Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID
- Stanford University, United States
- Howard Hughes Medical Institute, United States
- Carnegie Institution for Science, United States
- Massachusetts Institute of Technology, United States
- Chan Zuckerberg Biohub, United States
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. …
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 …
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 …
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 …
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 …
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 …
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. …
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 …
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 …
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) …
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 …
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 …
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 …
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 4–6. …
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) …
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. …
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 …
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, …
Figure 4—figure supplement 4
Confirming successful labeling of proteins in the PL experiment.
For the PL experiments shown in Figures 4–6, 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 …
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) …
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 …
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 …
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) …
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 …
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 …
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 …
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 …
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 …
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 …
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…
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 …
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 …
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 …
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 …
Tables
Table 1
FAMA complex candidates from Figure 5.
https://doi.org/10.7554/eLife.47864.025| Enriched at time point | AGI | Gene name | Functional annotation | Subcellular localization | ||
|---|---|---|---|---|---|---|
| 0 h | 0.5 h | 3 h | ||||
| Y | Y | AT3G26744 | ICE1, SCRM | bHLH transcription factor | N | |
| Y | Y | AT2G46510 | AIB, JAM1 | bHLH transcription factor | N | |
| Y | AT4G16430 | JAM3 | bHLH transcription factor | N, C | ||
| Y | AT5G08130 | BIM1 | bHLH transcription factor | N | ||
| Y | AT1G75080 | BZR1 | Transcription factor | N, C | ||
| Y | Y | AT5G11060 | KNAT4 | Homeobox transcription factor | N, C | |
| Y | AT2G41900 | OXS2, TCF7 | Zinc finger transcription factor | (N), C | ||
| Y | AT1G79000 | HAC1, PCAT2 | Transcriptional co-activator (histone acetyltransferase) | N, C | ||
| Y | AT4G04920 | MED16, SFR6 | Transcriptional co-activator (mediator complex) | N | ||
| Y | AT1G43850 | SEU | Transcriptional co-repressor adapter | N | ||
| Y | AT4G32551 | LUG, RON2 | Transcriptional co-repressor | N | ||
| Y | AT2G32700 | LUH, MUM1 | Transcriptional co-repressor | N | ||
| Y | AT3G15880 | TPR4, WSIP2 | Transcriptional co-repressor | N | ||
| Y | AT5G27030 | TPR3 | Transcriptional co-repressor | N | ||
| Y | AT5G02500 | HSP70-1 | HSP70 chaperone | N, C | ||
| Y | Y | AT5G02490 | HSP70-2 | HSP70 chaperone | N, C | |
| Y | AT3G09440 | HSP70-3 | HSP70 chaperone | N, C | ||
| Y | AT3G12580 | HSP70-4 | HSP70 chaperone | N, C | ||
| Y | AT5G22060 | J2 | HSP70 co-chaperone | N | ||
| Y | AT3G44110 | J3 | HSP70 co-chaperone | N, C, MA | ||
| Y | AT1G62740 | HOP2 | HSP90/70 co-chaperone | (N), C | ||
| Y | AT3G25230 | FKBP62, ROF1 | HSP90/70 co-chaperone | (N), C | ||
| Y | AT4G22670 | HIP1, TPR11 | HSP90/70 co-chaperone | N, C | ||
| Y | Y | Y | AT4G02450 | P23-1 | HSP90 co-chaperone | N, C |
