Phenotypic of ScUba4 mutant yeast strains in response to rapamycin .

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Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.


MS / MS spectrum of K202 - ε - GGH containing peptide from CtUba4C202K . A representative annotated fragmentation spectrum is shown with b - y - ions marked in red blue , respectively . precursor ion at m / z 591 , 98 is labelled in green . peptide is shown at top with collision - induced fragmentation pattern . Urm1 conjugation site was identified by detection of GGH remnant motif generated by chymotryptic digestion .

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MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.


of covalent adduct formation between CtUba4 WT or Cys202 mutants thiocarboxylated ( - SH ) CtUrm1 in presence or absence of TBH HA , respectively .

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Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.


( A ) Genome - wide ubiquitin binding profiles identify numerous regions of H2B - ubiquitylation in WT distinct sites of non - H2B ubiquitylation ( ' ubiquitin - hotspot ' , ub - hotspot , ub - HS ) which persist in h2b - K123R rad6 ∆ strains , increase in cdc48 mutants ( cdc48 - 6 ) . A 90 kb stretch of chromosome XIII ( ChrXIII ) is depicted . immunoprecipitation was performed using FK2 ubiquitin antibody ( see also Appendix Fig . S1A ) enriched was analyzed on NimbleGen arrays ( Chip - chip ) . from non - specific IgG - ChIP experiments served as background control . Significantly enriched regions are marked by bars above respective ChIP - chip tracks are summarized in Dataset EV1 . Data represent means from two independent replicates , except for ubiquitin ( FK2 ) in rad6 ∆ IgG - ChIP in WT ( n = 1 ) . experiments , including those using cdc48 - 6 other - sensitive ( ts ) alleles , were performed at 30 ° C ( semipermissive for ts - alleles ) unless stated otherwise .

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(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.


( B ) Seven ub - HSs show strong correlation between ubiquitin binding Slx8 enrichment ( Slx8 - 9myc ChIP ) . 16 - kb - windows of indicated regions centered around ub - HSs are depicted for ubiquitin Slx8 binding profiles . Ubiquitin ( FK2 ) data for WT cdc48 - 6 are from same experiment as depicted in ( A ) . Data represent means from two independent replicates .

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(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.


( D ) Slx8 Slx5 are recruited to ub - HSs . from Slx8 - 9myc ( top ) or 3HA - Slx5 ( bottom ) ChIP experiments of indicated strains was analyzed by quantitative real - time PCR ( ChIP - qPCR ) at selected ub - HSs . Data represent mean ± standard deviation ( SD , n = 2 ) . Consistent with a previous study , we did not observe any Slx8 enrichment at centromeres ( van de Pasch et al , 2013 ) , however , we note that we could not confirm reported centromere binding of Slx5 ( see also Appendix Fig . S2E ) .

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(D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E).


( E ) K48 - linked ubiquitin chains accumulate in a cdc48 mutant ( cdc48 - 3 ) . Ub - K48 ChIP followed by qPCR for same loci as in ( D ) are depicted . See also Fig . EV1C - D . Data represent means ± SD ( n = 2 ) .

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(E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2).


( F ) Slx5 Slx8 are required for ub - HS formation . Ub - K48 ChIP - qPCR in strains lacking one subunit of Slx5 / Slx8 ( slx5 ∆ , slx8 ∆ ) . Data represent means ± SD ( n = 3 ) .

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(F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3).


( G ) Ufd1 Npl4 act in concert with Cdc48 to remove ubiquitylated from ub - HS sites . Ub - K48 ChIP for indicated strains grown at semi - permissive of 30 ° C . Data represent means ± SD ( n = 2 ) .

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(G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).


B ) A 39 bp fragment of ub - HS4 is sufficient to drive ectopic ub - HS formation . Fragments of ub - HS4 were integrated ectopically ub - K48 ChIP - qPCR was performed for endogenous ub - HS5 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 bioinformatic prediction identify a similar ub - HS - motif . Comparison between experimentally mapped ub - HS4 F7 ( B ) consensus motif of ub - HSs identified by 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 ub - HS - motif in a Y1H . Gal4 - AD - or Gal4 - AD - Euc1 - encoding plasmids were transformed into a Y1H reporter as described in ( D ) . Serial dilutions were spotted on control plates plates lacking histidine with 20mM 3 - - triazole ( 3AT ) . 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 indicated strains enriched 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 . Euc1 constructs under endogenous EUC1 promoter were transformed in a reporter as described for Fig . 2D serial dilutions were spotted on control or selective media to HIS3 - activation . 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 ) of HIS3 mRNA from strains used in ( C ) . were grown in liquid media with selection for transformed plasmids ( SC - Leu ) , harvested in logarithmic growth phase total mRNA was prepared . After reverse transcription , HIS3 mRNA were quantified using qPCR ( RT - qPCR ) , normalized first to ACT1 mRNA then to empty vector control . Data represent means ± SD ( n = 4 ) . p = 2 . 43 * 10 - 5 ( Student ' s t - ) .

