1. Cell Biology
Download icon

In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

  1. Alison K Gillingham  Is a corresponding author
  2. Jessie Bertram
  3. Farida Begum
  4. Sean Munro  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
Tools and Resources
Cite this article as: eLife 2019;8:e45916 doi: 10.7554/eLife.45916
9 figures, 1 table and 6 additional files

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 and the of chimeric version used for MitoID. (B) Confocal images of HeLa cells expressing mitochondrially targeted forms of Rab5A-BirA* as in (A), and stained for the HA tag in the chimera as well as markers for the mitochondria and Golgi. The Rab5A chimeras have either the Q79L or the S34N versions that lock them in the GTP- or GDP-bound states respectively. (C) Confocal images of HeLa cells expressing mitochondrial Rab5A(Q79L)-BirA*, or untransfected, and labeled for the chimera and with biotin-binding neutravidin and a Golgi marker. (D) As (C) except that the cells were incubated with fluorescent transferrin for 45 min at 37°C prior to fixation to label endosomes, and then also stained for cation-independent mannos-6-phosphate receptor (CI-MPR) that recycles through endosomes. Neither endosomal marker is relocated to the Rab5A(Q69L)-covered mitochondria. (E, F). Confocal micrographs of cells expressing mitochondrial forms of Rab2A-BirA* in either the GTP-form (Q65L) or the GDP-form (S20N) and stained for mitochondria, the Golgi or with neutravidin as indicated. The Rab2A chimeras accumulate on mitochondria along with biotin, but not the Golgi markers.

https://doi.org/10.7554/eLife.45916.002
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 tag in the Rab chimera and a Golgi marker. The endosomes are not relocated to the Rab5A(Q69L)-covered mitochondria. (B) Confocal images of HeLa cells expressing mitochondrially targeted forms of the indicated GTPases, and stained for the HA tag in the chimera as well as markers for the mitochondria and Golgi.

https://doi.org/10.7554/eLife.45916.003
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 non-specific background band that is marked with an asterisk and also present in the lysate from untransfected cells (untrans.).

https://doi.org/10.7554/eLife.45916.004
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 cells were also treated with 10 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for three hours prior to fixation. The mitochondrial uncoupling agent CCCP induces translocation of the parkin stress sensor but this is not seen with the GTPase chimeras.

https://doi.org/10.7554/eLife.45916.005
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 forms were either GTP-bound (Q79L) or GDP-bound (S34N), and the counts represent means of triplicate biological repeats. The most abundant proteins found with both are Rab5A and the endogenous biotin-containing proteins Acetyl-CoA carboxylase (ACACA) and pyruvate carboxylase (PC). (B) As (A) except only the region in the dashed box in (A) is shown. Known Rab5 effectors and exchange factors are labeled as indicated by their gene names. The effectors are specific for the GTP form (Q79L), whilst the exchange factors are found with the GDP form (S34N). The proteins encoded by RABEP1 and RABGEF1 are found with both forms, but they are known to form a heterodimer that has Rab5 GEF activity but is also a Rab5 effector. (C) Spectral counts for known Rab5 interactors obtained with the two mitochondrial forms of Rab5A shown in (A) and BirA* alone as a control. Values from three biological replicates are shown, with the area of the circle proportional to the number of counts. For full list of values for all panels see Supplementary file 1.

https://doi.org/10.7554/eLife.45916.006
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 by their highest spectral counts with the GTP form of Rab5A (Rab5A(Q79L)). Only the top 34 proteins are shown with the full list in Supplementary file 1. Known Rab5 effectors are shown red text and Rab5A with blue. (B) As (A) except that the spectral counts have been converted to WD scores and the proteins ranked by their highest WD score with Rab5A(Q79L). Known Rab5 effectors now dominate the highest positions in the list. For the full list of values see Supplementary file 1. (C) Volcano plot comparing the spectral intensities from MitoID with Rab5A(Q79L) to MitoID with the GDP-bound forms of Rab5A and sixteen other GTPases (background). Known Rab5 effectors are marked red. Values are in Supplementary file 3. (D) Bubble-volcano plot as in (C), but with the area of each point proportional to the root of the WD score. The root was used to ensure that the full range of bubble sizes was visible on the plot.

https://doi.org/10.7554/eLife.45916.007
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 for which no known or plausible valid interaction was obtained (shown also in Figure 6). Also plotted are proteins that have not previously been reported to be effectors for the relevant Rab, but which were found as specific hits in an affinity-chromatography based screen in Drosophila and so seem highly likely to be valid (Gillingham et al., 2014). All values are in Supplementary file 5.

