Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins

Abstract

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.

Figure 1. Mapping the interaction between SNX5 and CI-MPR.

(A) Co-immunoprecipitation of GFP-tagged SNX-BARs transiently transfected in HEK293T cells. (B) Alignment of yeast Vps17 and Vps5, together with SNX1, SNX5, SNX6, SNX32, and the SNX5 homologues from different species. The α’ and α” helices that compose the unique helix-turn-helix extension are highlighted in green. (C) Co-immunoprecipitation of GFP-tagged SNX1, SNX5 and a SNX1 chimera generated by replacement of the SNX1 PX domain with that of SNX5. (D) Co-immunoprecipitation of GFP-tagged CI-MPR tail deletion mutants transiently transfected in HEK293T cells. (E) CI-MPR peptide was injected into either SNX5, SNX5(F136D), SNX6 or SNX32 PX domains and binding measured by ITC. Top panels show the raw data and bottom panels represent the integrated and normalized data fit with a 1:1 binding model. Binding of CI-MPR with SNX5 or SNX6 was measured over three independent experiments, binding of CI-MPR with SNX32 was measured once. ITC binding parameters, including Standard deviation (SD) where calculated, are provided in Table S1. In each case 1A, 1C and 1D are representative blots of three independent GFP traps and the unprocessed original scans of immunoblots are shown in Supplementary Figure 3.

Figure 2. Crystal structure of the SNX5:CI-MPR complex.

(A and B) The SNX5–CI-MPR Form 1 crystal structure (spacegroup P2₁2₁2₁) (A) and the SNX5–CI-MPR Form 2 crystal structure (spacegroup C222₁) (B) are shown in ribbon diagram (left panels), with a close-up of the bound CI-MPR peptide shown in stick representation (right panels). (C) Binding of SNX5 to CI-MPR peptide and CI-MPR βA-strand mutants Y2351N and Y2351D. Top panels show the raw data and bottom panels represent the integrated and normalized data fit with a 1:1 binding model. (D) The SNX5–CI-MPR Form 1 (spacegroup P2₁2₁2₁) and Form 2 (spacegroup C222₁) crystal structures are shown with corresponding CI-MPR peptide fo-fc omit maps in the left panels (grey, 3σ level), while the right panels show the anomalous difference maps for the Se atoms in the SeMet labeled protein (orange, 3σ level). This identifies the Met2371 side-chain unambiguously in each structure. (E) Binding of SNX5 to CI-MPR βB-strand mutants L2370D, M2371D and WLM-AAA by ITC. Top panels show the raw data and bottom panels represent the integrated and normalized data fit with a 1:1 binding model. Binding of SNX5 with CI-MPR Wt was measured over three independent experiments, binding of SNX5 with CI-MPR βA/βB-strand mutants was measured once. ITC binding parameters, including Standard deviation (SD) where calculated, are provided in Table S1

Figure 3. The interaction between SNX5 and the CI-MPR β-hairpin structure is required for CI-MPR retrograde trafficking.

