Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

  1. Andrea Mair
  2. Shou-Ling Xu
  3. Tess C Branon
  4. Alice Y Ting
  5. Dominique C Bergmann  Is a corresponding author
  1. Stanford University, United States
  2. Howard Hughes Medical Institute, United States
  3. Carnegie Institution for Science, United States
  4. Massachusetts Institute of Technology, United States
  5. Chan Zuckerberg Biohub, United States
6 figures, 2 tables and 4 additional files

Figures

Figure 1 with 5 supplements
TbID and mTb exhibit robust biotinylation activity in N. benthamiana and Arabidopsis.

(A) Overview of the experimental setup. UBQ10pro::BirA*/TbID/mTb-YFP constructs with an NLS or NES for nuclear or cytosolic localization were used for transient and stable transformation of N. …

https://doi.org/10.7554/eLife.47864.003
Figure 1—figure supplement 1
Subcellular localization of biotin ligase constructs in transiently transformed N. benthamiana leaves.

Epifluorescence images of N. benthamiana leaves transformed with UBQ10pro::BirA-YFPNLS or UBQ10pro::BirA-NESYFP expression vectors (BirA = BirA*, mTb or TbID) 2 days after transformation. Shown are …

https://doi.org/10.7554/eLife.47864.004
Figure 1—figure supplement 2
Subcellular localization of biotin ligase constructs in stable Arabidopsis lines.

Confocal microscopy images of the cotyledon epidermis of 5-day-old Arabidopsis seedlings transformed with UBQ10pro::BirA-YFPNLS or UBQ10pro::BirA-NESYFP expression vectors (BirA = BirA*, mTb or …

https://doi.org/10.7554/eLife.47864.005
Figure 1—figure supplement 3
TbID and mTb are highly active in the cytosol and nucleus of transiently transformed N. benthamiana leaves.

N. benthamiana leaves were transformed with UBQ10pro::BirA-YFPNLS or UBQ10pro::BirA-NESYFP expression vectors (BirA = BirA*, mTb or TbID). Two days after infiltration, leaf discs expressing either …

https://doi.org/10.7554/eLife.47864.006
Figure 1—figure supplement 4
TbID is more active than mTb, but also produces more background labeling in Arabidopsis.

Five-day-old Arabidopsis seedlings expressing UBQ10pro::BirA-YFPNLS or UBQ10pro::BirA-NESYFP constructs (BirA = BirA*, mTb or TbID) were submerged in 250 µM biotin, briefly vacuum infiltrated and …

https://doi.org/10.7554/eLife.47864.007
Figure 1—figure supplement 5
Generating a toolbox of gateway-compatible vectors for PL in plants.

Schematic overview over cloning steps involved in the generation of the PL toolbox and over the available vectors (highlighted as orange boxes). Analogous constructs for different biotin ligase …

https://doi.org/10.7554/eLife.47864.008
Figure 2 with 3 supplements
TbID and mTb work quickly and tolerate a range of experimental conditions in Arabidopsis seedlings.

(A–D) Dependency of TbID and mTb activity on labeling time, temperature, biotin concentration and biotin application. Four- to 5-day-old seedlings were treated with biotin as described below. …

https://doi.org/10.7554/eLife.47864.009
Figure 2—figure supplement 1
Biotinylation by TbID in Arabidopsis increases over time.

Labeling time course with TbID in 4-day-old Arabidopsis seedlings expressing the UBQ10pro::TbID-YFPNLS construct (TbID-YFPNLS). Seedlings were submerged in 50 μM biotin, briefly vacuum infiltrated …

https://doi.org/10.7554/eLife.47864.010
Figure 2—figure supplement 2
TbID and mTb are active from 22°C to 37°C in N. benthamiana.

Streptavidin-HRP (SA) and anti-GFP immunoblots (IB) showing the activity and expression of TbID and mTb at room temperature (22°C), 30°C and 37°C. N. benthamiana leaves were transformed with …

https://doi.org/10.7554/eLife.47864.011
Figure 2—figure supplement 3
Quantification of TbID and mTb activity in Arabidopsis at different biotin concentrations.