https://doi.org/10.7554/eLife.45916.008
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 GDP-locked/empty forms of all seventeen GTPases, with the size of the bubbles proportional to the WD scores. Indicated are known effectors (red) and GAPs (blue). For each plot the Rab is omitted and all values are in Supplementary file 3.

https://doi.org/10.7554/eLife.45916.009
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 the size of the bubbles proportional to the WD scores. Indicated are known effectors (red) and GAPs (blue). For each plot the Rab is omitted and all values are in Supplementary file 3.

https://doi.org/10.7554/eLife.45916.010
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 GDP-locked forms of seventeen GTPases, with the size of the bubbles proportional to the WD scores. Indicated are known effectors (red) and GEFs (yellow). Note that some of the lower scoring hits were also found with the GTP-bound form of the respective Rabs (eg TBC1D25 for Rab2A, FBXL4 and SPAG9 for Rab5A). In one case (PI4KB with Rab11A) the interaction has been reported to be nucleotide-independent, and so such hits seem more likely to be similar nucleotide-independent effectors rather than GEFs (Burke et al., 2014). For each plot the Rab is omitted and all values are in Supplementary file 3.

https://doi.org/10.7554/eLife.45916.011
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 proteins. Lysates were from HEK293 cells and probed for STAMBPL1, or from rat brain and probed for ARFGEF3. (B) Confocal micrographs of MEFs expressing either GFP-STAMPBL1 alone and probed for a Golgi marker, or co-expressing the mitochondrial Rab2A(Q65L)-BirA* as indicated. (C) As (B) except that the cells were expressing GFP-ARFGEF3. (D) Coomassie blue stained gel showing affinity chromatography of purified recombinant GFP-STAMBPL1 using GST fusions to the indicate GTP-locked (QL) or GDP-locked (SN) Rabs. The recombinant GFP-STAMBPL1 binds specifically to the GTP form of Rab2A. (E) Affinity chromatography of HeLa 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 proteins. TBCK, OSBPL9 and RELCH show specific binding to the GTP form of Rab5, with RELCH also binding to Rab11-GTP as previously reported (Sobajima et al., 2018). (F) Confocal micrographs of cells expressing GFP-TBCK alone, or with co-expression of mitochondrial Rab5A(Q79L)-BirA* as indicated. (G) Coomassie blue stained gel showing affinity chromatography of lysate from E. coli expressing His6-Nde1 (input) using GST fusions to GTP-locked (Q66L) or GDP-locked (S21N) Rab9A. The samples were also immunoblotted for the His6 tag. The recombinant His6-Nde1 binds specifically to the GTP form of Rab9A. (H) Affinity chromatography of lysate from HEK293 cells expressing FLAG-HPS3 (Input) using GST-fusions to GTP-locked (QL) or GDP-locked (SN) Rab9A. The bound proteins were eluted and probed for the FLAG tag. (I) Confocal micrographs of HeLa cells expressing FLAG-HPS3 and mitochondrial Rab9A(Q66L)-BirA* (GTP-locked) or Rab9A(S21N)-BirA* (GDP-locked). The GTP-form specifically recruits HPS3 to mitochondria. (J) Affinity chromatography of HEK293 cell lysate (Input) using GTP-locked (QL) or GDP-locked (SN) GST-Rab11A. Bound proteins were eluted and probed for ALS2/alsin.

https://doi.org/10.7554/eLife.45916.012
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 each case the GTPase is compared to a background comprising the GDP-locked/empty forms of seventeen GTPases, with the size of the bubbles proportional to the WD scores. Indicated are known effectors (red), GAPs (blue) and GEFS (yellow). All values are in Supplementary file 4.

https://doi.org/10.7554/eLife.45916.013
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 the GTPase is compared to a background comprising the GDP-locked/empty forms of seventeen GTPases, with the size of the bubbles proportional to the WD scores. Indicated are known effectors (red), GAPs (blue) and GEFS (yellow). All values are in Supplementary file 4.

https://doi.org/10.7554/eLife.45916.014
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 the GTPase is compared to a background comprising the GDP-locked/empty forms of seventeen GTPases. Known effectors and GAPs are indicated along with proteins whose GO annotations include the term DNA-binding or RNA-binding. The removal of the basic stretch reduces contamination with the latter proteins. (C) Immunoblot of strepatividin precipitates from cells in which MitoID had been performed with the indicated versions of Rab5A(Q79L). A representative lysate, and the precipitates were blotted for the indicated Rab5 effectors. Mutation of Ser84 reduces biotinylation of OSBPL9, but has no effect on that of two other effectors, EEA1 and PIK3R4.

