated using dashed and continuous lines , respectively . Scale bar = 10µm .

O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

F, G. Analysis of HERP1-induced ERQC domain localization as a function of vimentin and RNF26. F. Representative confocal fluorescence images of control cells, RNF26 KO cells, or vimentin KO#2 cells, transfected with HA-HERP1 and immunostained for HA. Zoom-ins highlight PN regions. G. Perinuclearity analysis of HA-HERP1 in the presence or absence of RNF26 or vimentin. Plot reports median (red line) and 95% confidence interval (error bar) of sample values (open circle), n Parental=79, n R26 KO=51, n Vim KO=45 technical replicates from three independent experiments (Mann-Whitney U test; ** p<0.01, *** p<0.001). Graphs report median (red line) and 95% confidence interval (error bars) of sample values (open circles). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.


C , D . Lysosomal processing of Sec62 - HALO - GFP over time in the presence or absence of RNF26 or vimentin . C . Parental , Vim KO # 2 or RNF26 KO U2OS cells were transfected with Sec62 - HALO - GFP overnight , incubated with 6 - chlorohexanol for 15 minutes , washed 3 times with PBS , pulsed with TAMRA HALOtag ligand ( 200nM ) for 1hr , and incubated with 6 - chlorohexanol with or without Bafilomycin A1 ( 100nM ) for the indicated times before cell lysis and analysis by SDS - PAGE . Shown is a representative in - gel fluorescence scan with indication of full - length Sec62 , protease - resistant HALO fragment and molecular weight markers ( MW ) , as well as a Vinculin blot ( loading control ) D . Quantification of Sec62 accumulation in LEs from ( B ) . Unprocessed and protease - resistant ( lysosomal ) band intensities in ( B ) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours . Error bars show mean + / - SD of n Parental = 6 , n R26 KO = 4 , n Vim KO = 6 independent experiments . Scale bar = 10µm . Graphs report mean ( in D of sample values ( open circles ) . Statistical analyses were performed using Students ' T - test ( D ( paired ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Lysosomal processing of Sec62-HALO-GFP over time in the presence or absence of RNF26 or vimentin. C. Parental, Vim KO#2 or RNF26 KO U2OS cells were transfected with Sec62-HALO-GFP overnight, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with TAMRA HALOtag ligand (200nM) for 1hr, and incubated with 6-chlorohexanol with or without Bafilomycin A1 (100nM) for the indicated times before cell lysis and analysis by SDS-PAGE. Shown is a representative in-gel fluorescence scan with indication of full-length Sec62, protease-resistant HALO fragment and molecular weight markers (MW), as well as a Vinculin blot (loading control) D. Quantification of Sec62 accumulation in LEs from (B). Unprocessed and protease-resistant (lysosomal) band intensities in (B) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours. Error bars show mean +/- SD of n Parental=6, n R26 KO=4, n Vim KO =6 independent experiments. Scale bar = 10µm. Graphs report mean (in D of sample values (open circles). Statistical analyses were performed using Students' T-test (D (paired) ; * p<0.05, ** p<0.01, *** p<0.001).