Quantification of the streptavidin-HRP (SA) immunoblots (IB) shown in Figure 2C. Five-day-old seedlings transformed with UBQ10pro::TbID-NESYFP (TbID-NESYFP) and UBQ10pro::mTb-NESYFP (mTb-NESYFP) …

https://doi.org/10.7554/eLife.47864.012
Figure 3 with 3 supplements
TbID works in different developmental stages and organs of Arabidopsis and does not require vacuum infiltration of biotin.

TbID activity in shoots and roots of 10-day-old plate-grown UBQ10pro::TbID-YFPNLS (TbID-YFP) seedlings, and in rosette leaves and unopened flower buds of mature soil-grown plants. Col-0 wild-type …

https://doi.org/10.7554/eLife.47864.013
Figure 3—figure supplement 1
Activity and background labeling of TbID are similar in seedlings ranging from 4 to 14 days of age.

Plate-grown Arabidopsis wild-type (WT) and UBQ10pro::TbID-YFPNLS (TbID-YFPNLS) seedlings of the indicated age (dpg = days post germination) were submerged in a 250 µM biotin solution, briefly vacuum …

https://doi.org/10.7554/eLife.47864.014
Figure 3—figure supplement 2
Activity and background labeling of TbID are similar in roots and shoots of 6- to 14-day-old seedlings.

Six, 10 and 14 days post germination (dpg), plate-grown Arabidopsis wild-type (WT) and UBQ10pro::TbID-YFPNLS (TbID-YFPNLS) seedlings were divided into a shoot and root section. Shoots and roots were …

https://doi.org/10.7554/eLife.47864.015
Figure 3—figure supplement 3
UBQ10pro::TbID-YFPNLS is expressed throughout the whole plant.

Epifluorescence microscopy images of plate-grown Arabidopsis seedlings and soil-grown mature tissues from the strong TbID-YFPNLS line used in Figures 2 and 3 and the PL experiments in Figures 46. …

https://doi.org/10.7554/eLife.47864.016
Figure 4 with 7 supplements
Testing TbID’s potential to label protein interactors and subcellular proteomes in a rare cell type in Arabidopsis.

(A) Plant lines generated for the ‘FAMA interactome’ and ‘nuclear proteome’ experiments. Line names and genotypes are given on the top, schematic expression of the TbID fusion proteins (yellow dots) …

https://doi.org/10.7554/eLife.47864.017
Figure 4—figure supplement 1
Expression of the TbID constructs in lines used for the ‘FAMA interactome’ and ‘nuclear proteome’ PL experiments.

Confocal microscopy images of the cotyledon epidermis of 5-day-old Arabidopsis seedlings. Shown are the YFP channel (yellow), the cell walls stained with propidium iodide (purple) and an overlay. …

https://doi.org/10.7554/eLife.47864.018
Figure 4—figure supplement 2
The FAMApro::FAMA-TbID-Venus construct rescues the fama-1 mutant phenotype.

(A) FAMA-TbID-Venus rescues the seedling-lethal growth phenotype of fama-1. Col-0 wild-type (WT), fama-1 -/- and the FAMA-TbID rescue line (FAMApro::FAMA-TbID-Venus in fama-1 - /- background) were …

https://doi.org/10.7554/eLife.47864.019
Figure 4—figure supplement 3
FAMA-TbID self-labeling is visible within 5 min of biotin treatment and increases over time, regardless of vacuum infiltration.

Time course to determine time points for the PL experiments with FAMA-TbID. Five-day-old wild-type (WT) and FAMApro::FAMA-TbID-Venus (FAMA-TbID-Venus) seedlings were submerged in 250 µM biotin, …

https://doi.org/10.7554/eLife.47864.020
Figure 4—figure supplement 4
Confirming successful labeling of proteins in the PL experiment.