| Y | Y | Y | AT5G56460 | Putative protein kinase | PM | |
| Y | AT5G35410 | SOS2, CIPK24 | Protein kinase | N, C, PM | ||
| Y | AT3G54170 | FIP37 | m6A methyltransferase complex component | N | ||
| Y | AT1G02140 | HAP1, MAGO | Exon-junction complex component | N, C | ||
| Y | AT5G41880 | POLA3, POLA4 | Putative DNA polymerase alpha subunit | N | ||
| Y | AT3G22380 | TIC | Nuclear clock regulation factor | N | ||
| Y | AT2G41100 | TCH3, CAL12 | Calcium-binding protein | N | ||
| Y | AT1G72390 | PHL | Nuclear receptor/co-activator | N, C | ||
| Y | Y | AT1G20110 | FREE1, FYVE1 | ESCRT-I complex component | C, ES, N | |
| Y | AT1G18660 | IAP1 | C3HC4-type RING-finger domain protein | MA, N | ||
| Y | Y | Y | AT1G12200 | FMO | Putative flavin monooxygenase | N/A |
| Y | Y | AT3G53260 | PAL2 | Phenylalanine ammonia-lyase | N, C, EX | |
| Y | AT3G23840 | CER26-LIKE | acyl‐CoA‐dependent acyltransferase | N/A | ||
| Y | AT5G13710 | CPH, SMT1 | C‐24 sterol methyl transferase | N | ||
| Y | AT1G63180 | UGE3 | UDP-Glucose 4-Epimerase | C* | ||
| Y | AT5G17990 | PAT1, TRP1 | Phosphoribosylanthranilate transferase | CP* | ||
| Y | AT1G15980 | NDH48, NDF1 | Chloroplast NAD(P)H dehydrogenase complex subunit | CP | ||
| Y | AT4G30720 | PDE327 | Putative oxidoreductase/electron carrier | CP | ||
| Y | AT1G50570 | Undescribed protein | N | |||
| Y | AT1G30070 | Undescribed protein | N | |||
| Y | AT5G15680 | Undescribed protein | N/A | |||
| Y | AT5G53330 | Undescribed protein | N | |||
| Y | AT4G25290 | Undescribed protein | N/A | |||
-
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 | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (E. coli - modified) | BirA* | Branon et al., 2018; DOI: 10.1038/nbt.4201 | R118G mutant of BirA; promiscuous bacterial biotin ligase | |
| Gene (E. coli - modified) | TurboID; TbID | Branon et al., 2018; DOI: 10.1038/nbt.4201 | more active variant of BirA* | |
| Gene (E. coli - modified) | miniTurbo; mTb | Branon et al., 2018; DOI: 10.1038/nbt.4201 | smaller and more active variant of BirA* | |
| Gene (Arabidopsis thaliana) | FAMA | NA | TAIR:AT3G24140 | transcription factor involved in stomatal development |
| Gene (Arabidopsis thaliana) | SPCH | NA | TAIR:AT5G53210 | transcription factor involved in stomatal development |
| Gene (Arabidopsis thaliana) | MUTE | NA | TAIR:AT3G06120 | transcription factor involved in stomatal development |
| Gene (Arabidopsis thaliana) | SEU | NA | TAIR:AT1G43850 | component of transcriptional co-repressor complex |
| Gene (Arabidopsis thaliana) | LUH | NA | TAIR:AT2G32700 | component of transcriptional co-repressor complex |
| Gene (Arabidopsis thaliana) | BZR1 | NA | TAIR:AT1G75080 | transcription factor involved in brassinosteroid signaling |
| Gene (Arabidopsis thaliana) | BIM1 | NA | TAIR:AT5G08130 | transcription factor involved in brassinosteroid signaling |
| Gene (Arabidopsis thaliana) | ICE1 | NA | TAIR:AT3G26744 | transcription factor involved in stomatal development and cold adaptation |
| Strain, strain background (E. coli) | TOP10 | other | chemically competent E. coli, can be obtained from Invitrogen | |
| Strain, strain background (Saccharomyces cerevisiae) | AH109 | Clontech | ||
| Strain, strain background (Agrobacterium thumefaciens) | GV3101 | other | electrocompetent A. thumefaciens | |
| Strain, strain background (Nicotiana benthamiana) | NB-1 | NA | standard lab strain | |
| Strain, strain background (Arabidopsis thaliana) | Columbia-0; Col-0 | ABRC | ABRC:CS28166 | can be obtained from ABRC |
| Genetic reagent (Arabidopsis thaliana) | fama-1 | Ohashi-Ito and Bergmann, 2006; DOI: 10.1105/tpc.106.046136 | ABRC:SALK_100073 | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::BirA*-YFPNLS | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::TbID-YFPNLS | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::mTb-YFPNLS | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::BirA*-NESYFP | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::TbID-NESYFP | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | UBQ10pro::mTb-NESYFP | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | FAMApro::FAMA-TbID-mVenus | This paper | in fama-1 - /- background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | FAMApro::TbID-YFPNLS | This paper | in Col-0 wild-type background; see Materials and methods for line generation | |