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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 in a yeast two - hybrid ( Y2H ) . A Y2H reporter ( PJ69 - 7a ) was transformed with Gal4 - binding domain ( BD ) Gal4 activation domain ( AD ) fusion constructs in indicated combinations . AD - SUMO - GG can be conjugated to SUMOylation substrates , while AD - SUMO - AA is conjugation - deficient . were spotted on control media or selective media ( - His ) grown for 3 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 Siz2 on K231 . Euc1 was immunoprecipitated from indicated strains eluates were probed by WB against SUMO ( top ) 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 3FLAGEuc1 constructs under control of an ADH promoter . Covalently SUMO - modified were enriched 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 K231R mutation here 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 indicated strains . ubx5 ∆ background was used to enhance 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 partly on Euc1 SUMOylation . Denaturing NiNTA - PDs as described for ( C ) but with strains expressing His - ubiquitin ( HisUbi ) 3FLAGEuc1 under control of ADH promoter . WBs were developed with a FLAG antibody to probe for 3FLAGEuc1 , a ubiquitin blot served as PD - control Dpm1 as input control . slx8 - CD allele carries 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 Slx8 to ub - HSs . ChIP against Slx5 in indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT 3HA - Slx5 in ( F )

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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 indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT 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 performed as in Fig . 4A . Serial dilutions were spotted for each plasmid combination were grown at 30 ° C for 2 days . Slx5 - RING ∆ construct is deleted for Slx5 C - terminus from beginning of 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 . lysates from an euc1 ∆ expressing 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 Euc1 antibodies , inputs were probed with Slx5 , FLAG Dpm1 antibodies ( top to bottom ) . Note that Euc1 - Slx5 interaction is independent of 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 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 were grown at 30 ° C for 2 days . See Appendix Fig . S6A for expression .

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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 - Euc1 constructs ( with or without K231R mutation ) FLAG IPs were performed as described in ( C ) . WB showed strong Slx5 - binding defects for SBM1 / SBM2 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 ) Slx5 middle domain ( Slx5 - Md ) is required for interaction with Euc1 . Y2H assays with AD - Euc181 - 183 BD - Slx5 constructs . Slx5 - Md : aa 201 - 335 , Slx5 - Md ∆ : ∆ aa 201 - 338 . Serial dilutions were spotted 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 Slx5 - Md . Y2H with 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 were enriched by denaturing NiNTA - PDs as described in Fig . 4E . 3FLAG - tagged Euc1 constructs were expressed from plasmids under control of ADH promoter . NiNTA - PD eluates were probed with FLAG ubiquitin ( P4D1 ) antibodies . Whole lysates ( input ) were probed with FLAG 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 ) Slx5 - SIMs Slx5 - Md are required for Euc1 ubiquitylation . NiNTA - PDs for His - ubiquitin as in ( A ) . strains expressed wild - type 3FLAGEuc1 from plasmids under ADH promoter His - ubiquitin . Slx5 constructs were expressed from plasmids under control of endogenous promoter . WBs were probed as described in ( A ) , Slx5 - 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 Slx5 - Md , which overlaps with a putative NLS ( Westerbeck et al , 2014 ) , an N - terminal NLS was fused to 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 recruitment of Slx5 to ub - HSs . ChIP - qPCR for ub - K48 ( top ) or Slx5 ( using Slx5 antibody , bottom ) in euc1 ∆ ubx5 ∆ expressing indicated constructs from plasmids under control of 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 Slx5 - Md are required for formation of ub - HSs recruitment of Slx5 . ChIP - qPCR for ub - K48 ( left ) or Slx5 ( anti - HA ChIP , right ) in ubx5 ∆ or slx5 ∆ ubx5 ∆ complemented with plasmids expressing indicated Slx5 - constructs under control of 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 isolated from WT euc1 ∆ was polyA - enriched sequenced ( RNAseq , triplicates ) . Significance testing was on Wald , see Materials Methods for details . See also Appendix Fig . S8A for of selected transcripts by qPCR .

