In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 3 supplements
MitoID: Expression of BirA*-tagged GTPases on the surface of mitochondria.
(A) Schematic of the MitoID approach in which a GTPase is expressed as a chimera with BirA* and a mitochondrial targeting sequence. Also shown is the structure of a typical Ras superfamily GTPase …
Figure 1—figure supplement 1
Expression of representative BirA*-tagged GTPases on the surface on mitochondria.
(A) Confocal images of HeLa cells expressing the mitochondrially targeted form Rab5A(QL)-BirA* and a GFP-fusion to a PX domain that binds PtdIns(3)P on endosomes. The cells were stained for the HA …
Figure 1—figure supplement 2
Expression levels of BirA*-tagged GTPases on mitochondria.
Immunoblots of cell lysates from HEK293 cells expressing mitochondrially targeted forms of the indicated GTPases, and stained for the HA tag in the chimera and α-tubulin (tub). The anti-HA blot has …
Figure 1—figure supplement 3
Expression of BirA*-tagged GTPases on mitochondria does not induce mitochondrial stress.
Confocal images of HeLa cells stably expressing mCherry-parkin and either untransfected or transfected with plasmids expressing the indicated GTPase chimeras and fixed after 48 hr. Untransfected …
Figure 2
Expression of mitochondrial Rab5A-BirA* chimeras leads to biotinylation of known Rab5A effectors and regulators.
(A) Plot of spectral counts obtained for individual proteins following mass-spectrometric analysis of streptavidin precipitations from cells expressing mitochondrial forms of Rab5A-BirA*. These …
Figure 3 with 1 supplement
Comparison of Rab5A MitoID data to that obtained from sixteen other GTPases.
(A) Spectral counts for the indicated proteins obtained from triple replicates of MitoID with the indicated GTPases. All GTPases are in either the GDP (D) or GTP (T) form and the proteins are ranked …
Figure 3—figure supplement 1
Relative distribution of specific and non-specific interactors on bubble-volcano plots.
(A,B) Comparison of bubble-volcano plots of the MitoID data for known effectors for mammalian Rab2A and Rab5A versus the non-specific interactions obtained with Rab6-GDP, a representative Rab form …
Figure 4
Application of MitoID to GTP-locked forms of Rab2A, Rab6A, Rab7A, Rab9A, Rab8A and Rab10.
(A–F). Bubble-volcano plots of MitoID with the indicated Rabs, each with a mutation predicted to lock it in the GTP-bound form. In each case the Rab is compared to a background comprising the …
Figure 5
Application of MitoID to GTP-locked forms of Rab11A, Rab18, Rab30, and Rab33B.
(A–D). Bubble-volcano plots of MitoID with the GTP-locked forms of the indicated Rabs. In each case the Rab is compared to a background comprising the GDP-locked forms of all seventeen GTPases, with …
Figure 6
Application of MitoID to GDP-locked/empty forms of Rab6A, Rab2A, Rab5A, and Rab11A.
(A–D). Bubble-volcano plots of MitoID with the indicated Rabs, each with a mutation predicted to lock it in the GDP-bound form. In each case the Rab is compared to a background comprising the …
Figure 7
Validation of putative effectors for Rab2A, Rab5A and Rab9A.
(A) Affinity chromatography of cell lysates (input) using GST-fusions to the indicated GTP-locked (QL) or GDP-locked Rabs. The bound proteins were eluted and probed with antibodies to the indicated …
Figure 8
Application of MitoID to N-Ras, RalB and Rheb.
(A–F). Bubble-volcano plots of MitoID with the indicated Ras family GTPases, each with mutations predicted to lock it into the GTP-bound form, with N-Ras also shown with the GDP-bound/empty form. In …
Figure 9 with 1 supplement
Application of MitoID to CDC42, RhoA and Rac1.
(A–F). Bubble-volcano plots of MitoID with the indicated Rho family GTPase, each with mutations predicted to lock it in to either the GTP-bound (A,C,E), or GDP-bound/empty form (B,D,G). In each case …
Figure 9—figure supplement 1
MitoID with sequence variants of GTPases.