For the PL experiments shown in Figures 46, 5-day-old wild-type (WT), FAMA-TbID, FAMAnucTbID and UBQnucTbID seedlings were submerged in a 50 μM biotin solution for 0, 0.5 and 3 h and washed …

https://doi.org/10.7554/eLife.47864.021
Figure 4—figure supplement 5
Affinity purification of biotinylated proteins in the PL experiment.

Five-day-old wild-type (WT), FAMA-TbID, FAMAnucTbID and UBQnucTbID seedlings were treated with biotin for 0, 0.5, and 3 h (see Figure 4—figure supplement 4) and used for affinity purification (AP) …

https://doi.org/10.7554/eLife.47864.022
Figure 4—figure supplement 6
Free biotin from biotin treatment out-competes biotinylated proteins for streptavidin bead binding.

Four-day-old wild-type (WT) and UBQ10pro::TbID-YFPNLS (TbID-YFPNLS) seedlings were submerged in H2O (-) or 50 μM biotin for 1 h and used for affinity purification of biotinylated proteins with …

https://doi.org/10.7554/eLife.47864.023
Figure 4—figure supplement 7
Comparison of different biotin depletion methods and bead concentrations for an effective pulldown of biotinylated proteins.

Five-day-old seedlings expressing the UBQ10pro::TbID-YFPNLS construct were submerged in a 50 μM biotin solution for 3 h and used for biotin depletion-affinity purification (AP) experiments. For …

https://doi.org/10.7554/eLife.47864.024
Figure 5 with 6 supplements
‘FAMA interactome’ experiment – PL with TbID reveals potential FAMA interactors involved in transcriptional regulation.

Workflow (left) and results (right) of the experimental setup and data filtering process. Biotinylated proteins from seedlings expressing FAMA-TbID or nuclear TbID in FAMA-stage cells (FAMAnucTbID) …

https://doi.org/10.7554/eLife.47864.026
Figure 5—figure supplement 1
FAMA-CFP AP-MS experiments identified ICE1, but no novel transcriptional regulators as putative FAMA partners.

(A) Simplified workflow of the AP-MS experiments. Four-day-old seedlings of a FAMA-CFP line (FAMApro::FAMA-CFP in fama-1) and two lines expressing nuclear GFP under stomatal lineage-specific …

https://doi.org/10.7554/eLife.47864.027
Figure 5—figure supplement 2
Clustering and PCA of samples for the ‘FAMA interactome’ PL experiment.

(A) Hierarchical clustering. Proteins that were identified in all three replicates of at least one time point of one genotype were used for average linkage clustering with Euclidian distance. Rows …

https://doi.org/10.7554/eLife.47864.028
Figure 5—figure supplement 3
Multi scatter plot of samples for the ‘FAMA interactome’ PL experiment.

Proteins that were identified in all three replicates of at least one time point of one genotype were used for a multi scatter plot. For each pair-wise comparison, LFQ values of individual proteins …

https://doi.org/10.7554/eLife.47864.029
Figure 5—figure supplement 4
Significantly enriched proteins in the FAMA-TbID and FAMAnucTbID lines.

For each time point, proteins that were significantly enriched in FAMA-TbID or FAMAnucTbID compared to wild-type (WT) were determined by two-sided t-tests with a permutation-based FDR for multiple …

https://doi.org/10.7554/eLife.47864.030
Figure 5—figure supplement 5
Validation of FAMA complex candidates.

Y2H (A–B) and pairwise proximity labeling assays (C) to test direct interaction and relative proximity of four putative FAMA interaction candidates: co-repressor complex components SEU and LUH and …

https://doi.org/10.7554/eLife.47864.031
Figure 5—figure supplement 6
Increase of biotinylation in the PL samples over time.

Proteins that were significantly enriched in wild-type (WT), FAMA-TbID or FAMAnucTbID samples after 0.5 and after 3 h of biotin treatment compared to untreated samples were determined by two-sided …

https://doi.org/10.7554/eLife.47864.032
Figure 6 with 4 supplements
‘Nuclear proteome’ experiment – PL with nuclear TbID results in identification of FAMA- and global nuclear proteomes with high organellar specificity.