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(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 of EUC1 overexpression as in ( A ) . ∆ euc1 with pGAL - EUC1 or pEUC1 - EUC1 ( control ) integrated at URA3 - locus ( YIplac211 ) were grown to mid - log phase 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 upon exposure to fluidizing drug benzylalcohol . For ( C , D , F ) , serial dilutions of indicated strains were spotted 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 binding transactivation . indicated EUC1 alleles with endogenous or galactose - inducible promoters were integrated at URA3 locus ( YIplac211 , euc1 ∆ background ) 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 binding . ChIP - qPCR of Euc1 after 3 hours galactose induction . Note that IP / input ratios of Euc1 signals are shown , also for 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 SLX8 rescue EUC1 toxicity . Experiment as in ( D ) , but with concomitant plasmid - borne overexpression ( ADH promoter ) of SLX5 SLX8 ( bottom panels ) on media selecting for SLX5 / SLX8 plasmids . See also Fig . EV4E 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 specific transcriptional regulation ( A ) in particular with members of Rpd3L histone deacetylase ( B ) . For ( A - D ) , serial dilutions of indicated strains were spotted grown on YPD control or selective media plates , or under conditions as indicated . Note that 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 Rpd3L histone deacetylase For , serial dilutions of indicated strains were spotted grown on YPD control or selective media plates , or under conditions as indicated . Note that 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 indicated strains were spotted grown on YPD control or selective media plates , or under conditions as indicated . Note that 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 indicated strains 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 indicated strains were spotted grown on YPD control or selective media plates , or under conditions as indicated . Note that strains used contained an extra copy of MED11 ( D ) EUC1 Rpd3S act in a common pathway which is redundant with Rpd3L . rco1 ∆ eaf3 ∆ ( Rpd3S ) show similar when paired with sds3 ∆ ( Rpd3L ) as euc1 ∆ 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 conditions as indicated . Data represent means ± SD ( n = 4 ) , see Appendix Fig . S11B for statistical .

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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.


ability to form ub - hotspots is crucial for endogenous EUC1 function . genetic interaction of EUC1 with SDS3 ( Rpd3L ) was rescued with plasmid - borne EUC1 - alleles 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 indicated strains were spotted on YPD incubated as indicated . See also Appendix Fig . S12C - D . Note that 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,


A . Raw 264 . 7 macrophage were treated with DMSO ( 0 . 05 % ) , 1 μM control antisense oligonucleotide ( ASO ) , or Rab10 - directed ASO ( 1 μM ) , for four days . Immunoblots are representative of three independent experiments , with similar results obtained . B . Calculated reduction of Rab10 ( n = 3 biologically independent experiments ) . Each dot in plot presents one independent experiment . Group are shown . Error bars represent ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * * representing P < 0 . 0001 .

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A. Raw 264.7 macrophage cells were treated with DMSO (0.05%), 1 μM control antisense oligonucleotide (ASO), or Rab10-directed ASO (1 μM), for four days. Immunoblots are representative of three independent experiments, with similar results obtained. B. Calculated reduction of Rab10 (n=3 biologically independent experiments). Each dot in the plot presents one independent experiment. Group are shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.


C . Raw 264 . 7 were treated with indicated ASO for four days , or 50 μM EIPA ( 5 - [ N - ethyl - N - isopropyl ] amiloride ) for 20 min , prior to incubation for 60 min with ( tetramethylrhodamine , TRITC ) - labeled dextran ( 70 kDa , shown as red color ) , transferrin , or IgG - conjugated agarose beads . Representative photomicrographs ( from 30 analyzed for each condition , from three biologically independent experiments ) are shown . were washed , fixed , stained with DAPI ( shown as blue color ) . Scale bars show 200 μm . D - F . Relative signals were calculated as a of control - ASO treated to indicate ( D ) dextran or ( E ) transferrin uptake level . ( F ) For of phagocytosis , average number of IgG - agarose beads internalized were calculated as a of control - ASO treated . 30 across three independent experiments were quantified for each condition , with each dot in plot representing results from one image . group means were calculated shown . Error bars represent ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * representing P < 0 . 0001 , N . S P > 0 . 05 .

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

C. Raw 264.7 cells were treated with the indicated ASO for four days, or 50 μM EIPA (5-[N-ethyl-N-isopropyl]amiloride) for 20 min, prior to incubation for 60 min with fluorescent (tetramethylrhodamine, TRITC)-labeled dextran (70 kDa, shown as red color), transferrin, or IgG-conjugated agarose beads. Representative photomicrographs (from 30 images analyzed for each condition, from three biologically independent experiments) are shown. Cells were washed, fixed, and stained with DAPI (shown as blue color). Scale bars show 200 μm. D-F. Relative fluorescent signals were calculated as a percent of control-ASO treated cells to indicate (D) dextran or (E) transferrin uptake level. (F) For quantification of phagocytosis, the average number of IgG-agarose beads internalized were calculated as a percent of control-ASO treated cells. 30 images across three independent experiments were quantified for each condition, with each dot in the plot representing results from one image. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P<0.0001, N.S P>0.05.