(A,B) Bubble-volcano plots of MitoID with the GTP-locked form of Rac1 (Q60L) or with the same GTPase from which a C-terminal stretch of six basic residues has been deleted (Δ183–188). In each case …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | BL21 GOLD (DE3) | Agilent Technologies | 230132 | Used for expression |
| Strain, strain background (E. coli) | Alpha-select gold | Bio-line | BIO-85027 | Used for cloning |
| Cell line (H. sapiens) | mCherry-Parkin, HeLa | (Lazarou et al., 2015) | ||
| Cell line (H. sapiens) | HEK293 | ATCC | ATCC CRL-11268 RRID:CVCL_1926 | |
| Cell line (H. sapiens) | HeLa | ATCC | ATCC CCL-2 RRID:CVCL_0030 | |
| Recombinant DNA reagent | pcDNA3.1 | Clontech | V79020 | |
| Recombinant DNA reagent | pGEX6p2 | GE Healthcare Life Sciences | GE28-9546-50 | |
| Detection reagent | Streptavidin-HRP | Cell Signaling Technology | 3999S RRID:AB_10830897 | WB: 1:300 |
| Antibody | Rat monoclonal anti-HA (3F10) | Roche | 867 423 001 RRID:AB_2314622 | IHC: 1:300 |
| Antibody | Goat polyclonal anti-giantin | Santa Cruz | sc-46993 RRID:AB_2279271 | IHC: 1:200 |
| Antibody | Rabbit polyclonal human COXIV (3E11) | New England Biolabs | 4850S RRID:AB_2085424 | IHC: 1:200 |
| Antibody | Rabbit polyclonal anti- ZFPL1 | Sigma | HPA014909 RRID:AB_1859055 | IHC: 1:300 |
| Antibody | Mouse monoclonal anti-TOM20 | BD Transduction Labs | 612278 RRID:AB_399595 | IHC: 1:200 |
| Antibody | Sheep polyclonal anti-TGN46 | ABD serotec | AHP500G RRID:AB_323104 | IHC: 1:300 |
| Antibody | Rabbit polyclonal anti-golgin-84 | Sigma | HPA000992 RRID:AB_1079009 | IHC: 1:300 |
| Antibody | Mouse monoclonal anti-HIS6 tag | Abcam | Ab18184 RRID:AB_444306 | WB: 1:1000 |
| Antibody | Rabbit polyclonal Anti-STAMBPL1 | Sigma | SAB4200146 RRID:AB_10622601 | WB: 1:1000 |
| Antibody | Rabbit polyclonal anti-NDE1 | Life Technologies | 711424 RRID:AB_2692258 | WB: 1:500 |
| Antibody | Rabbit polyclonal anti-TBCK | Cambridge Bioscience | HPA039951 RRID:AB_10795300 | WB: 1:500 |
| Antibody | Rabbit polyclonal anti-RELCH/KIAA1468 | Cambridge Bioscience | HPA040038 RRID:AB_10793860 | WB: 1:1000 |
| Antibody | Rabbit monoclonal anti-OSBPL9 | Abcam | ab151691 | WB: 1:1000 |
| Antibody | Mouse monoclonal anti-FLAG (M2) | Sigma | F1804 RRID:AB_262044 | WB: 1:5000 |
| Antibody | Rabbit polyclonal anti-ARFGEF3 | ThermoFisher Scientific | PA5-57623 RRID:AB_2638119 | WB: 1:1000 |
| Antibody | Alexa 488 Donkey polyclonal anti-Rat IgG | ThermoFisher Scientific | A21208 RRID:AB_2535794 | IHC: 1:400 |
| Antibody | Alexa 647 Donkey polyclonal anti-Goat IgG | ThermoFisher Scientific | A32849 RRID:AB_2762840 | IHC: 1:400 |
| Antibody | Alexa 555 Donkey polyclonal anti-Rabbit IgG | ThermoFisher Scientific | A32794 RRID:AB_2762834 | IHC: 1:400 |
| Antibody | Alexa 647 Donkey polyclonal anti-Rabbit IgG | ThermoFisher Scientific | A32795 RRID:AB_2762835 | IHC: 1:400 |
| Antibody | Alexa 555Donkey polyclonal anti-mouse IgG | ThermoFisher Scientific | A32773 RRID:AB_2762848 | IHC: 1:400 |
| Recombinant DNA reagent | STAMBPL1 | Addgene | #22559 | |
| Recombinant DNA reagent | TBCK | Insight Biotechnology | RC203605 | |
| Recombinant DNA reagent | HPS3 | Insight Biotechnology | RC204569 | |
| Recombinant DNA reagent | NDE1 | Stratech Scientific | HG15586 | |
| Recombinant DNA reagent | Rab2AQ65L-BirA*-HA-MAO | This study | pN40 | Deposited at Addgene |
| Recombinant DNA reagent | Rab2AS20N-BirA*-HA-MAO | This study | JB2 | Deposited at Addgene |
| Recombinant DNA reagent | Rab5AQ79L-BirA*-HA-MAO | This study | JB28 | Deposited at Addgene |
| Recombinant DNA reagent | Rab5AS34N-BirA*-HA-MAO | This study | JB40 | Deposited at Addgene |