(A) Schematic of interactions reported to overlap with CI-MPR β-hairpin structure. (B) Co-immunoprecipitation of GFP-tagged CI-MPR tail constructs transiently transfected in HEK293T cells; summary of relative binding to indicated proteins. Band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression presented as average fraction of GFP-CI-MPR Wt control (C) Bound CI-MPR peptide in stick representation highlighting Val2349 position. (D) Re-expression of full-length CI-MPR and CI-MPR(V2349D) mutant in HeLa CI-MPR KO clonal line. CI-MPR levels analyzed by quantitative fluorescence-based western blotting. Band intensities measured using Odyssey software and normalized to β-Actin before calculating percentage of protein compared with full-length CI-MPR control. Bars represent mean of n=4 independent experiments. (E and F) HeLa CI-MPR KO clonal line transiently transfected with full-length CI-MPR or CI-MPR(V2349D) mutant. Steady state colocalization of CI-MPR with EEA1, Golgin-97 and TGN46, analyzed using Pearsons correlation. Cell numbers analysed for CI-MPR vs Golgin-97 colocalisation (E): CI-MPR KO+CI-MPR: 64 cells; CI-MPR KO+CI-MPR(V2349D): 56 cells across n=3 independent experiments. Pearsons coefficient values compared with +CI-MPR using two-tailed Unpaired t-test (**P<0.01; P=0.0036). Cell numbers analysed for CI-MPR vs EEA1 Pearsons correlation: CI-MPR KO+CI-MPR: 69 cells; CI-MPR KO+CI-MPR(V2349D): 49 cells across n=3 independent experiments. Pearsons coefficient values compared with +CI-MPR using two-tailed Unpaired t-test (**P<0.05; P=0.0385). Cell numbers analysed for CI-MPR vs TGN46 colocalisation (F): CI-MPR KO+CI-MPR: 70 cells; CI-MPR KO+CI-MPR(V2349D): 66 cells across n=3 independent experiments. Pearsons coefficient values compared with +CI-MPR using two-tailed Unpaired t-test (**P<0.01; P=0.0019). (G) HeLa CI-MPR KO clonal line transfected with full length CI-MPR or CI-MPR(V2349) mutant and CI-MPR colocalization with TGN46, analyzed after 40 min chase of surface CI-MPR. Cell numbers analysed: CI-MPR KO+CI-MPR: 45 cells; CI-MPR KO+CI-MPR(V2349D): 53 cells across n=3 independent experiments. Pearson’s coefficient values were compared with +CI-MPR using Unpaired t test (*P<0.05; P=0.0133). (E-G) Scale bars 20 μm (micrographs) and 10 μm (insets). (B, D-F), Bars, mean; error bars, SEM, circles represent individual data points. Statistics source data: Supplementary Table 4. Unprocessed original scans of immunoblots: Supplementary Figure 3.

Figure 4. Interaction between CI-MPR and the hydrophobic groove of SNX5 PX domain is required for CI-MPR retrograde trafficking.