https://doi.org/10.7554/eLife.45916.015

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (E. coli)BL21 GOLD (DE3)Agilent Technologies230132Used for expression
Strain, strain background (E. coli)Alpha-select goldBio-lineBIO-85027Used for cloning
Cell line (H. sapiens)mCherry-Parkin, HeLa(Lazarou et al., 2015)
Cell line (H. sapiens)HEK293ATCCATCC CRL-11268
RRID:CVCL_1926
Cell line (H. sapiens)HeLaATCCATCC CCL-2
RRID:CVCL_0030
Recombinant DNA reagentpcDNA3.1ClontechV79020
Recombinant
DNA reagent
pGEX6p2GE Healthcare Life SciencesGE28-9546-50
Detection reagentStreptavidin-HRPCell Signaling Technology3999S
RRID:AB_10830897
WB: 1:300
AntibodyRat monoclonal anti-HA (3F10)Roche867 423 001
RRID:AB_2314622
IHC: 1:300
AntibodyGoat polyclonal anti-giantinSanta Cruzsc-46993
RRID:AB_2279271
IHC: 1:200
AntibodyRabbit polyclonal human COXIV (3E11)New England Biolabs4850S
RRID:AB_2085424
IHC: 1:200
AntibodyRabbit polyclonal anti- ZFPL1SigmaHPA014909
RRID:AB_1859055
IHC: 1:300
AntibodyMouse monoclonal anti-TOM20BD Transduction Labs612278
RRID:AB_399595
IHC: 1:200
AntibodySheep polyclonal anti-TGN46ABD serotecAHP500G
RRID:AB_323104
IHC: 1:300
AntibodyRabbit polyclonal anti-golgin-84SigmaHPA000992
RRID:AB_1079009
IHC: 1:300
AntibodyMouse monoclonal anti-HIS6 tagAbcamAb18184
RRID:AB_444306
WB: 1:1000
AntibodyRabbit polyclonal
Anti-STAMBPL1
SigmaSAB4200146
RRID:AB_10622601
WB: 1:1000
AntibodyRabbit polyclonal anti-NDE1Life Technologies711424
RRID:AB_2692258
WB: 1:500
AntibodyRabbit polyclonal anti-TBCKCambridge BioscienceHPA039951
RRID:AB_10795300
WB: 1:500
AntibodyRabbit polyclonal anti-RELCH/KIAA1468Cambridge BioscienceHPA040038
RRID:AB_10793860
WB: 1:1000
AntibodyRabbit monoclonal anti-OSBPL9Abcamab151691WB: 1:1000
AntibodyMouse monoclonal anti-FLAG (M2)SigmaF1804
RRID:AB_262044
WB: 1:5000
AntibodyRabbit polyclonal anti-ARFGEF3ThermoFisher
Scientific
PA5-57623
RRID:AB_2638119
WB: 1:1000
AntibodyAlexa 488 Donkey polyclonal anti-Rat IgGThermoFisher
Scientific
A21208
RRID:AB_2535794
IHC: 1:400
AntibodyAlexa 647 Donkey polyclonal anti-Goat IgGThermoFisher
Scientific
A32849
RRID:AB_2762840
IHC: 1:400
AntibodyAlexa 555 Donkey polyclonal anti-Rabbit IgGThermoFisher
Scientific
A32794
RRID:AB_2762834
IHC: 1:400
AntibodyAlexa 647 Donkey polyclonal anti-Rabbit IgGThermoFisher
Scientific
A32795
RRID:AB_2762835
IHC: 1:400
AntibodyAlexa 555Donkey polyclonal anti-mouse IgGThermoFisher
Scientific
A32773
RRID:AB_2762848
IHC: 1:400
Recombinant DNA reagentSTAMBPL1Addgene#22559
Recombinant DNA reagentTBCKInsight BiotechnologyRC203605
Recombinant DNA reagentHPS3Insight BiotechnologyRC204569
Recombinant DNA reagentNDE1Stratech ScientificHG15586
Recombinant DNA reagentRab2AQ65L-BirA*-HA-MAOThis studypN40Deposited at Addgene
Recombinant DNA reagentRab2AS20N-BirA*-HA-MAOThis studyJB2Deposited at Addgene
Recombinant DNA reagentRab5AQ79L-BirA*-HA-MAOThis studyJB28Deposited at Addgene
Recombinant DNA reagentRab5AS34N-BirA*-HA-MAOThis studyJB40Deposited at Addgene
Recombinant DNA reagentRab6AQ72L-BirA*-HA-MAOThis studypO40Deposited at Addgene
Recombinant DNA reagentRab6AT27N-BirA*-HA-MAOThis studyJB4Deposited at Addgene
Recombinant DNA reagentRab7AQ67L-BirA*-HA-MAOThis studyJB93Deposited at Addgene
Recombinant DNA reagentRab7AT22N-BirA*-HA-MAOThis studyJB94Deposited at Addgene