| Recombinant DNA reagent | Rab6AQ72L-BirA*-HA-MAO | This study | pO40 | Deposited at Addgene |
| Recombinant DNA reagent | Rab6AT27N-BirA*-HA-MAO | This study | JB4 | Deposited at Addgene |
| Recombinant DNA reagent | Rab7AQ67L-BirA*-HA-MAO | This study | JB93 | Deposited at Addgene |
| Recombinant DNA reagent | Rab7AT22N-BirA*-HA-MAO | This study | JB94 | Deposited at Addgene |
| Recombinant DNA reagent | Rab8AQ67L-BirA*-HA-MAO | This study | JB24 | Deposited at Addgene |
| Recombinant DNA reagent | Rab8AT22N-BirA*-HA-MAO | This study | JB23 | Deposited at Addgene |
| Recombinant DNA reagent | Rab9AQ66L-BirA*-HA-MAO | This study | JB84 | Deposited at Addgene |
| Recombinant DNA reagent | Rab9AS21N-BirA*-HA-MAO | This study | JB85 | Deposited at Addgene |
| Recombinant DNA reagent | Rab10Q68L-BirA*-HA-MAO | This study | JB81 | Deposited at Addgene |
| Recombinant DNA reagent | Rab10T23N-BirA*-HA-MAO | This study | JB82 | Deposited at Addgene |
| Recombinant DNA reagent | Rab11AQ69L-BirA*-HA-MAO | This study | JB20 | Deposited at Addgene |
| Recombinant DNA reagent | Rab11AS25N-BirA*-HA-MAO | This study | JB19 | Deposited at Addgene |
| Recombinant DNA reagent | Rab11BQ70L-BirA*-HA-MAO | This study | pX49 | Deposited at Addgene |
| Recombinant DNA reagent | Rab11BS25N-BirA*-HA-MAO | This study | pY49 | Deposited at Addgene |
| Recombinant DNA reagent | Rab18Q67L-BirA*-HA-MAO | This study | JB11 | Deposited at Addgene |
| Recombinant DNA reagent | Rab18S22N-BirA*-HA-MAO | This study | JB10 | Deposited at Addgene |
| Recombinant DNA reagent | Rab30Q68L-BirA*-HA-MAO | This study | JB15 | Deposited at Addgene |
| Recombinant DNA reagent | Rab30T23N-BirA*-HA-MAO | This study | JB3 | Deposited at Addgene |
| Recombinant DNA reagent | Rab33BQ92L-BirA*-HA-MAO | This study | JB26 | Deposited at Addgene |
| Recombinant DNA reagent | Rab33BT47N-BirA*-HA-MAO | This study | JB25 | Deposited at Addgene |
| Recombinant DNA reagent | nRasG12D-BirA*-HA-MAO | This study | JB95 | Deposited at Addgene |
| Recombinant DNA reagent | nRasS17N-BirA*-HA-MAO | This study | JB96 | Deposited at Addgene |
| Recombinant DNA reagent | Rac1Q60L-BirA*-HA-MAO | This study | JB42 | Deposited at Addgene |
| Recombinant DNA reagent | Rac1T17N-BirA*-HA-MAO | This study | JB54 | Deposited at Addgene |
| Recombinant DNA reagent | RalBG23V-BirA*-HA-MAO | This study | JB97 | Deposited at Addgene |
| Recombinant DNA reagent | RalBS29A-BirA*-HA-MAO | This study | JB98 | Deposited at Addgene |
| Recombinant DNA reagent | RhoAQ63L-BirA*-HA-MAO | This study | JB68 | Deposited at Addgene |
| Recombinant DNA reagent | RhoAT19N-BirA*-HA-MAO | This study | JB55 | Deposited at Addgene |
| Recombinant DNA reagent | Cdc42G12V-BirA*-HA-MAO | This study | JB48 | Deposited at Addgene |
| Recombinant DNA reagent | Cdc42T17N-BirA*-HA-MAO | This study | JB57 | Deposited at Addgene |
| Recombinant DNA reagent | RhebQ64L-BirA*-HA-MAO | This study | JB87 | Deposited at Addgene |
| Recombinant DNA reagent | RhebS20N-BirA*-HA-MAO | This study | JB88 | Deposited at Addgene |
| Peptide, recombinant protein | 3X FLAG peptide | Sigma | F4799 | |
| Commercial assay or kit | Amersham ECL Prime detection agent | GE Healthcare Lifesciences | RPN2232 | Western blots |
| Commercial assay or kit | Supersignal West Femto Maximum Sensitivity Substrate | Thermo Scientific | 34095 | Western blots |
| Chemical compound | Biotin | Sigma | B4501 | |
| Software, algorithm | Perseus | (Tyanova et al., 2016). | RRID:SCR_015753 | |
| Other | Dynabeads MyOne Streptavidin C1 | Invitrogen | 65002 | Isolation of biotinylated proteins |
| Other | Anti-FLAG M2 affinity resin | Sigma | A2220 RRID:AB_10063035 | Anti-FLAG immunoprecipitation |