Workflow (left) and results (right) of the experimental setup and data filtering process. Biotinylated proteins from seedlings expressing nuclear TbID under the FAMA (FAMAnucTbID) and UBQ10 (UBQnucTb…

https://doi.org/10.7554/eLife.47864.033
Figure 6—figure supplement 1
Clustering and PCA of samples for the ‘nuclear proteome’ PL experiment.

(A) Hierarchical clustering. Proteins that were identified in all three replicates of at least one genotype were used for average linkage clustering with Euclidian distance. Rows and columns …

https://doi.org/10.7554/eLife.47864.034
Figure 6—figure supplement 2
Multi scatter plot of samples for the ‘nuclear proteome’ PL experiment.

Proteins that were identified in all three replicates of at least one genotype were used for a multi scatter plot. For each pair-wise comparison, LFQ values of individual proteins in sample A …

https://doi.org/10.7554/eLife.47864.035
Figure 6—figure supplement 3
Significantly enriched proteins in FAMA-expressing and all nuclei.

Proteins that were significantly enriched in FAMAnucTbID or UBQnucTbID compared to wild-type (WT) were determined by two-sided t-tests with a permutation-based FDR for multiple sample correction …

https://doi.org/10.7554/eLife.47864.036
Figure 6—figure supplement 4
GO terms enriched in global and FAMA nuclear proteomes.

GO terms enriched for global and FAMA nuclear proteins (Figure 6) were determined with AgriGO v2 and visualized with REViGO. Shown is the TreeMap plot of enriched ‘biological processes’ in global …

https://doi.org/10.7554/eLife.47864.037

Tables

Table 1
FAMA complex candidates from Figure 5.
https://doi.org/10.7554/eLife.47864.025
Enriched at time pointAGIGene nameFunctional annotationSubcellular localization
0 h0.5 h3 h
YYAT3G26744ICE1, SCRMbHLH transcription factorN
YYAT2G46510AIB, JAM1bHLH transcription factorN
YAT4G16430JAM3bHLH transcription factorN, C
YAT5G08130BIM1bHLH transcription factorN
YAT1G75080BZR1Transcription factorN, C
YYAT5G11060KNAT4Homeobox transcription factorN, C
YAT2G41900OXS2, TCF7Zinc finger transcription factor(N), C
YAT1G79000HAC1, PCAT2Transcriptional co-activator (histone acetyltransferase)N, C
YAT4G04920MED16, SFR6Transcriptional co-activator (mediator complex)N
YAT1G43850SEUTranscriptional co-repressor adapterN
YAT4G32551LUG, RON2Transcriptional co-repressorN
YAT2G32700LUH, MUM1Transcriptional co-repressorN
YAT3G15880TPR4, WSIP2Transcriptional co-repressorN
YAT5G27030TPR3Transcriptional co-repressorN
YAT5G02500HSP70-1HSP70 chaperoneN, C
YYAT5G02490HSP70-2HSP70 chaperoneN, C
YAT3G09440HSP70-3HSP70 chaperoneN, C
YAT3G12580HSP70-4HSP70 chaperoneN, C
YAT5G22060J2HSP70 co-chaperoneN
YAT3G44110J3HSP70 co-chaperoneN, C, MA
YAT1G62740HOP2HSP90/70 co-chaperone(N), C
YAT3G25230FKBP62, ROF1HSP90/70 co-chaperone(N), C
YAT4G22670HIP1, TPR11HSP90/70 co-chaperoneN, C
 YYYAT4G02450P23-1HSP90 co-chaperoneN, C
 YYYAT5G56460Putative protein kinasePM
YAT5G35410SOS2, CIPK24Protein kinaseN, C, PM
YAT3G54170FIP37m6A methyltransferase complex componentN
YAT1G02140HAP1, MAGOExon-junction complex componentN, C
YAT5G41880POLA3, POLA4Putative DNA polymerase alpha subunitN
YAT3G22380TICNuclear clock regulation factorN
YAT2G41100TCH3, CAL12Calcium-binding proteinN
YAT1G72390PHLNuclear receptor/co-activatorN, C
YYAT1G20110FREE1, FYVE1ESCRT-I complex componentC, ES, N
YAT1G18660IAP1C3HC4-type RING-finger domain proteinMA, N
 YYYAT1G12200FMOPutative flavin monooxygenaseN/A
YYAT3G53260PAL2Phenylalanine ammonia-lyaseN, C, EX
YAT3G23840CER26-LIKEacyl‐CoA‐dependent acyltransferaseN/A
YAT5G13710CPH, SMT1C‐24 sterol methyl transferaseN
YAT1G63180UGE3UDP-Glucose 4-EpimeraseC*
YAT5G17990PAT1, TRP1Phosphoribosylanthranilate transferaseCP*
YAT1G15980NDH48, NDF1Chloroplast NAD(P)H dehydrogenase complex subunitCP
YAT4G30720PDE327Putative oxidoreductase/electron carrierCP
YAT1G50570Undescribed proteinN
YAT1G30070Undescribed proteinN
YAT5G15680Undescribed proteinN/A
YAT5G53330Undescribed proteinN
YAT4G25290Undescribed proteinN/A
  1. Column labels: Enriched at time point: time points at which a protein was significantly enriched are marked with Y. AGI: Arabidopsis gene identifier. Subcellular localization: as described for fluorescent protein fusions in literature unless marked with * (localization inferred from functional annotation): N, nucleus; (N), nucleus under heat or other stress; C, cytosol; EX, extracellular; PM, plasma membrane; ES, endosomes; MA, membrane (associated); CP, chloroplast; N/A, localization unknown (no experimental evidence found and localization cannot be clearly inferred from function).