G . mouse bone marrow - derived macrophage ( BMDM ) from adult C57BL / 6J mice were treated with murine cytokines to polarize towards a monocytic lineage ( mBMDM ) , dendritic lineage ( mBMDC ) , or microglia - like lineage ( mMicroglia , see Methods section ) . were incubated with TRITC - dextran for 30 min prior to washing , fixing , with DAPI . Representative photomicrographs from 30 analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . H - J . Relative signals were calculated as a of control - ASO treated . 30 across 3 independent experiments were quantified for each condition , with each dot in dotted plot representing 1 image quantified . group means were calculated shown . Error bars represent ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * * representing P < 0 . 0001 , N . S P > 0 . 05 .

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O B-CELL_TYPE O O B-CELL_TYPE O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE 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 O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male C57BL/6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage (mBMDM), dendritic lineage (mBMDC), or microglia-like lineage (mMicroglia, see Methods section). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. H-J. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the dotted plot representing 1 image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with ****representing P <0.0001, N.S P>0.05.


K . Human monocytes purified from venous blood draws from healthy adult volunteers were treated with human cytokines towards a monocytic lineage ( hMDM ) , dendritic lineage ( hMDC ) , or microglia - like lineage ( hMicroglia ) . were incubated with TRITC - dextran for 30 min prior to washing , fixing , with DAPI . Representative photomicrographs from 30 analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . L - N . Relative signals were calculated as a of control - ASO treated . 30 across 3 independent experiments were quantified for each condition , with each dot in plot representing one image quantified . group means were calculated shown . Error bars represent ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * * representing P < 0 . 0001 .

O O B-ORGANISM B-CELL_TYPE O O O B-TISSUE O O O O O O O O B-ORGANISM O O O B-CELL_TYPE O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage (hMDM), dendritic lineage (hMDC), or microglia-like lineage (hMicroglia). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. L-N. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the plot representing one image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.


A . Raw 264 . 7 macrophage were transfected with FLAG ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker ) , or mkate2 ( N - term ) - 2xTAPP1 - PH domain ( PI ( 3 , 4 ) P2 marker ) . Representative photomicrographs ( from 25 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . bounding boxes show " High Mag " panels that magnify representative vesicles . colocalization of Rab10 indicated markers in bounding boxes were analyzed using Image J as described in method shown in " CoLoc " panels . colocalized pixels are shown in with same . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate representative Rab10 vesicles positively colocalized with indicated marker . B . At least 25 across three independent experiments were analyzed for each condition ( see Materials Methods section ) . % of Rab10 vesicles positive with indicated marker within each was quantified . Each dot represents mean value from one analyzed . Group means are given , with error bars representing ± .

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

A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM.


C . Raw 264 . 7 macrophage were transfected with FLAG ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) treated with wortmannin ( 1 μM ) for one hour . Representative photomicrographs ( from 25 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . Scale bars are 10 μm , or 1 μm in " Hig Mag " panel . D . of treated with or without wortmannin harboring Rab10 vesicles was calculated from > 30 across five independent experiments . Each dot on plot represents mean values from one experiment . Error bars represent ± significance was assessed by Mann - Whitney ( owing to non - normal distributions ) with * * indicating P < 0 . 01 .

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

C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01.


E . Raw 264 . 7 were co - transfected with eGFP ( N - term ) - Rab10 mRuby ( N - term ) - Rab5 for 48 hours , with live - recordings for ~ 10 minutes . A representative vesicle is shown over presented time - lapse . Scale bar represents 1 μm . F . intensities of vesicles are calculated over time . Dots show mean values calculated from n = 8 recorded vesicles from from n = 3 biologically independent experiments . Error bars show ± .

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

E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm. F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , were incubated with ( A ) TRITC - dextran ( 70 kDa , shown as red signal ) Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , were incubated with ( B ) DQ - ovalbumin , for 30 min before washing fixation . Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . ( C , D ) were further stained for ( C ) EEA1 , or ( D ) Lamp1 , which was detected with Cy5 dye ( show as red signal ) . Representative photomicrographs are from 25 each analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are magnified in " High Mag " panels that show vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels . arrow heads indicate Rab10 vesicles colocalized with indicated marker .

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

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


E ) 25 across three independent experiments were analyzed for each condition . % of Rab10 vesicles positive with indicated marker in each were quantified . Each dot represents mean results from vesicles analyze in one . group means of % Rab10 vesicles positive with marker are shown , with error bars representing ± . Significance was assessed by Mann - Whitney for each marker , where * * representing p < 0 . 01 , * * * * p < 0 . 0001 N . S p > 0 . 05 .