| Other | Fugene 6 | Promega | E2691 | Transfection |
| Other | Neutravidin-FITC | Invitrogen | 31006 | |
| Other | Glutathione sepharose 4B | GE Healthcare Lifesciences | 17075601 | GST affinity chromatography |
| Other | Vectashield | Vector Laboratories Inc | H-1000 | Mounting media |
| Other | Novex 4–20% Tris-Glycine gels | Thermofisher | XP04202BOX | Pre-cast gels |
| Other | Gibco Opti-MEM | Fisher Scientific | 31985070 | Media |
Additional files
-
Supplementary file 1
Analysis of mass-spectral data from GTPase MitoID based on spectral counts.
Proteins identified by MitoID with at least one GTPase. Total spectral counts for biological triplicates of GTP-bound (T) and GDP-bound forms (D) are in Sheet S1A. Spectral counts were converted into D scores and WD scores based on the CompPASS platform. D scores in Sheet S1B, mean D scores in Sheet S1C, WD scores in Sheet S1D and mean WD scores in Sheet S1E (see tabs at bottom of sheet). Excel (.xlsx) file.
- https://doi.org/10.7554/eLife.45916.016
-
Supplementary file 2
Analysis of mass-spectral data from GTPase MitoID based on peak intensities.
Label free quantitation (LFQ) intensities from MaxQuant are shown for all proteins identified by MitoID with at least one GTPase. Excel (.xlsx) file.
- https://doi.org/10.7554/eLife.45916.017
-
Supplementary file 3
Volcano plot data from mass spectral peak intensities from MitoID with GTPases of the Rab family.
For each Rab form indicated in the tabs, the LFQ intensities of every protein found in at least two of the triplicates was compared to the values obtained with the GDP forms of all GTPases by using the Perseus platform. The difference, expressed as log2 of the ratio, and the P-value for the significance of the difference are shown, with these being used to generate the volcano plots shown in the figures. Also shown are the WD score for each interaction as obtained from analysis of spectral counts. Excel (.xlsx) file.
- https://doi.org/10.7554/eLife.45916.018
-
Supplementary file 4
Volcano plot data from mass spectral peak intensities from MitoID with GTPases of the Rho and Ras families.
For each GTPase form indicated in the tabs, the LFQ intensities of every protein found in at least two of the triplicates was compared to the values obtained with the GDP forms of all GTPases by using the Perseus platform. The difference, expressed as log2 of the ratio, and the P-value for the significance of the difference are shown, with these being used to generate the volcano plots shown in the figures. Also shown are the WD score for each interaction as obtained from analysis of spectral counts. Excel (.xlsx) file.
- https://doi.org/10.7554/eLife.45916.019
-
Supplementary file 5
Coverage of previously reported effectors of mammalian Rab2A and Rab5A by MitoID and S2 cell affinity chromatography.
Previously reported effectors for mammalian Rab2 and Rab5 are listed along with their coverage by MitoID and by a previous screen for Rab effectors based on affinity chromatography of Drosophila S2 cell lysates (Gillingham et al., 2014). Additional tables show for the MitoID interactions the comparisons of LFQ intensities and the WD scores as in Supplementary file 4. This illustrates typical such scores for bona fide effectors. The FAM71 family (also called the GARI family) have also been reported to bind human Rab2 (Fukuda et al., 2008). However, this interaction is specific for Rab2B and not the Rab2A used for MitoID, and the family is only present in vertebrates, and so it is not included in the comparison. Excel (.xlsx) file.
- https://doi.org/10.7554/eLife.45916.020
-
Transparent reporting form
- https://doi.org/10.7554/eLife.45916.021