    For further information on the candidate proteins and selected references see Supplementary file 2 – Table 4.

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (E. coli - modified)BirA*Branon et al., 2018; DOI: 10.1038/nbt.4201R118G mutant of BirA; promiscuous bacterial biotin ligase
Gene (E. coli - modified)TurboID; TbIDBranon et al., 2018; DOI: 10.1038/nbt.4201more active variant of BirA*
Gene (E. coli - modified)miniTurbo; mTbBranon et al., 2018; DOI: 10.1038/nbt.4201smaller and more active variant of BirA*
Gene (Arabidopsis thaliana)FAMANATAIR:AT3G24140transcription factor involved in stomatal development
Gene (Arabidopsis thaliana)SPCHNATAIR:AT5G53210transcription factor involved in stomatal development
Gene (Arabidopsis thaliana)MUTENATAIR:AT3G06120transcription factor involved in stomatal development
Gene (Arabidopsis thaliana)SEUNATAIR:AT1G43850component of transcriptional co-repressor complex
Gene (Arabidopsis thaliana)LUHNATAIR:AT2G32700component of transcriptional co-repressor complex
Gene (Arabidopsis thaliana)BZR1NATAIR:AT1G75080transcription factor involved in brassinosteroid signaling
Gene (Arabidopsis thaliana)BIM1NATAIR:AT5G08130transcription factor involved in brassinosteroid signaling
Gene (Arabidopsis thaliana)ICE1NATAIR:AT3G26744transcription factor involved in stomatal development and cold adaptation
Strain, strain background (E. coli)TOP10otherchemically competent E. coli, can be obtained from Invitrogen
Strain, strain background (Saccharomyces cerevisiae)AH109Clontech
Strain, strain background (Agrobacterium thumefaciens)GV3101otherelectrocompetent A. thumefaciens
Strain, strain background (Nicotiana benthamiana)NB-1NAstandard lab strain
Strain, strain background (Arabidopsis thaliana)Columbia-0; Col-0ABRCABRC:CS28166can be obtained from ABRC
Genetic reagent
(Arabidopsis thaliana)
fama-1Ohashi-Ito and Bergmann, 2006; DOI: 10.1105/tpc.106.046136ABRC:SALK_100073
Genetic reagent (Arabidopsis thaliana)UBQ10pro::BirA*-YFPNLSThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)UBQ10pro::TbID-YFPNLSThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)UBQ10pro::mTb-YFPNLSThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)UBQ10pro::BirA*-NESYFPThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)UBQ10pro::TbID-NESYFPThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)UBQ10pro::mTb-NESYFPThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)FAMApro::FAMA-TbID-mVenusThis paperin fama-1 - /- background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)FAMApro::TbID-YFPNLSThis paperin Col-0 wild-type background; see Materials and methods for line generation
Genetic reagent (Arabidopsis thaliana)FAMApro::FAMA-CFPWeimer et al., 2018; DOI: 10.1242/dev.160671
Genetic reagent (Arabidopsis thaliana)SPCHpro::GFPNLSAdrian et al. (2015); DOI: 10.1016/j.devcel.2015.01.025
Genetic reagent (Arabidopsis thaliana)MUTEpro::GFPNLSAdrian et al. (2015); DOI: 10.1016/j.devcel.2015.01.025
AntibodyStreptavidin-HRPThermo Fisher ScientificThermo Fisher Scientific:S9110.2 µg/ml; 5% BSA in TBS-T
AntibodyRat monoclonal anti-GFP antibodyChromotekChromotek:3H91:2000; 1–5% skim milk in TBS-T
AntibodyAnti-HA High Affinity from rat IgG1RocheRoche:118674230011:1000; 3–5% skim milk in TBS-T
AntibodyMyc-Tag (71D10) Rabbit mAbCell SignalingCell Signaling:2278S1:1000; 5% BSA in TBS-T