O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR 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 B-GENEPROD 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

E) 25 cells across three independent experiments were analyzed for each condition. % of Rab10 vesicles positive with the indicated marker in each cell were quantified. Each dot represents the mean results from vesicles analyze in one cell. The group means of % Rab10 vesicles positive with the marker are shown, with error bars representing ± SEM. Significance was assessed by Mann-Whitney test for each marker, where ** representing p<0.01, **** p<0.0001 and N.S p>0.05.


A . Raw 264 . 7 macrophage were transfected with Flag ( N - term ) - Rab10 eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker , epifluorescence , shown in blue ) followed by anti - Flag immunofluorescence ( shown in green ) pT73 - Rab10 antibody ( shown in red ) . To evaluate colocalization of phospho - Rab10 with early endosomes or late endosome / lysosome markers , immunofluorescence detection was performed for endogenous EEA1 or Lamp1 ( shown in blue ) . Raw 264 . 7 macrophage were further fed 0 . 05 mg mL - 1 of 70 kDa TRITC - conjugated dextran ( epifluorescence , shown in blue ) or 0 . 05 mg mL - 1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence . Representative were selected from 25 analyzed for each condition from n = 3 biologically independent experiments . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc ( pRab10 / Rab10 ) ' ' CoLoc ( pRab10 / Marker ) ' columns are indicated . arrow heads highlight vesicles scored as co - positive between pT73 - Rab10 Rab10 or indicated marker . B . 25 across three independent experiments were analyzed for each condition , with each dot representing mean of vesicle across . group means of % pT73 - Rab10 vesicles positive for indicated marker are given , with error bars showing ± .

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

A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in the "CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker. B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM.


A . Raw264 . 7 were transfected with Flag ( N - term ) - LRRK2 eGFP ( N - term ) - Rab10 for 24 hours treated with or without LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from > 20 analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) , immuno - stained for pT73 - Rab10 ( shown as red signal ) LRRK2 ( shown as blue signal ) . bounding boxes are magnified in " High Mag " panels that show vesicles . Scale bars represent 10 μm or 1 μm in " Hig Mag " panels .

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

A. Raw264.7 cells were transfected with Flag(N-term)-LRRK2 and eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells with eGFP(N-term)-Rab10 epifluorescence (shown as green signal), and immuno-stained for pT73-Rab10 (shown as red signal) and LRRK2 (shown as blue signal). White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm or 1 μm in "Hig Mag" panels.


B . Raw 264 . 7 macrophage transfected with eGFP ( N - term ) - Rab10 Flag ( N - term ) - LRRK2 were sequentially lysed mechanically into buffer to create a ' soluble ' fraction , then insoluble material lysed into triton X - 100 buffer to create a " triton x - 100 " fraction . " soluble " triton X - 100 buffer solubilized fractions were analyzed using SDS - PAGE ( TGX gels ) , representative immunoblots from n = 3 independent experiments are shown .

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

B. Raw 264.7 macrophage cells transfected with eGFP(N-term)-Rab10 and Flag(N-term)-LRRK2 were sequentially lysed mechanically into buffer to create a 'soluble' protein fraction, and then insoluble material lysed into triton X-100 buffer to create a "triton x-100" fraction. The "soluble" and triton X-100 buffer solubilized fractions were analyzed using SDS-PAGE (TGX gels), and representative immunoblots from n=3 independent experiments are shown.


C . " triton X - 100 " fraction was used as input for LRRK2 immunoprecipitation , with subsequent detection of total Rab10 pT73 - Rab10 in LRRK2 : Rab10 immunocomplexes using SDS - PAGE ( TGX gels ) . To measure stoichiometry of pT73 - Rab10 to total Rab10 , same immunocomplexes were analyzed using phos - tag SDS - PAGE method , with unphosphorylated eGFP ( N - term ) - Rab10 migrating below phospho - Rab10 . Immunoblots are representative of three independent experiments , with similar results obtained .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O

C. The "triton X-100" fraction was used as input for LRRK2 immunoprecipitation, with subsequent detection of total Rab10 and pT73-Rab10 proteins in LRRK2:Rab10 immunocomplexes using SDS-PAGE (TGX gels). To measure the stoichiometry of pT73-Rab10 to total Rab10, the same immunocomplexes were analyzed using phos-tag SDS-PAGE method, with unphosphorylated eGFP(N-term)-Rab10 migrating below the phospho-Rab10 protein. Immunoblots are representative of three independent experiments, with similar results obtained.


