Abstract
Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex ‘retriever’; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17–retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.
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Acknowledgements
We thank the Wolfson Bioimaging Facility at the University of Bristol and also the Life Imaging Center (LIC) of the University of Freiburg for their support. P.J.C. is supported by the Medical Research Council (MR/K018299/1 and MR/P018807/1) and the Wellcome Trust (089928 and 104568). Funding to B.M.C. from the Australian Research Council (ARC) (DP160101743) and National Health and Medical Research Council (NHMRC) (APP1058734) is acknowledged. The NIH (R01DK073639, R01DK107733) supported E.B. and (R01AI65474) D.D.B. L.B. gratefully acknowledges research support from the Associazione Italiana per la Ricerca sul Cancro. L.B. acknowledges research support from the Associazione Italiana per la Ricerca sul Cancro grant no. 18578. Wellcome Trust PhD Studentships from the Dynamic Cell Biology programme (083474) supported K.E.M. and M.G. An NHMRC Dementia Fellowship (APP1097185) supported R.G., and B.M.C. is supported by an NHMRC Career Development Fellowship (APP1061574). F.S. is funded by the German Research Council (DFG) Emmy Noether grant (STE2310/1-1).
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Initial conceptualization, K.E.M., F.S. and P.J.C. Evolution of conceptualization, K.E.M., F.S., I.B., D.D.B., E.B. and P.J.C. Formal analysis, R.S. Investigation, K.E.M., F.S., R.F., M.G., K.J.H., D.P. and R.G. Writing—original draft, K.E.M. and P.J.C. Writing—review and editing, all authors. Funding acquisition, F.S., L.B., B.M.C., D.D.B., E.B. and P.J.C. Resources, P.L., N.P., C.M.D., L.L.M., B.L.O., A.S. and H.N. Supervision, F.S., L.B., B.M.C., D.D.B., E.B. and P.J.C.
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Supplementary Figure 1 SNX17 and VPS29 rescue experiments.
(A) Lower magnification images of Figure 1D. Scale bars = 20 μm. Representative images from 3 independent experiments. (B) Parental, VPS35 KO, VPS29 KO and VPS29 KO HeLa cells where VPS29 has been re-expressed (VPS29 KO rescue), were treated with 10 μg/ml cycloheximide for 0, 4 or 8 hours before lysis. Lysates were then analysed by Western blot for the indicated proteins. Representative blot from 3 independent experiments. (C,D) Quantification of B. Band intensities were measured using the Odyssey software. Band intensities of integrin α5 or integrin β1 are represented as a % of the band intensity at 0 hours cycloheximide treatment for each of the four conditions. % of integrin in KO cells were compared to parental HeLa at each time point using two-tailed t-test. Points represent the mean of n = 3 independent experiments. Error bars represent s.d. ∗∗ p < 0.01, ∗ p < 0.05. Unprocessed original scans of immunoblots are shown in Supplementary figure 8. See statistics source data for precise p values.
Supplementary Figure 2 Production of recombinant human retriever complex using MultiBac44.
(A) Strep-DSCR3, untagged C16orf62 and VPS29-6xHis were cloned into MultiBac plasmids pACEBac1, pIDC and pIDK, respectively. The resulting expression constructs were conjoined by Cre/loxP reaction44, giving rise to a co-expression construct combining all three genes. This construct was inserted into the EMBacY baculoviral genome and virus prepared as described44. Expression in Sf21 insect cells was followed by protein purification using immobilized metal affinity chromatography IMAC) and size exclusion chromatography (SEC), resulting in highly purified retriever complex. (B) SEC profile of the recombinant retriever complex. Profile from one size exclusion chromatography experiment. (C) Analysis of the second peak in SEC by Coomassie-stained SDS-PAGE evidences excess VPS29-6xHis. Unprocessed original scans of the Coomassie gel are shown in Supplementary figure 8.
Supplementary Figure 3 DSCR3, C16orf62 and VPS29 localise on retromer positive endosomes.
(A) GFP-SNX17 transduced RPE-1 cells were fixed and stained for endogenous VPS35 and either C16orf62 or FAM21. Representative field of view from 3 independent fixations and stainings. Scale bars represent 20 μm. (B) RPE-1 cells were transduced with GFP-VPS29. Antibodies against endogenous SNX17 or C16orf62 stained endosomes which were positive for GFP-VPS29 and VPS35. Images are a representative field from 3 independent experiments. Scale bars represent 20 μm. (C) RPE-1 cells were transduced to express low levels of GFP-DSCR3. GFP-DSCR3 labelled endosomes stained positive for endogenous C16orf62 and VPS35. Representative field of view from 3 independent experiments. Scale bars represent 20 μm.