AntibodyAffiniPure Donkey Anti-Rat IgG-HRPJackson Immuno Research LaboratoriesJackson Immuno Research Laboratories:712-035-1531:10000; 1–5% skim milk in TBS-T
AntibodyRabbit Anti-Rat IgG-HRPSigmaSigma:A57951:10000; 1–5% skim milk in TBS-T
AntibodyGoat anti-Rabbit IgG (H and L), HRP conjugatedAgriseraAgrisera:AS09 6021:20000; 3–5% skim milk in TBS-T
Recombinant DNA reagentR4pGWB601 (plasmid)Nakamura et al., 2010; DOI: 10.1271/bbb.100184RIKEN BRC:pdi00133obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm)
Recombinant DNA reagentR4pGWB613 (plasmid)Nakamura et al., 2010; DOI: 10.1271/bbb.100184RIKEN BRC:pdi00099obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm)
Recombinant DNA reagentR4pGWB616 (plasmid)Nakamura et al., 2010; DOI: 10.1271/bbb.100184RIKEN BRC:pdi00102obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm)
Recombinant DNA reagentpB7m34GW,0 (plasmidKarimi et al., 2005; DOI: 10.1016/j.tplants.2005.01.008
Recombinant DNA reagentpK7m34GW,0 (plasmid)Karimi et al., 2005; DOI: 10.1016/j.tplants.2005.01.008
Recombinant DNA reagentpENTR5'/TOPO (plasmid)InvitrogenGateway entry vector for promoters
Recombinant DNA reagentpENTR/D-TOPO (plasmidInvitrogenGaterway entry vector for tags/genes
Recombinant DNA reagentpDONR-P2R-P3 (plasmid)InvitrogenGateway entry vector for tags/genes
Recombinant DNA reagentpGADT7-GW (plasmid)Lu et al., 2010; DOI: 10.1111/j.1365-313X.2009.04048.xAddgene:61702Gateway compatible Y2H prey vector (Gal4 activation domain)
Recombinant DNA reagentpXDGATcy86 (plasmid)Ding et al., 2007;DOI: 10.1385/1-59259-966-4:85Gateway compatible Y2H bait vector (Gal4 DNA-binding domain)
Recombinant DNA reagentV5-hBirA(R118G)-NES_pCDNA3 (plasmid)Branon et al., 2018; DOI: 10.1038/nbt.4201obtained from Ting lab
Recombinant DNA reagentV5-hBirA-Turbo-NES_pCDNA3 (plasmid)Branon et al., 2018; DOI: 10.1038/nbt.4201Addgene:107169obtained from Ting lab
Recombinant DNA reagentV5-hBirA-miniTurbo-NES_pCDNA3 (plasmid)Branon et al., 2018; DOI: 10.1038/nbt.4201Addgene:107170obtained from Ting lab
Recombinant DNA reagentR4pGWB601_UBQ10p_BirA(R118G)-NES-YFP (plasmid)This paperAddgene:127363UBQ10 promoter (2 kb), BirA* (cDNA) with nuclear export signal, YFP in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentR4pGWB601_UBQ10p_BirA(R118G)-YFP-NLS (plasmid)This paperAddgene:127365UBQ10 promoter (2 kb), BirA* (cDNA), YFP with nuclear import signal in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentR4pGWB601_UBQ10p_Turbo-NES-YFP (plasmid)This paperAddgene:127366UBQ10 promoter (2 kb), TurboID (cDNA) with nuclear export signal, YFP in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentR4pGWB601_UBQ10p_Turbo-YFP-NLS (plasmid)This paperAddgene:127368UBQ10 promoter (2 kbA), TurboID (cDNA), YFP with nuclear import signal in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentR4pGWB601_UBQ10p_miniTurbo-NES-YFP (plasmid)This paperAddgene:127369UBQ10 promoter (2 kb), miniTurbo (cDNA) with nuclear export signal, YFP in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentR4pGWB601_UBQ10p_miniTurbo-YFP-NLS (plasmid)This paperAddgene:127370UBQ10 promoter (2 kb), miniTurbo (cDNA), YFP with nuclear import signal in Gateway vector R4pGWB601; see Materials and methods for cloning and Addgene for vector map