| Genetic reagent (Arabidopsis thaliana) | FAMApro::FAMA-CFP | Weimer et al., 2018; DOI: 10.1242/dev.160671 | ||
| Genetic reagent (Arabidopsis thaliana) | SPCHpro::GFPNLS | Adrian et al. (2015); DOI: 10.1016/j.devcel.2015.01.025 | ||
| Genetic reagent (Arabidopsis thaliana) | MUTEpro::GFPNLS | Adrian et al. (2015); DOI: 10.1016/j.devcel.2015.01.025 | ||
| Antibody | Streptavidin-HRP | Thermo Fisher Scientific | Thermo Fisher Scientific:S911 | 0.2 µg/ml; 5% BSA in TBS-T |
| Antibody | Rat monoclonal anti-GFP antibody | Chromotek | Chromotek:3H9 | 1:2000; 1–5% skim milk in TBS-T |
| Antibody | Anti-HA High Affinity from rat IgG1 | Roche | Roche:11867423001 | 1:1000; 3–5% skim milk in TBS-T |
| Antibody | Myc-Tag (71D10) Rabbit mAb | Cell Signaling | Cell Signaling:2278S | 1:1000; 5% BSA in TBS-T |
| Antibody | AffiniPure Donkey Anti-Rat IgG-HRP | Jackson Immuno Research Laboratories | Jackson Immuno Research Laboratories:712-035-153 | 1:10000; 1–5% skim milk in TBS-T |
| Antibody | Rabbit Anti-Rat IgG-HRP | Sigma | Sigma:A5795 | 1:10000; 1–5% skim milk in TBS-T |
| Antibody | Goat anti-Rabbit IgG (H and L), HRP conjugated | Agrisera | Agrisera:AS09 602 | 1:20000; 3–5% skim milk in TBS-T |
| Recombinant DNA reagent | R4pGWB601 (plasmid) | Nakamura et al., 2010; DOI: 10.1271/bbb.100184 | RIKEN BRC:pdi00133 | obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm) |
| Recombinant DNA reagent | R4pGWB613 (plasmid) | Nakamura et al., 2010; DOI: 10.1271/bbb.100184 | RIKEN BRC:pdi00099 | obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm) |
| Recombinant DNA reagent | R4pGWB616 (plasmid) | Nakamura et al., 2010; DOI: 10.1271/bbb.100184 | RIKEN BRC:pdi00102 | obtained from the Nakagawa lab (http://shimane-u.org/nakagawa/gbv.htm) |
| Recombinant DNA reagent | pB7m34GW,0 (plasmid | Karimi et al., 2005; DOI: 10.1016/j.tplants.2005.01.008 | ||
| Recombinant DNA reagent | pK7m34GW,0 (plasmid) | Karimi et al., 2005; DOI: 10.1016/j.tplants.2005.01.008 | ||
| Recombinant DNA reagent | pENTR5'/TOPO (plasmid) | Invitrogen | Gateway entry vector for promoters | |
| Recombinant DNA reagent | pENTR/D-TOPO (plasmid | Invitrogen | Gaterway entry vector for tags/genes | |
| Recombinant DNA reagent | pDONR-P2R-P3 (plasmid) | Invitrogen | Gateway entry vector for tags/genes | |
| Recombinant DNA reagent | pGADT7-GW (plasmid) | Lu et al., 2010; DOI: 10.1111/j.1365-313X.2009.04048.x | Addgene:61702 | Gateway compatible Y2H prey vector (Gal4 activation domain) |
| Recombinant DNA reagent | pXDGATcy86 (plasmid) | Ding et al., 2007;DOI: 10.1385/1-59259-966-4:85 | Gateway compatible Y2H bait vector (Gal4 DNA-binding domain) | |
| Recombinant DNA reagent | V5-hBirA(R118G)-NES_pCDNA3 (plasmid) | Branon et al., 2018; DOI: 10.1038/nbt.4201 | obtained from Ting lab | |
| Recombinant DNA reagent | V5-hBirA-Turbo-NES_pCDNA3 (plasmid) | Branon et al., 2018; DOI: 10.1038/nbt.4201 | Addgene:107169 | obtained from Ting lab |
| Recombinant DNA reagent | V5-hBirA-miniTurbo-NES_pCDNA3 (plasmid) | Branon et al., 2018; DOI: 10.1038/nbt.4201 | Addgene:107170 | obtained from Ting lab |
| Recombinant DNA reagent | R4pGWB601_UBQ10p_BirA(R118G)-NES-YFP (plasmid) | This paper | Addgene:127363 | UBQ10 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 reagent | R4pGWB601_UBQ10p_BirA(R118G)-YFP-NLS (plasmid) | This paper | Addgene:127365 | UBQ10 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 reagent | R4pGWB601_UBQ10p_Turbo-NES-YFP (plasmid) | This paper | Addgene:127366 | UBQ10 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 reagent | R4pGWB601_UBQ10p_Turbo-YFP-NLS (plasmid) | This paper | Addgene:127368 | UBQ10 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 reagent | R4pGWB601_UBQ10p_miniTurbo-NES-YFP (plasmid) | This paper | Addgene:127369 | UBQ10 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 reagent | R4pGWB601_UBQ10p_miniTurbo-YFP-NLS (plasmid) | This paper | Addgene:127370 | UBQ10 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 reagent | pB7m34GW,0_FAMAp_gFAMA-Turbo-Venus (plasmid) | This paper | FAMA promoter (2.4 kb), FAMA (genomic DNA), TurboID, Venus in Gateway vector pB7m34GW,0; see Materials and methods for cloning | |