(A) CI-MPR peptide bound to SNX5 hydrophobic groove highlighting Phe136 position. (B) Co-immunoprecipitation of GFP-tagged SNX5 and SNX5(F136D) transiently transfected in HEK293T cells, summary of relative binding. Quantification of band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression, presented as average fraction of GFP-SNX5 control. GFP-SNX5(F136D) mutant compared with GFP-SNX5 using two-tailed Unpaired t-test (***P<0.001; P values for SNX1: P=0.3655, SNX2: P=0.7646; CI-MPR: P=0.0006). (C) F136D mutation in SNX5 PX domain does not affect SNX5 endosomal localization. HeLa cells lentivirally transduced with GFP-SNX5 or GFP-SNX5(F136D) viral particles. Transduced cells fixed and stained for EEA1 and SNX1. Cell numbers analysed for colocalization between GFP and EEA1: GFP-SNX5: 50 cells; GFP-SNX5(F136D): 57 cells across n=3 independent experiments. Pearson’s coefficient values compared using two-tailed Unpaired t-test (P=0.1862). Cell numbers analysed for colocalization between GFP and SNX1: GFP-SNX5: 51 cells; GFP-SNX5(F136D): 59 cells across n=3 independent experiments. Pearson’s coefficient values compared using Unpaired t-test (P=0.1771). Scale bars 20 μm (non-zoom images) and 10 μm (insets). (D) GFP-SNX5 and GFP-SNX5(F136D) reintroduced in SNX5+SNX6 KO cells at levels comparable to endogenous. HeLa SNX5+SNX6 KO clonal line lentivirally transduced with GFP-SNX5 or GFP-SNX5(F136D) viral particles and SNX5-levels analyzed by quantitative fluorescence-based western blotting. Band intensities measured using Odyssey software and normalized to β-Actin before calculating percentage of protein compared with parental HeLa control. Bars represent mean of n=3 independent experiments. (E, F and G) Distribution of endogenous CI-MPR in HeLa cells, HeLa SNX5+SNX6 KO clonal line and HeLa SNX5+SNX6 KO clonal line transduced with GFP-SNX5 or GFP-SNX5(F136D). Colocalization analysis between CI-MPR and EEA1, CI-MPR and TGN46, and CI-MPR and Golgin-97. Cell numbers analysed across n=3 independent experiments for CI-MPR and EEA1 colocalisation (E): HeLa: 129 cells, SNX5+SNX6 KO: 144 cells, KO+GFP-SNX5: 159 cells; KO+GFP-SNX5(F136D): 122 cells. Pearson’s coefficient values compared with HeLa control using one-way ANOVA and Dunnett’s test (***P<0.001, **P<0.01; SNX5+SNX6 KO: P=0.0007, KO+GFP-SNX5: P=0.2886, KO+GFP-SNX5(F136D): P=0.0010). Cell numbers analysed across n=3 independent experiments for CI-MPR and TGN46 colocalisation (F): HeLa: 126 cells, SNX5+SNX6 KO: 111 cells, KO + GFP-SNX5: 121 cells; KO+GFP-SNX5(F136D): 110 cells. Pearson’s coefficient values compared with HeLa control using one-way ANOVA and Dunnett’s test (***P<0.001; SNX5+SNX6 KO: P=0.0005, KO+GFP-SNX5: P=0.6897, KO+GFP-SNX5(F136D): P=0.0006). Cell numbers analysed across n=3 independent experiments for CI-MPR and Golgin-97 colocalisation (G): HeLa: 105 cells, SNX5+SNX6 KO: 127 cells, KO+GFP-SNX5: 122 cells; KO+GFP-SNX5(F136D): 131 cells. Pearson’s coefficient values compared with HeLa control using one-way ANOVA and Dunnett’s test ****P<0.0001, ***P<0.001; SNX5+SNX6 KO: P=<0.0001, KO+GFP-SNX5: P=0.3482, KO+GFP-SNX5(F136D): P=0.0002). Scale bars 20 μm (micrographs) and 5 μm (insets). (B-D,G) Bars, mean; error bars, SEM; circles represent individual data points. Unprocessed original scans of immunoblots: Supplementary Figure 3. Statistics source data: Supplementary Table 4.