D . Calculated of total LRRK2 : Rab10 with without LRRK2 kinase inhibition . Data are from n = 3 biologically independent experiments with error bars showing ± . Significance was assessed by a two - t - with N . S representing P > 0 . 05 .

O O O O 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 O 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. Calculated levels of total LRRK2:Rab10 protein complex with and without LRRK2 kinase inhibition. Data are from n=3 biologically independent experiments with error bars showing ± SEM. Significance was assessed by a two-tailed t-test with N.S representing P>0.05.


E . signal intensities from pT73 - Rab10 unphosphorylated Rab10 in input lysate co - immunoprecipitated with LRRK2 antibodies . stoichiometry of pT73 - Rab10 was calculated by dividing pT73 - Rab10 signal by total Rab10 signal ( total Rab10 signal equivalent to pT73 - Rab10 signal + unphosphorylated Rab10 signal ) . Data are from n = 3 biologically independent experiments with error bars showing ± ; Significance was assessed by two - t - with * * * representing P < 0 . 0005 .

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

E. The signal intensities from pT73-Rab10 and unphosphorylated Rab10 in the input lysate co-immunoprecipitated with LRRK2 antibodies. The stoichiometry of pT73-Rab10 was calculated by dividing the pT73-Rab10 signal by total Rab10 signal (total Rab10 signal equivalent to pT73-Rab10 signal+ unphosphorylated Rab10 signal). Data are from n=3 biologically independent experiments with error bars showing ± SEM; Significance was assessed by two-tailed t-test with *** representing P<0.0005.


A , B . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - LRRK2 mBAC transgenic mice were pre - incubated with FITC - conjugated dextran ( 70 kDa , green signal ) prior to washing , fixing , immunostaining with total - Rab10 ( shown as red signal ) , or pT73 - Rab10 antibody ( shown as red signal ) . Alternatively , were co - stained with antibodies to Rab5 , Rab7 or Lamp1 ( shown as green signal in separate ) . Representative photomicrographs ( from > 30 analyzed for each condition from n = 3 biologically independent experiments ) are shown . bounding boxes are " High Mag " panels that show vesicles analyzed . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 indicated marker . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels " CoLoc " panels .

O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR 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 B-EXP_ASSAY O O O

A,B. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were pre-incubated with FITC-conjugated dextran (70 kDa, green signal) prior to washing, fixing, and immunostaining with total-Rab10 (shown as red signal), or pT73-Rab10 antibody (shown as red signal). Alternatively, cells were co-stained with antibodies to Rab5, Rab7 or Lamp1 (shown as green signal in separate images). Representative photomicrographs (from >30 images analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels.


C . of 20 across three independent experiments are given for each indicated co - localization , where % of Rab10 or pT73 - Rab10 vesicles positive with indicated markers was calculated for each ( with signal inside of vesicles analyzed for TRITC - dextran DQ - ovalbumin ) . Each dot represents mean value of vesicles analyzed inside one . group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated . Error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * * * * representing p < 0 . 0001 N . S representing p > 0 . 05 .

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 B-GENEPROD O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD 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

C. Analysis of 20 cells across three independent experiments are given for each indicated co-localization, where % of Rab10 or pT73-Rab10 vesicles positive with indicated markers was calculated for each cell (with signal inside of vesicles analyzed for TRITC-dextran and DQ-ovalbumin). Each dot represents the mean value of vesicles analyzed inside one cell. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated. Error bars represent ± SEM. Significance was assessed by Mann-Whitney tests for each marker with **** representing p<0.0001 and N.S representing p>0.05.


D . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - LRRK2 mBAC transgenic mice were treated with 0 . 001 % DMSO or 100 nM MLi2 ( i . e . , IC90 ) for two hours prior to immunostaining with total Rab10 or pT73 - Rab10 antibody ( shown as red signal ) , anti - Rab5 or anti - Rab7antibody ( shown as green signal ) . Representative photomicrographs ( from 20 analyzed for each condition from n = 3 biologically independent experiments ) are shown . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 indicated marker .

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O

D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were treated with 0.001% DMSO or 100 nM MLi2 (i.e., IC90 concentration) for two hours prior to immunostaining with total Rab10 or pT73-Rab10 antibody (shown as red signal), and anti-Rab5 or anti-Rab7antibody (shown as green signal). Representative photomicrographs (from 20 cells analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker.


E . Calculated of double - positive vesicles ( with > 50 % of vesicle boundary demarcated by signal in colocalization ) from 20 counted from n = 3 biologically independent experiments are shown . Each dot represents 1 in dotted plot . group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated shown . Error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * representing p < 0 . 05 , * * P < 0 . 01 .