| Recombinant DNA reagent | R4pGWB601_FAMAp_Turbo-YFP-NLS (plasmid) | This paper | FAMA promoter (2.4 kb), TurboID, YFP with nuclear import signal in Gateway vector R4pGWB601; see Materials and methods for cloning | |
| Recombinant DNA reagent | pK7m34GW,0_UBQ10p_cFAMA-TbID-Venus (plasmid) | This paper | UBQ10 promoter (2 kb), FAMA (cDNA), TurboID, Venus in Gateway vector pB7m34GW,0; see Materials and methods for cloning | |
| Recombinant DNA reagent | R4pGWB613_UBQ10p_ICE1-3xHA (plasmid) | This paper | UBQ10 promoter (2 kb), ICE1 (cDNA) in Gateway vector R4pGWB613; see Materials and methods for cloning | |
| Recombinant DNA reagent | R4pGWB613_UBQ10p_MUTE-3xHA (plasmid) | This paper | UBQ10 promoter (2 kb), MUTE (cDNA) in Gateway vector R4pGWB616; see Materials and methods for cloning | |
| Recombinant DNA reagent | R4pGWB313_UBQ10p_SEU-4xmyc (plasmid) | This paper | UBQ10 promoter (2 kb), SEU (cDNA) in Gateway vector R4pGWB616; see Materials and methods for cloning | |
| Recombinant DNA reagent | R4pGWB613_UBQ10p_LUH-3xHA (plasmid) | This paper | UBQ10 promoter (2 kb), LUH (cDNA) in Gateway vector R4pGWB613; see Materials and methods for cloning | |
| Recombinant DNA reagent | pXDGATcy86-FAMA (plasmid) | This paper | FAMA (cDNA) in Gateway compatible Y2H bait vector pXDGATcy86; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-ICE1 (plasmid) | This paper | ICE1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-MUTE (plasmid) | This paper | MUTE (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-SEU (plasmid) | This paper | SEU (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-LUH (plasmid) | This paper | LUH (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-BZR1 (plasmid) | This paper | BZR1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | pGADT7-GW-BIM1 (plasmid) | This paper | BIM1 (cDNA) in Gateway compatible Y2H prey vector pGADT7-GW; see Materials and methods for cloning | |
| Recombinant DNA reagent | Additional plasmids | for 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 reagent | Primers | see primer table in Materials and methods | ||
| Peptide, recombinant protein | Biotin powder | Sigma | Sigma:B4639 | |
| Commercial assay or kit | BioRad protein assay | BioRad | BioRad:5000006 | |
| Commercial assay or kit | Novex Colloidal blue staining kit | Invitrogen | Invitrogen: LC6025 | |
| Chemical compound, drug | Dynabeads MyOne Streptavidin C1 | Invitrogen | Thermo Fisher:65002 | |
| Chemical compound, drug | Dynabeads MyOne Streptavidin T1 | Invitrogen | Thermo Fisher:65601 | |
| Chemical compound, drug | GFP-Trap_MA beads | ChromotTek | ChromoTek:gtma-20 | |
| Software, algorithm | MaxQuant | Tyanova et al., 2016a; DOI: 10.1038/nprot.2016.136 | version 1.6.2.6 | |
| Software, algorithm | Perseus | Tyanova et al., 2016b; DOI: 10.1038/nmeth.3901 | version 1.6.2.3 | |
| Software, algorithm | Normalyzer | Chawade et al., 2014; DOI: 10.1021/pr401264n | version 1.1.1.1 (web interface: http://normalyzer.immunoprot.lth.se/) | |
| Software, algorithm | R studio | RStudio Team, 2016 | ||
| Software, algorithm | SUBA4 | Hooper et al., 2017; DOI: 10.1093/nar/gkw1041 | web interface: http://suba.live/ | |
| Software, algorithm | AgriGO v2 | Tian et al., 2017; DOI: 10.1093/nar/gkx382 | web interface: http://systemsbiology.cau.edu.cn/agriGOv2/ | |
| Software, algorithm | REViGO | Supek et al., 2011; DOI: 10.1371/journal.pone.0021800 | web interface:http://revigo.irb.hr/ | |
| Other | PD-10 Sesalting Column | GE-Healthcare | Fisher Scientific:45-000-148 |
Additional files
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Supplementary file 1
Non-cropped immunoblots.
- https://doi.org/10.7554/eLife.47864.038
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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
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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
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Transparent reporting form
- https://doi.org/10.7554/eLife.47864.041