Figure 5. CI-MPR retrograde trafficking requires functional SNX5 heterodimers and co-incidence detection of multiple membrane features.

(A) Model of SNX5:SNX1 heterodimer. (B) Co-immunoprecipitation of GFP-tagged SNX5 and SNX5(S226E) transiently transfected in HEK293T cells and summary of relative binding. Quantification of band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression, presented as average fraction of GFP-SNX5 control. GFP-SNX5(S226E) mutant compared with GFP-SNX5 using two-tailed Unpaired t-test (*P<0.05; P values for SNX1: P=0.0193, SNX2: P=0.0324; CI-MPR: P=0.1160). (C) Co-immunoprecipitation of mCherry and mCherry-SNX5 transiently transfected in HEK293T cells alongside GFP, GFP-SNX1, GFP-SNX1(K214A), GFP-SNX1(KKR-EEE) and GFP-SNX1(ΔAH); summary of relative binding. Quantification of GFP and CI-MPR band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to mCherry expression, presented as average fraction of the mCherry-SNX5+GFP-SNX1 control. Interactions of mCherry-SNX5+GFP-SNX1 mutants were compared with mCherry-SNX5+wild type GFP-SNX1 using one-way ANOVA and Dunnett’s test (GFP P values for SNX5+GFP-SNX1(K214A): 0.9969, SNX5+GFP-SNX1(KKR-EEE): P=0.8473, SNX5+GFP-SNX1(ΔAH): P=0.2983; CI-MPR P values for SNX5 + GFP-SNX1(K214A): 0.4109, SNX5+GFP-SNX1(KKR-EEE): P=0.6042, SNX5+GFP-SNX1(ΔAH): P=0.9648. (D) Distribution of endogenous CI-MPR in HeLa cells, HeLa SNX5+SNX6 KO clonal line and HeLa SNX5+SNX6 KO clonal line transfected with GFP, GFP-SNX5 or GFP-SNX5(S226E). Colocalization analysis between CI-MPR and the TGN marker Golgin-97. Cell numbers analysed: HeLa: 55 cells, SNX5+SNX6 KO+GFP: 62 cells, KO+GFP-SNX5: 63 cells; KO+GFP-SNX5(S226E): 68 cells across n=3 independent experiments. Pearson’s coefficient values compared with HeLa control using one-way ANOVA and Dunnett’s test, ****P<0.0001 (P values for SNX5+SNX6 KO+GFP: P=<0.0001, KO+GFP-SNX5: P=0.9173, KO+GFP-SNX5(S226E): P=<0.0001). (E) Distribution of endogenous CI-MPR in HeLa cells, HeLa SNX1+SNX2 KO clonal line and HeLa SNX1+SNX2 KO clonal line transfected with GFP, GFP-SNX1, GFP-SNX1(K214A), GFP-SNX1(KKR-EEE) and GFP-SNX1(ΔAH). Colocalization analysis between CI-MPR and Golgin-97. Cell numbers analysed: HeLa: 60 cells, SNX1+SNX2 KO +GFP: 53 cells, KO+GFP-SNX1: 58 cells; KO+GFP-SNX1(K214A): 72 cells, KO+GFP-SNX1(KKR-EEE): 77 cells, KO+GFP-SNX1(ΔAH): 73 cells across n=3 independent experiments. Pearson’s coefficient values compared with HeLa control using one-way ANOVA and Dunnett’s test, *P<0.05,*P<0.01 (P values for SNX1+SNX2 KO+GFP: P=0.0055, KO+GFP-SNX1: P=0.9752; KO+GFP-SNX1(K214A): P=0.0106, KO+GFP-SNX1(KKR-EEE): P=0.0126, KO+GFP-SNX1(ΔAH): P=0.0132). (F) Distribution of endogenous SNX6 in HeLa cells, HeLa SNX1+SNX2 KO clonal line and HeLa SNX1+SNX2 KO clonal line transfected with GFP, GFP-SNX1, GFP-SNX1(K214A), GFP-SNX1(KKR-EEE) and GFP-SNX1(ΔAH). SNX6 colocalization with EEA1. Figure representative of three independent experiments with similar results. (G) Model for how SNX5/6:SNX1/2 heterodimers sense multiple features of endosomal membranes, including presence of specific phosphoinositides, local membrane curvature, and cytosolic tails of cargoes. By co-incident sensing these feature SNX5/6:SNX1/2 heterodimers assemble into functional membrane-associated complexes that couple cargo recognition with membrane remodeling for formation of cargo-enrich transport carriers. (B,C) Bars, mean; error bars, SEM. Circles represent individual data points. Statistics source data: Supplementary Table 4. Unprocessed original scans of immunoblots: Supplementary Figure 3.

Figure 6. Mechanism of SEMA4C and IGF1R cargo binding to SNX5.