O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR 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 B-GENEPROD O O O B-GENEPROD 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

E. Calculated percentage of double-positive vesicles (with >50% of the vesicle boundary demarcated by white signal in the colocalization analysis) from 20 cells counted from n=3 biologically independent experiments are shown. Each dot represents 1 cell in the dotted plot. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated and shown. Error bars represent± SEM. Significance was assessed by Mann-Whitney test for each marker with * representing p<0.05, ** P<0.01.


F . Raw 264 . 7 macrophage were transfected with eGFP ( N - term ) - Rab10 for 24 hours treated with or without LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from 25 analyzed for each condition across n = 3 biologically independent experiments ) are shown with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) pT73 - Rab10 ( shown as red signal ) , with EEA1 ( shown as blue signal ) . bounding boxes are " High Mag " panels that show vesicles analyzed . Scale bars represent 10 μm 1 μm for " High Mag " . Positive colocalization signal ( depicted as ) in " CoLoc " panels are given with arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 Rab10 indicated marker . G . Calculated of colocalized vesicles , with each dot representing mean vesicle frequency in each analyzed . group mean of % Rab10 vesicles positive with EEA1 were calculated , error bars represent ± . Significance was assessed by Mann - Whitney for each marker with * * * representing P < 0 . 005 .

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

F. Raw 264.7 macrophage cells were transfected with eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from 25 images analyzed for each condition across n=3 biologically independent experiments) are shown with eGFP(N-term)-Rab10 epifluorescence (shown as green signal) and pT73-Rab10 (shown as red signal), with EEA1 (shown as blue signal). White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 Rab10 and the indicated marker. G. Calculated percentage of colocalized vesicles, with each dot representing mean vesicle frequency in each cell analyzed. The group mean of % Rab10 vesicles positive with EEA1 were calculated, and error bars represent ± SEM. Significance was assessed by Mann-Whitney test for each marker with *** representing P<0.005.


A . Raw264 . 7 were transfected with Flag - Rab10 or control empty vector . transfected were lysed in presence of 2 mM GTPγS or 2 mM GDP . Flag - Rab10 was immunoprecipitated with Flag - resin followed by extensive washing elution of complexes with 200 μg mL - 1 3xFlag - tide . Eluates were analyzed by SDS - PAGE followed by immuno - blotting with anti - Flag - HRP antibody .

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

A. Raw264.7 cells were transfected with Flag-Rab10 or control empty vector. The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP. The Flag-Rab10 complex was immunoprecipitated with Flag-resin followed by extensive washing and elution of complexes with 200 μg mL-1 3xFlag-tide. Eluates were analyzed by SDS-PAGE followed by immuno-blotting with anti-Flag-HRP antibody.


C . Flag ( N - term ) - Rab10 was purified from HEK293 - FT transfected with Flag ( N - term ) - Rab10 plasmids immobilized on Flag - resin in lysis buffer containing 2 mM GTPγS , or 2 mM GDP , as indicated . Beads were mixed with lysates from transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to generate Rab10 : EHBP1L1 complexes . Total Rab10 on beads was detected using anti - Flag antibody , EHBP1L1 immobilized to total Rab10 detected with an anti - Myc antibody . Representative immunoblots from n = 3 biologically independent experiments are shown . D . of relative EHBP1L1 pulled down . Data are from n = 3 biologically independent experiments . Error bars represent ± . . .

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

C. Flag(N-term)-Rab10 protein was purified from HEK293-FT cells transfected with Flag(N-term)-Rab10 plasmids and immobilized on Flag-resin in lysis buffer containing 2 mM GTPγS, or 2 mM GDP, as indicated. Beads were mixed with lysates from cells transfected with Myc-mkate2(N-term)-EHBP1L1 to generate Rab10:EHBP1L1 complexes. Total Rab10 on the beads was detected using anti-Flag antibody, and EHBP1L1 immobilized to total Rab10 detected with an anti-Myc antibody. Representative immunoblots from n=3 biologically independent experiments are shown. D. Quantification of relative EHBP1L1 pulled down. Data are from n=3 biologically independent experiments. Error bars represent ± SEM...


