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TBC1D23 is a bridging factor for endosomal vesicle capture by golgins at the trans-Golgi

Nature Cell Biology volume 19, pages 1424–1432 (2017)Cite this article

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An Author Correction to this article was published on 08 January 2018

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Abstract

The specificity of membrane traffic involves tethers at destination organelles that selectively capture incoming transport vesicles to allow SNAREs on opposing membranes to then assemble and drive fusion1,2. Tethers include both protein complexes and long coiled-coil proteins, although how they contribute to specificity remains unclear3,4. The golgin coiled-coil proteins at the Golgi apparatus capture vesicles from different origins, but the vesicle-specific molecular cues that they recognize are unknown5,6,7,8. Vesicle tethering is typically a transient process and therefore is challenging to interrogate in vivo. Thus, we have used a system in which an ectopic golgin causes vesicles to accumulate in a tethered state. By applying proximity biotinylation to the golgin-captured vesicles, we identify TBC1D23, an apparently catalytically inactive member of a family of Rab GTPase-activating proteins (GAPs), as a vesicle–golgin adaptor that is required for endosome-to-Golgi trafficking. The Rab GAP domain of TBC1D23 binds to a conserved motif at the tip of golgin-245 and golgin-97 at the trans-Golgi, while the C terminus binds to the WASH complex on endosome-derived vesicles. Thus, TBC1D23 is a specificity determinant that links the vesicle to the target membrane during endosome-to-Golgi trafficking.

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Figure 1: BioID screens to identify putative vesicle receptors recognized by golgin-97 and golgin-245 reveal TBC1D23 and FAM91A1.
Figure 2: TBC1D23 binds directly to the N termini of golgin-97 and golgin-245 to mediate vesicle capture.
Figure 3: TBC1D23 captures vesicles, golgin-97, golgin-245, FAM91A1 and WASH complex subunits.
Figure 4: TBC1D23 links golgin-97 and golgin-245 to the FAM21A subunit of the WASH complex.
Figure 5: TBC1D23 is required for endosome-to-Golgi trafficking.

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Change history

  • 08 January 2018

    In the version of Supplementary Table 1 originally published with this Article, in the sheet relating to Fig. 3c, all values in the ‘golgin-97-mito’ column were 1.3 times larger than the actual values, which was due to author error when generating the Supplementary Table. These errors did not affect the graph in Fig. 3c, which was plotted with the correct values. Supplementary Table 1 has now been replaced so that it contains the correct values.

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Acknowledgements

We are indebted to R. Hegde and B. Nichols for comments on the manuscript, to T. Stevens for phylogenetic profiling, to E. Derivery (MRC Laboratory of Molecular Biology (LMB), UK), A. Gautreau (Ecole Polytechnique Palaiseau, France), O. Perisic (MRC LMB, UK), F. Randow (MRC LMB, UK) and M. Seaman (Cambridge Institute of Medical Research, UK) for reagents, and to M. Robinson for communicating results prior to publication. Funding was from the Medical Research Council (MRC file reference number MC_U105178783), and by a European Molecular Biology Organisation long-term fellowship to J.J.H.S.

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  1. John J. H. Shin and Alison K. Gillingham: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    John J. H. Shin, Alison K. Gillingham, Farida Begum, Jessica Chadwick & Sean Munro

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Contributions

S.M., A.K.G. and J.J.H.S. devised and planned the study. A.G. and J.J.H.S. performed all the experiments except for electron microscopy (J.C.) and mass-spectrometric protein identification (F.B.). S.M. wrote the manuscript.

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Correspondence to Sean Munro.

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Integrated supplementary information

Supplementary Figure 1 Analysis of Golgin-mito chimeras containing BirA∗ and effect on TBC1D23 and FAM91A1 of deleting the golgins golgin-97 and golgin-245.

(a) Immunoblot of whole cell lysates from stable HEK-293T cells expressing either no construct (control) or the indicated doxycycline-inducible BirA∗-golgin-mito chimeras and treated with 1 μg ml−1 doxycycline and 50 μM biotin for 24 h. The blot was labeled for the HA-tag in the chimeras or actin as a loading control. (b) Confocal micrographs of HeLa cells expressing a BirA∗-golgin-mito chimera with or without 50 μM biotin in the medium, and stained for its HA epitope, Neutravidin for biotinylated proteins and Golgi marker giantin. Scale bars, 10 μm. (c–e) Immunofluorescence of COS-7 cells transfected with the designated golgin-mito constructs containing BirA∗ stained for their HA epitope and co-stained for endogenous CD-MPR (endosomal vesicle cargo) and ZFPL1 (Golgi marker). Scale bars, 10 μm. (f) Confocal micrographs of HeLa cells labeled for TBC1D23 or FAM91A1, with both co-localizing with golgin-97 or golgin-245 (giantin, medial-Golgi). (g) Confocal micrographs of HeLa cells showing that targeting of endogenous TBC1D23 and FAM91A1 to the Golgi is not affected by Δgolgin-97 or Δgolgin-245 single mutants. (h) Confocal micrographs of HeLa cells showing that transfection of myc-tagged golgin-245 rescues targeting of TBC1D23 and FAM91A1 to the Golgi in a Δgolgin-97/Δgolgin-245 double mutant. Scale bars, 10 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 6. Experiments were repeated 3 times except (b) which was performed twice.

Supplementary Figure 2 Δtbc1d23 and Δfam91a1 CRISPR knock-out mutants.