Recombinant DNA reagentRab8AQ67L-BirA*-HA-MAOThis studyJB24Deposited at Addgene
Recombinant DNA reagentRab8AT22N-BirA*-HA-MAOThis studyJB23Deposited at Addgene
Recombinant DNA reagentRab9AQ66L-BirA*-HA-MAOThis studyJB84Deposited at Addgene
Recombinant DNA reagentRab9AS21N-BirA*-HA-MAOThis studyJB85Deposited at Addgene
Recombinant DNA reagentRab10Q68L-BirA*-HA-MAOThis studyJB81Deposited at Addgene
Recombinant DNA reagentRab10T23N-BirA*-HA-MAOThis studyJB82Deposited at Addgene
Recombinant DNA reagentRab11AQ69L-BirA*-HA-MAOThis studyJB20Deposited at Addgene
Recombinant DNA reagentRab11AS25N-BirA*-HA-MAOThis studyJB19Deposited at Addgene
Recombinant DNA reagentRab11BQ70L-BirA*-HA-MAOThis studypX49Deposited at Addgene
Recombinant DNA reagentRab11BS25N-BirA*-HA-MAOThis studypY49Deposited at Addgene
Recombinant DNA reagentRab18Q67L-BirA*-HA-MAOThis studyJB11Deposited at Addgene
Recombinant DNA reagentRab18S22N-BirA*-HA-MAOThis studyJB10Deposited at Addgene
Recombinant DNA reagentRab30Q68L-BirA*-HA-MAOThis studyJB15Deposited at Addgene
Recombinant DNA reagentRab30T23N-BirA*-HA-MAOThis studyJB3Deposited at Addgene
Recombinant DNA reagentRab33BQ92L-BirA*-HA-MAOThis studyJB26Deposited at Addgene
Recombinant DNA reagentRab33BT47N-BirA*-HA-MAOThis studyJB25Deposited at Addgene
Recombinant DNA reagentnRasG12D-BirA*-HA-MAOThis studyJB95Deposited at Addgene
Recombinant DNA reagentnRasS17N-BirA*-HA-MAOThis studyJB96Deposited at Addgene
Recombinant DNA reagentRac1Q60L-BirA*-HA-MAOThis studyJB42Deposited at Addgene
Recombinant DNA reagentRac1T17N-BirA*-HA-MAOThis studyJB54Deposited at Addgene
Recombinant DNA reagentRalBG23V-BirA*-HA-MAOThis studyJB97Deposited at Addgene
Recombinant DNA reagentRalBS29A-BirA*-HA-MAOThis studyJB98Deposited at Addgene
Recombinant DNA reagentRhoAQ63L-BirA*-HA-MAOThis studyJB68Deposited at Addgene
Recombinant DNA reagentRhoAT19N-BirA*-HA-MAOThis studyJB55Deposited at Addgene
Recombinant DNA reagentCdc42G12V-BirA*-HA-MAOThis studyJB48Deposited at Addgene
Recombinant DNA reagentCdc42T17N-BirA*-HA-MAOThis studyJB57Deposited at Addgene
Recombinant DNA reagentRhebQ64L-BirA*-HA-MAOThis studyJB87Deposited at Addgene
Recombinant DNA reagentRhebS20N-BirA*-HA-MAOThis studyJB88Deposited at Addgene
Peptide, recombinant protein3X FLAG peptideSigmaF4799
Commercial assay or kitAmersham ECL Prime detection agentGE Healthcare LifesciencesRPN2232Western blots
Commercial assay or kitSupersignal West Femto Maximum Sensitivity SubstrateThermo Scientific34095Western blots
Chemical compoundBiotinSigmaB4501
Software, algorithmPerseus(Tyanova et al., 2016).RRID:SCR_015753
OtherDynabeads MyOne Streptavidin C1Invitrogen65002Isolation of biotinylated proteins
OtherAnti-FLAG M2 affinity resinSigmaA2220
RRID:AB_10063035
Anti-FLAG immunoprecipitation
OtherFugene 6PromegaE2691Transfection
OtherNeutravidin-FITCInvitrogen31006
OtherGlutathione sepharose 4BGE Healthcare Lifesciences17075601GST affinity chromatography
OtherVectashieldVector Laboratories IncH-1000Mounting media
OtherNovex 4–20% Tris-Glycine gelsThermofisherXP04202BOXPre-cast gels
OtherGibco Opti-MEMFisher Scientific31985070Media

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

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)