(A) Co-immunoprecipitation of GFP-tagged SNX5 and SNX5(F136D) transiently transfected in HEK293T cells; summary of relative binding. Quantification of band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression, presented as average fraction of GFP-SNX5 control. GFP-SNX5(F136D) mutant compared with GFP-SNX5 using two-tailed Unpaired t-test, ***P<0.001, ****P<0.0001 (P values for IGF1R: P=0.0001, SEMA4C: P=<0.0001). (B) Co-immunoprecipitation of GFP-tagged IGF1R tail truncation mutants transiently transfected in HEK293T cells. Representative blot of three independent GFP traps. (C) Co-immunoprecipitation of GFP-tagged SEMA4C tail truncation mutants transiently transfected in HEK293T cells. Representative blot of three independent GFP traps. (D and E) Binding of SNX5, SNX6 and SNX32 PX domain to IGF1R peptide (D) and SEMA4C peptide (E) by ITC. Top panels show raw data; bottom panels represent integrated and normalized data fit with a 1:1 binding model. Binding of IGF1R and SEMA4C with SNX5 measured over three independent experiments, binding of IGF1R and SEMA4C with SNX6 or SNX32 measured once. ITC binding parameters, including Standard deviation (SD) where calculated, provided in Table S1. (F) SNX5–SEMA4C crystal structure shown in ribbon diagram (left panel), with bound SEMA4C peptide shown in stick representation (right panel). (G) Co-immunoprecipitation of GFP-tagged SEMA4C tail constructs transiently transfected in HEK293T cells. Band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression, presented as average fraction of GFP-SEMA4C wild-type control. GFP-SEMA4C mutants compared with GFP-SEMA4C wild-type control using one-way ANOVA and Dunnett’s test; **P<0.01, ***P<0.001, ****P<0.0001 (P values for V734: P=0.0045,G735D: P=0.0929, Y736D: P=0.0003, Y737D: P=<0.0001, Y738D: P=<0.0001, S739D=0.0003). (H) Co-immunoprecipitation of GFP-tagged IGF1R tail constructs transiently transfected in HEK293T cells. Quantification of band intensities measured from n=4 independent experiments using Odyssey software. Band intensities normalized to GFP expression, presented as average fraction of GFP-IGF1R wild-type control. GFP-IGF1R mutants compared with GFP-IGF1R wild-type control using one-way ANOVA and Dunnett’s test; *P<0.05, **P<0.01 (P values for V1277D: P=0.4031, S1278D: P=0.0049, F1279D: P=<0.0101, Y1280D: P=0.0107, Y1281D: P=0.0941, S1282D: P=0.9996). (A, G, H) Bars, mean; error bars, SEM; circles represent individual data points. Unprocessed original scans of immunoblots: Supplementary Figure 3. Statistics source data: Supplementary Table 4.

Figure 7. Interaction between the IGF1R cytosolic tail and the SNX5 PX domain hydrophobic groove is required for the endosome-to-plasma membrane recycling of the receptor.

(A) Co-immunoprecipitation of myc-tagged full length IGF1R and IGF1R(F1279D) in HEK293T cells. Representative blot of three independent myc-IPs. (B) HeLa cells, HeLa IGF1R KO line un-transduced, or transduced with full length IGF1R or IGF1R(F1279D), serum starved and treated with 10 nM IGF-1 for indicated periods. Level of IGF1R and p-ERK analyzed by immunoblotting. Representative blot of three independent experiments. (C) HeLa cells, IGF1R KO line, un-transfected or transfected with full length IGF1R or IGF1R(F1279D) serum starved and treated with 10 nM IGF-1 for indicated periods and level of IGF1R analyzed by quantitative fluorescence-based western blotting. Quantification of IGF1R band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GAPDH expression, presented as average fraction of the IGF1R signal relative to time point 0. IGF1R(F1279D) levels compared with the levels of IGF1R using two-way ANOVA and Sidak test. *P<0.05 (P values for 3h: P=0.4742, P values for 6h: P=0.0219). (D) HeLa SNX5+SNX6 KO clonal line un-transduced or transduced with GFP-SNX5 or GFP-SNX5(F136D) serum starved and treated with 10 nM IGF-1 for indicated periods and level of endogenous IGF1R analyzed by quantitative fluorescence-based western blotting. Quantification of endogenous IGF1R band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to β-actin expression and expressed as relative fraction of the IGF1R signal to timepoint 0. Levels of IGF1R in different cell lines were compared with levels of IGF1R in HeLa control using two-way ANOVA and Dunnett’s test. ***P<0.001, ****P<0.0001 (P values for 3h KO: P=0.1418, 3h KO+GFP-SNX5: P=0.0919, 3h KO+GFP-SNX5(F136D) P=0.0663, 6h KO: P=0.0001, 6h KO+GFP-SNX5: P=0.9622, 6h KO+GFP-SNX5(F136D) P=<0.0001). (E) Distribution of mCherry-IGF1R in HeLa SNX5+SNX6 KO clonal line un-transduced or transduced with GFP-SNX5 or GFP-SNX5(F136D). Cells were serum starved and treated with 10 nM IGF-1 for 2 hours, prior to fixation and immunostaining. (C,D) Circle data points, mean; error bars, SEM. Representative image of three independent experiments. Unprocessed original scans of immunoblots: Supplementary Figure 3. Statistics source data: Supplementary Table 4.