E . HEK293 - FT were transfected with Flag ( N - term ) - Rab10 Myc ( N - term ) - LRRK2R1441C to induce Rab10 phosphorylation . bound Flag ( N - term ) - Rab10 was extracted with Flag - resin to generate ' pRab10 beads , ' or extracted from treated with MLi2 ( 100 nM ) to generate ' Rab10 beads . ' Beads were mixed with lysates from HEK293 - FT transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to form on - bead Rab10 : EHBP1L1 complexes . total Rab10 pT73 - Rab10 on beads was detected using anti - Flag antibody pT73 - Rab10 antibody . EHBP1L1 in lysates immobilized on beads was detected using anti - Myc tag antibody . Representative immunoblots are shown . F . relative EHBP1L1 pulled down , normalized to total Rab10 on - bead , was determined from n = 3 biologically independent experiments . Error bars represent ± . Each dot represents one experiment , with significance assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc P < 0 . 0001 .

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

E. HEK293-FT cells were transfected with Flag(N-term)-Rab10 and Myc(N-term)-LRRK2R1441C to induce Rab10 phosphorylation. Membrane bound Flag(N-term)-Rab10 protein was extracted with Flag-resin to generate 'pRab10 beads,' or extracted from cells treated with MLi2 (100 nM) to generate 'Rab10 beads.' Beads were mixed with lysates from HEK293-FT cells transfected with Myc-mkate2(N-term)-EHBP1L1 to form on-bead Rab10:EHBP1L1 complexes. The total Rab10 and pT73-Rab10 on the beads was detected using anti-Flag antibody and pT73-Rab10 antibody. The EHBP1L1 in the lysates and immobilized on the beads was detected using anti-Myc tag antibody. Representative immunoblots are shown. F. The relative EHBP1L1 pulled down, normalized to total Rab10 protein on-bead, was determined from n=3 biologically independent experiments. Error bars represent ± SEM. Each dot represents one experiment, with significance assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001.


G . Raw 264 . 7 were co - transfected with eGFP ( N - term ) - Rab10 ( epifluorescence shown in green ) Myc - mkate2 ( N - term ) - EHBP1L1 ( epifluorescence shown in blue ) , or Flag ( C - term ) - RILPL2 ( anti - Flag signal shown in blue ) . were stained for pT73 - Rab10 ( shown in red ) . Representative photomicrographs of vesicles ( from > 10 analyzed for each condition from n = 3 biologically independent experiments ) are shown . Scale bar represents 1 μm . H . Pearson ' s colocalization coefficient for Rab10 pT73 - Rab10 , EHBP1L1 RILPL2 on vesicle bodies tubular vesicles . Data are from n = 20 vesicles with tubular structures positive for pT73 - Rab10 , n = 12 vesicles with tubular structures positive for EHBP1L1 , or RILPL2 , calculated from from three independent experiments . Each dot represents one vesicle or tubular structure , with significance assessed by Kruskal - Wallis followed by Dunn ' s multiple comparison with * * * representing P < 0 . 0005 * * * * P < 0 . 0001 . Error bars represent ± . .

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

G. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence shown in green) and Myc-mkate2(N-term)-EHBP1L1 (epifluorescence shown in blue), or Flag(C-term)-RILPL2 (anti-Flag signal shown in blue). Cells were stained for pT73-Rab10 (shown in red). Representative photomicrographs of individual vesicles (from >10 cells analyzed for each condition from n=3 biologically independent experiments) are shown. Scale bar represents 1 μm. H. Pearson's colocalization coefficient for Rab10 and pT73-Rab10, EHBP1L1 and RILPL2 on vesicle bodies and tubular vesicles. Data are from n=20 vesicles with tubular structures positive for pT73-Rab10, and n=12 vesicles with tubular structures positive for EHBP1L1, or RILPL2, calculated from cells from three independent experiments. Each dot represents one vesicle body or tubular structure, with significance assessed by Kruskal-Wallis test followed by Dunn's multiple comparison test with *** representing P<0.0005 and **** P<0.0001. Error bars represent ± SEM..


I . Raw264 . 7 were co - transfected with Flag ( N - term ) - Rab10 , Flag ( N - term ) - LRRK2R1441C , Myc ( N - term ) - EHBP1L1 , with representative immunoblots shown . J . Relative pT73 - Rab10 were calculated as fold of transfected with Flag ( N - term ) - Rab10 Flag ( N - term ) - LRRK2 only . Data are from n = 3 biologically independent experiments . Each dot represents one independent experiment . Significance was assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc P < 0 . 0001 .

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

I. Raw264.7 cells were co-transfected with Flag(N-term)-Rab10, Flag(N-term)-LRRK2R1441C, and Myc(N-term)-EHBP1L1, with representative immunoblots shown. J. Relative pT73-Rab10 levels were calculated as fold of cells transfected with Flag(N-term)-Rab10 and Flag(N-term)-LRRK2 only. Data are from n=3 biologically independent experiments. Each dot represents one independent experiment. Significance was assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001.