ARTICLES A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport Florian Steinberg1, Matthew Gallon1, Mark Winfield2, Elaine Thomas1, Amanda J. Bell1, Kate J. Heesom3, Jeremy M. Tavaré1 and Peter J. Cullen1,4 The PDZ-domain-containing sorting nexin 27 (SNX27) promotes recycling of internalized transmembrane proteins from endosomes to the plasma membrane by linking PDZ-dependent cargo recognition to retromer-mediated transport. Here, we employed quantitative proteomics of the SNX27 interactome and quantification of the surface proteome of SNX27- and retromer-suppressed cells to dissect the assembly of the SNX27 complex and provide an unbiased global view of SNX27-mediated sorting. Over 100 cell surface proteins, many of which interact with SNX27, including the glucose transporter GLUT1, the Menkes disease copper transporter ATP7A, various zinc and amino acid transporters, and numerous signalling receptors, require SNX27–retromer to prevent lysosomal degradation and maintain surface levels. Furthermore, we establish that direct interaction of the SNX27 PDZ domain with the retromer subunit VPS26 is necessary and sufficient to prevent lysosomal entry of SNX27 cargo. Our data identify the SNX27–retromer as a major endosomal recycling hub required to maintain cellular nutrient homeostasis. Following internalization, endocytosed transmembrane proteins are direct endosome-to-plasma membrane recycling6. Here, we have delivered to early endosomes. Here, cargo proteins are sorted for employed quantitative proteomic analysis of the SNX27 interactome degradation in lysosomes or alternatively retrieved and recycled combined with quantification of the surface proteome of SNX27- to the plasma membrane or the trans-Golgi network (TGN). The and VPS35-deficient cells to obtain a global view of SNX27–retromer sorting nexins (SNX) of the SNX17–SNX27–SNX31 subfamily are assembly and cargo specificity. This has identified SNX27 as a core early endosome-associated proteins that promote the retrieval and component of a multi-protein complex defined by the independent recycling of specific cargo1. Whereas SNX17 sorts cargoes containing association with the WASH complex and retromer SNX–BARs, and a NPxY–NxxY motifs such as LRP1 (ref. 2) and β1-integrins3,4, the direct PDZ-domain-mediated interaction with the VPS26 component PDZ-domain-containing SNX27 recycles the β2-adrenergic receptor of the VPS26–VPS29–VPS35 trimer. Moreover, our global approach (β2AR) through direct binding to a PDZ ligand located at the carboxy has established SNX27–retromer as a major retrieval and recycling hub terminus of the receptor5. for a variety of transmembrane proteins, many of which play crucial SNX27-mediated retrieval and recycling of the β2AR has been roles during organism growth and cellular homeostasis. proposed to depend on the SNX–BAR-retromer through an indirect interaction with the retromer-associated Wiskott-Aldrich syndrome RESULTS protein and SCAR homologue WASH complex6. In mammals, Proteomic analysis of SNX27 and retromer cargo specificity the SNX–BAR-retromer comprises a stable VPS26–VPS29–VPS35 To globally identify SNX27 and retromer-dependent cargo, we trimer that is loosely associated with heterodimers composed of developed a two-tiered proteomics approach