Supplementary Figure 4 The WASH complex co-evolved with retriever and is required for its endosomal localisation.
(A) Lower magnification images of figure 4C. Scale bars = 20 μm. (B) Validation of FAM21 knock-out in HeLa cells by immunofluorescence. Scale bars = 10 μm. Representative field of view from 2 independent experiments. (C) Quantification of Figure 4F. Pearson’s and colocalisation coefficients were calculated from 9 individual fields of view in each of the n = 2 independent experiments (18 fields of view in total). Bars = mean. (D) The evolutionary conservation of C16orf62 was mapped using the CLIME algorithm (Li et al., 2014). The pattern of C16orf62 conservation created an evolutionary conserved module. All genes in the genome were then compared to this evolutionary conserved module, searching for genes which share similar patterns of conservation and are therefore in inferred to have co-evolved with C16orf62. Proteins which are inferred to have co-evolved with C16orf62 have a LLR higher than 0, with a higher score indicating increased likeliness of co-evolution. PG = paralogue group, LLR = log-likelihood ratio scale.
Supplementary Figure 5 Characterisation of DSCR3 knock-out HeLas.
(A) Endogenous SNX17 IPs were performed in parental or CCDC22 knock-out HeLa cells. Representative blot from 2 independent experiments. (B) Lysates from DSCR3 knock-out HeLas were analysed by Western blotting for knock-out and effects on the levels of other proteins. Representative blot from 4 independent experiments. (C) Endogenous CCDC22 immunoprecipitations in parental or DSCR3 KO HeLa cells. This experiment was performed once. (D) Immunofluorescence of indicated endogenous proteins in parental HeLa cells. Representative field of view from 3 independent experiments. Scale bars = 10 μm for zoomed out image, 5 μm for zoomed in images. (E) Same as D but staining performed in DSCR3 knock-out HeLas. Scale bars = 20 μm for a field of cells, 5 μm for zoomed in images. (F) Parental HeLas and knock-out DSCR3 HeLa cells were analysed by immunofluorescence for their distribution of integrin α5. Bottom panels represent zoomed images of the white boxes. Scale bars = 10 μm for a field of cells, 5 μm for zoomed in images. (G) Parental or knock-out DSCR3 HeLa cells were either untransfected or transfected with Bafilomycin A to prevent lysosomal acidification. Lysates were then analysed by Western blotting against the indicated proteins. Representative blot from 3 independent cell lysis events. Unprocessed original scans of immunoblots are shown in Supplementary figure 8.
Supplementary Figure 6 The C terminal leucine of SNX17 and SNX31 is essential in engaging retriever and the CCC complex.
(A) GFP traps of HEK 293 cells expressing SNX17 mutants indicated in figure 4B. Representative Western blot from three GFP trap experiments. (B) GFP-SNX17 or the indicated mutants were transiently transfected into RPE-1 cells. Cells were fixed and stained for EEA1 and DAPI. Representative field of cells from three independent experiments. Scale bars = 20 μm. (C) GFP traps and Western analysis of GFP-SNX17 or GFP-SNX31 and their equivalent loss of function mutations (L470G and L490G). Representative blot from three independent experiments. Unprocessed original scans of immunoblots are shown in Supplementary figure 8.
Supplementary Figure 7 Characterisation of SNX17 knock-out HeLa cells, SNX17 knock-downs for cell surface proteomics and pseudo-virion infection.
(A) Parental HeLa cells or populations of SNX17 cells which have been targeted by CRISPR-Cas9 against either exon 1 or exon 2. Cells were fixed and stained for integrin α5 and the lysosomal marker LAMP1. Scale bars = 10 μm. Representative field of view from one experiment. (B) Cells in (A) were lysed and analysed by Western blot for knock-out of SNX17 and total levels of integrin α5. This experiment was performed once. (C) Knockdowns of SNX17 in SILAC labelled HeLa cells used for cell surface proteomics. 3 independent knock-down experiments used for SILAC proteomics analysed on one blot. (D) Steady-state cell surface analysis of indicated cargo proteins and their dependency on SNX17, retriever and retromer. HeLa cells were knocked-out for the indicated proteins using CRISPR/Cas9. Cell surface proteins were randomly biotinylated with a membrane-impermeable biotin conjugate. Cells were lysed and biotinylated proteins isolated with streptavidin sepharose followed by Western blotting. This experiment was performed once. (E) siRNA knockdowns of cells used in pseudovirion infection assay. Representative blot of 3 independent knock-down experiments. Unprocessed original scans of immunoblots are shown in Supplementary figure 8.
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McNally, K., Faulkner, R., Steinberg, F. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat Cell Biol 19, 1214–1225 (2017). https://doi.org/10.1038/ncb3610
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DOI: https://doi.org/10.1038/ncb3610
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