Recombinant DNA reagentpB7m34GW,0_FAMAp_gFAMA-Turbo-Venus (plasmid)This paperFAMA promoter (2.4 kb), FAMA (genomic DNA), TurboID, Venus in Gateway vector pB7m34GW,0; see Materials and methods for cloning
Recombinant DNA reagentR4pGWB601_FAMAp_Turbo-YFP-NLS (plasmid)This paperFAMA promoter (2.4 kb), TurboID, YFP with nuclear import signal in Gateway vector R4pGWB601; see Materials and methods for cloning
Recombinant DNA reagentpK7m34GW,0_UBQ10p_cFAMA-TbID-Venus (plasmid)This paperUBQ10 promoter (2 kb), FAMA (cDNA), TurboID, Venus in Gateway vector pB7m34GW,0; see Materials and methods for cloning
Recombinant DNA reagentR4pGWB613_UBQ10p_ICE1-3xHA (plasmid)This paperUBQ10 promoter (2 kb), ICE1 (cDNA) in Gateway vector R4pGWB613; see Materials and methods for cloning
Recombinant DNA reagentR4pGWB613_UBQ10p_MUTE-3xHA (plasmid)This paperUBQ10 promoter (2 kb), MUTE (cDNA) in Gateway vector R4pGWB616; see Materials and methods for cloning
Recombinant DNA reagentR4pGWB313_UBQ10p_SEU-4xmyc (plasmid)This paperUBQ10 promoter (2 kb), SEU (cDNA) in Gateway vector R4pGWB616; see Materials and methods for cloning
Recombinant DNA reagentR4pGWB613_UBQ10p_LUH-3xHA (plasmid)This paperUBQ10 promoter (2 kb), LUH (cDNA) in Gateway vector R4pGWB613; see Materials and methods for cloning
Recombinant DNA reagentpXDGATcy86-FAMA (plasmid)This paperFAMA (cDNA) in Gateway compatible Y2H bait vector pXDGATcy86; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-ICE1 (plasmid)This paperICE1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-MUTE (plasmid)This paperMUTE (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-SEU (plasmid)This paperSEU (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-LUH (plasmid)This paperLUH (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-BZR1 (plasmid)This paperBZR1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentpGADT7-GW-BIM1 (plasmid)This paperBIM1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning
Recombinant DNA reagentAdditional plasmidsfor list of Gateway compatible vectors to generate N- and C-terminal fusions with TbID or mTb ('PL toolbox') see table in Materials and methods section
Sequence-based reagentPrimerssee primer table in Materials and methods
Peptide, recombinant proteinBiotin powderSigmaSigma:B4639
Commercial assay or kitBioRad protein assayBioRadBioRad:5000006
Commercial assay or kitNovex Colloidal blue staining kitInvitrogenInvitrogen: LC6025
Chemical compound, drugDynabeads MyOne Streptavidin C1InvitrogenThermo Fisher:65002
Chemical compound, drugDynabeads MyOne Streptavidin T1InvitrogenThermo Fisher:65601
Chemical compound, drugGFP-Trap_MA beadsChromotTekChromoTek:gtma-20
Software, algorithmMaxQuantTyanova et al., 2016a; DOI: 10.1038/nprot.2016.136version 1.6.2.6
Software, algorithmPerseusTyanova et al., 2016b; DOI: 10.1038/nmeth.3901version 1.6.2.3
Software, algorithmNormalyzerChawade et al., 2014; DOI: 10.1021/pr401264nversion 1.1.1.1 (web interface: http://normalyzer.immunoprot.lth.se/)
Software, algorithmR studioRStudio Team, 2016
Software, algorithmSUBA4Hooper et al., 2017; DOI: 10.1093/nar/gkw1041web interface: http://suba.live/
Software, algorithmAgriGO v2Tian et al., 2017; DOI: 10.1093/nar/gkx382web interface: http://systemsbiology.cau.edu.cn/agriGOv2/
Software, algorithmREViGOSupek et al., 2011; DOI: 10.1371/journal.pone.0021800web interface:http://revigo.irb.hr/
OtherPD-10 Sesalting ColumnGE-HealthcareFisher Scientific:45-000-148