Figure 8. Identification of a ФxΩxФ consensus motif for SNX5-mediated cargo recruitment.

(A) Analysis of comparative interactome of wild-type SNX5 vs SNX5(F136D) mutant across n=3 independent experiments using One-sample t-test and Benjamini-Hochberg FDR. (B) Co-immunoprecipitation of GFP-tagged SNX5 and SNX5(F136D) transiently transfected in HEK293T cells and summary of relative binding. Band intensities measured from n=3 independent experiments using Odyssey software. Band intensities normalized to GFP expression and presented as the relative fraction of the GFP-SNX5 control. Bars, mean; error bars, SEM; circles represent individual data points. Unprocessed scans of immunoblots: Supplementary Figure 3. (C) Alignment of the cytosolic tail of SNX5 cargoes identified through comparative interactome of wild-type SNX5 vs SNX5(F136D) mutant. (D) Comparative proteomic analysis of transmembrane proteins that rely on the ability to engage the SNX5 hydrophobic groove for their plasma-membrane localisation. Analysis was performed across n=3 independent experiments using One-sample t-test and Benjamini-Hochberg FDR. (E) List of most represented GO of the SNX5 cargoes, established by comparative surfaceosome analysis. (F) Alignment of the cytosolic tail of the SNX5 cargoes that require the SNX5 hydrophobic groove for their endosomal retrieval and recycling as established by comparative surfaceosome analysis. (G) Drosophila Snx1 and Snx6 promote axon growth across the midline. Representative images of stage 17 Drosophila embryos stained with anti-fasciclin II (FasII) antibodies to reveal a subset of ipsilaterally projecting interneurons in the CNS. All embryos are heterozygous for mutations in slit and robo. Arrows indicate segments in which axon bundles are abnormally crossing the midline. In embryos that are also heterozygous for either snx1, snx6 or both snx1 and snx6 the percentage of segments showing ectopic midline crossing is reduced compared to control embryos. (H) Representative images of stage 16 embryos stained with anti-GFP to reveal the Eagle subset of commissural interneurons, comprised of Eg axons, which cross the midline in the anterior commissure and Eg axons, which cross in the posterior commissure. Asterisks (*) indicate segments where the EW axons have failed to cross the midline. All embryos selectively express the Fra∆C transgene, which results in the failure of a portion of EW axons to cross the midline. In embryos heterozygous for mutations in both snx1 and snx6 there is a significant reduction in the percentage of EW axons that cross the midline. (I) Quantification of ectopic midline crossing in the indicated genotypes; n=number embryos; wild-type control (n=26), snx6/+ (n=26), snx1/+ (n=24), snx1 (n=23), snx6/+ (n=23). 11 segments were scored in each embryo. Statistical significance was determined using one-way ANOVA (followed by Tukey’s test), **P<0.01, ***P<0.001, ****P<0.0001. (J) Quantification of failed midline crossing in the indicated genotypes; n=number embryos; wild-type control (n=26), snx6/+ (n=24), snx1/+ (n=16), snx1 (n=17), snx6/+ (n=17) and UASSnx1 (n=22). The eight abdominal segments were scored in each embryo. Statistical significance was determined using one-way ANOVA (followed by Tukey’s test), **P<0.01, ***P<0.001, ****P<0.0001. In (I) and (J) each embryo (in which segments were quantified) was considered an independent trial. Scale bar in (G) and (H) 10 microns. (K) A model of the role of the ESCPE-1 complex, which consists of heterodimeric combinations of either SNX5 or SNX6 dimerised to either SNX1 or SNX2, in retrieving and recycling transmembrane cargo protein on the cytosolic facing surface of endosomes. The cartoon also represents the other machineries involved in the retrograde transport of cargoes to the TGN or in their recycling to the plasma membrane. Known sorting motifs within the cytosolic domain of the prototypical cargoes are listed in the figure legend. (H, J) Bars, mean; error bars, SEM; circles represent individual data points. Statistics source data: Supplementary Table 4. Proteomic datasets: Supplementary Table 3.