(a, b) Comparison by immunofluorescence (a) or immunoblot (b) of the effect on TBC1D23 levels in HeLa cells of siRNA treatment against TBC1D23 or deleting the gene using CRISPR/Cas9. The TBC1D23 antisera also labels centrosomes, but this is apparently nonspecific as it is unaffected by either CRISPR gene disruption or siRNA knockdown. (c,d) Comparison by immunofluorescence (c) or immunoblot (d) of FAM91A1 in wild-type HAP1 cells versus Δfam91a1 HAP1 cells. (e) Immunofluorescence of wild-type HAP1 cells versus Δfam91a1 HAP1 cells to show that removal of FAM91A1 does not effect the ability of golgin-97-mito to capture either TBC1D23 or the endosome-to-Golgi cargo TGN46 (ZFPL1, Golgi marker). Scale bars, 5 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 6. Experiments were repeated 3 times.

Supplementary Figure 3 TBC1D23-mito is sufficient to relocate cytosolic golgin-97 and golgin-245 to the mitochondria.

(a) Immunofluorescence of HeLa cells expressing TBC1D23-mito and labeled for the HA tag in the chimera and markers for the mitochondria (mitochondrially-encoded cytochrome c oxidase II, MTCO2) or the trans-Golgi (GCC88) along with the cis-Golgi marker (ZFPL1). (b) Immunofluorescence of HeLa cells transfected with golgin-97(ΔCterm)-GFP and golgin-245(ΔCterm)-GFP with or without TBC1D23-Mito and labeled for the HA tag in the chimera and GM130 (Golgi Marker). (c) Confocal micrographs showing TBC1D23-mito relocates endogenous CI-MPR, TGN46 and Vti1a to mitochondria in HeLa cells (ZFPL1, Golgi marker). (d) Immunofluorescence of wild-type and Δfam91a1 HAP1 cells expressing TBC1D23-mito (HA epitope tag) and co-stained for CI-MPR (endosome-to-Golgi cargo) and ZFPL1 (Golgi marker). Scale bars: 10 μm (a–c), 5 μm (d). Experiments were repeated 3 times except (b,d) which were performed twice.

Supplementary Figure 4 TBC1D23 and golgin-97/golgin-245 are required for Golgi-to-endosome traffic.

(a) Binding of FAM91A1(1-328)-His6 expressed in E. coli to beads coated with fragments of TBC1D23 fused to GST. The FAM91A1 fragment binds directly to residues 514-558. (b) Binding of WASH complex to beads coated with as in a. The WASH complex was purified from a mammalian cell line expressing His-PC-TEV-WASH1 (ref. 1), and applied to the beads. Salt-eluates were probed for WASH1, and subsequent SDS eluates stained to show the GST fusions. (c) Immunoblots of HeLa cell extracts quantifying TGN46 and CI-MPR upon deletion of the indicated genes. Two independent replicates of Fig. 8b, d illustrate the robustness of the effects. Deletion of TBC1D23, or both golgin-97 and golgin-245, but not FAM91A1 results in degradation of TGN46 but not CI-MPR, consistent with previous reports that when CI-MPR does not return to the TGN it recycles to the surface in contrast to TGN46 which proceeds to lysosomes2,3. A previous study also observed a differential effect on the two proteins when TGN function was disrupted4. (d) Quantitation of TGN46 in HeLa cell Golgi. The intensity of immunofluorescent labeling of TGN46 in the Golgi was divided by the intensity of the Golgi marker ZFPL1. Mean ± s.e.m., n = 250 cells (wild-type), 196 cells (Δgolgin-97/Δgolgin-245), and 175 cells (Δtbc1d23). Both mutants show a statistically significant reduction in Golgi TGN46 levels (both P values < 0.0001, unpaired, two-tailed Mann-Whitney test). Source data in Supplementary Table 1. (e) Confocal micrographs of Δgolgin-97/Δgolgin-245 HeLa cells, a subset of which transiently express the indicated myc-tagged golgin-245 constructs. The rescue of Golgi accumulation of TGN46 requires residues 2-21 in golgin-97 that bind TBC1D23. Scale bars 10 μm. (f) Immunoblots of HeLa extracts to quantify TGN46 in cells treated with CRISPR/Cas9 directed at FAM21A. Despite using a cloned line there is residual FAM21, probably reflecting it being encoded by two near identical genes and the antibody recognising both. The reduction of TGN46 is consistent with previous reports that endosome-to-Golgi traffic is perturbed by inhibiting FAM21 activity or depleting WASH subunits1,5,6. Unprocessed original scans of blots are shown in Supplementary Fig. 6. Experiments were repeated 3 times except (a,b,f) which were performed twice.

Supplementary Figure 5 Evolutionary conservation of TBC1D23 and the subunits of the FAM91A and WASH complexes.

Coloured dots indicate the presence of a predicted orthologue to the human protein for each species. Orthologues were detected using HMMER3 to search with an HMM profile in each species7. The HMM profile was initially constructed using the phmmr reciprocal-best-hits to the human query, and then expanded (using a diminishing bit-score threshold) in stages as further orthologues were detected using hmmsearch, accepting sequences that reciprocally hit the human query or a close paralogue (>97% sequence identity) until no additional sequences were detected. All profile alignments were constructed with Clustal Omega8, accepting only positions a maximum gap representation of 90%. The species dendrogram represents known consensus phylogenetic relationships but branch lengths do not carry meaning.

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Shin, J., Gillingham, A., Begum, F. et al. TBC1D23 is a bridging factor for endosomal vesicle capture by golgins at the trans-Golgi. Nat Cell Biol 19, 1424–1432 (2017). https://doi.org/10.1038/ncb3627

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