Additional files

Supplementary file 1

Non-cropped immunoblots.

https://doi.org/10.7554/eLife.47864.038
Supplementary file 2

XLS file containing the following additional protein lists for the ‘FAMA interactome’ experiment (Figure 5 and supplements).

Table 1, ‘identified proteins’: Proteins identified in the ‘FAMA interactome’ experiment; Table 2, ‘FAMA-TbID enriched’: Proteins significantly enriched in FAMA-TbID vs. WT and FAMAnucTbID samples and in FAMAnucTbID vs. WT; Table 3, ‘enrichment over time’: Proteins significantly enriched in WT, FAMA-TbID, and FAMAnucTbID after biotin treatment compared to the no-biotin control; Table 4, ‘FAMA complex candidates’: FAMA interaction candidates from Figure 5 and Table 1; Table 5, ‘FAMA AP-MS’: Proteins significantly enriched in AP-MS experiments with FAMA-CFP

https://doi.org/10.7554/eLife.47864.039
Supplementary file 3

XLS file containing the following additional protein lists for the ‘nuclear proteome’ experiment (Figure 6 and supplements).

Table 1, ‘identified proteins’: Proteins identified in the 'nuclear proteome' experiment; Table 2, ‘enriched with UBQnucTbID’: Proteins significantly enriched in UBQnucTbID vs. WT samples; Table 3, ‘enriched with FAMAnucTbID’: Proteins significantly enriched in FAMAnucTbID vs. WT and FAMAnucTbID vs. UBQnucTbID samples; Table 4, ‘published nuclear proteomes’: Proteins found in proteomics studies of purified nuclei or sub-nuclear compartments by mass spectrometry or in localization studies with fluorescent protein fusions; Table 5, ‘localization prediction’: Protein localization data used for pie charts in Figure 6; Table 6, ‘published GC proteome’: Proteins found in published guard cell proteomics experiments; Table 7, ‘GO term enrichment’: Enriched GO terms of nuclear proteins in Figure 6 and supplements; Table 8, ‘nuclear compartments’: Selected marker proteins and protein classes for different nuclear compartments and domains identified in the UBQnucTbID and FAMAnucTbID dataset

https://doi.org/10.7554/eLife.47864.040
Transparent reporting form
https://doi.org/10.7554/eLife.47864.041

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