based on stable SNX1–SNX2 and SNX5–SNX6 (refs 7–11). The best characterized isotope labelling with amino acids in culture (SILAC). First, role of this complex is in the retrieval and recycling of cargo from we employed high-resolution interactome analysis of GFP-trap- endosomes back to the TGN (refs 7–11). The proposed link with precipitated GFP–SNX27 versus GFP to identify new SNX27 interactors. SNX27 therefore suggests another role for SNX–BAR-retromer in Second, we comparatively quantified the biotinylated surface proteome 1The Henry Wellcome Integrated Signalling Laboratories, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. 2School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK. 3Proteomics Facility, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. 4Correspondence should be addressed to P.J.C. (e-mail: [email protected]) Received 6 November 2012; accepted 1 March 2013; published online 7 April 2013; DOI: 10.1038/ncb2721 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2013 Macmillan Publishers Limited. All rights reserved. ARTICLES a c HS6ST2 siRNA EMP3 Lost from surface MUC1 FADS2 ERMP1 PLXNA1 Scramble SNX27 of SNX27- SLC9A6 ITGA5 CYB561 PTPRF ITGA7 ANPEP CD81 0247 0247 h Cu2+ depleted cells TGFBR1 ROR1 NETO2 CACHD1 TSPAN6 LRRC15 PLXND1 TMEM87A ATP7A PSCA ADCY9 PAR1 PTPRB CD109 NRP1 Surface SLITRK5 TMEM2 ATRN FLVCR1 SLC2A14 TfnR SLC25A29 SLC12A4 IL13RA1 ANTXR2 CSPG4 PTPRG LAMP1 FZD6 IL6ST HTRA2 PTPRE LRP5 ACVR1B DCBLD2 CADM4 Lysate Actin CRIM1 SLC25A20 SLC4A2 IL1RAP TNFRSF12A TM9SF3 KCNN4 TACSTD2 ZDHHC20 SEMA4B ATP11C Control GOLT1B ICAM2 TMEM206 B7H6 200 ATP11A CD70 MEGF9 β2AR ITGB8 DDR1 LRP6 SNX27KD VLDLR TMEM30A SGCE LGALS3 SLC31A1 160 TMTC3 HEG1 BCAM NT5E SCD ITGA11 PTPRJ CXADR APP PLXNA2 SLC35B2 PLXNA2 PDGFRb LHFPL2 120 PRRT3 GGT1 TMEM41B SLC35F2 TRAILR1 AMIGO2 FAP * METTL7A BMPR1A SLCO2A1 CLDN4 * ZIP6 ZIP10 CD320 CD276 80 * * MUC16 CD97 ZnT7 TPBG GPR172A GPR180 STEAP3 LSR MCT1 LRP10 PVRL3 PTK7 40 SLC23A2 GJA1 control at 0 h ZnT1 C9orf5 ITGB5 SLC1A4 in percentage of PTGFRN CD83 TMEM9 SLC4A7 IFNGR1 ITGB3 0 SLC1A3 TNFR1 Surface abundance LRFN4 SLC30A9 SLC7A1 KIDINS220 ITGA3 LTBR 0 h 2 h 4 h 7 h XPR1 KIAA1161 THY1 ICAM1 PROCR SLC44A1 PTPRF SLC29A1 ANO6 TfnR CDCP1 d GLUT1 SCARB1 KIAA1549 CD97 ATP7A SDC1 LRP8 QSOX2 SDC4 RDH11 EPHB2 100 SDC2 GOLM1 SLITRK4 CYBRD1 SLC22A5 SLC1A1 IFITM2 SGPL1 80 SLC03A1 Isotype ITGAV ADAM22 GPR56 SLC39A14 SLC12A7 CA9 60 FDFT1 ITGB4 Scrambled ESYT2 SLC6A6 CD46 FAT4 CD63 FZD2 TNFRSF10D 40 SNX27KD PODXL2 F3 CPD Basigin MUC18 OSMR 20 SLC12A2 Lost from surface VPS35KD SMC2 PODXL SLC5A6 Number of cells 0 STEAP4 ADAM17 of VPS35- 0102 103 104 105 depleted cells MMP14 Plexin-B2 SLC4A8 CD109 Alexa-488 intensity PMCA4 VANGL2 CELSR1 PMCA1 Neuroplastin SLC27A4 SLC1A5 SLC38A2 PAR4 PTPRJ MRP4 SLC6A8 100 CELSR2 KIRREL Robo1 ECE1 TMEM200 TMEM109 Enriched in 80 VANGL1 NLGN2 SEMA4C FAT1 60 STEAP2 MCTP2 SNX27 FZD1 c1ORF43 interactome 40 20 b Number of cells siRNA 0 0102 103 104 105 Alexa-488 intensity TRAILR1 Control-1Control-2SNX27-1SNX27-2VPS35-1VPS35-2 GLUT1 EGFR CSPG4 100 N-cadherin 120 120 120 80 100 100 100 60 EGFR 80 80 80 40 60 * 60 60 NG2/CSPG4 40 * 40 40 * 20 of control 20 20 20 Number of cells 0 MCT1 in percentage 0 0 0 0102 103 104 105 Contr.27 35 Contr.27 35 Contr. 27 35 Surface abundance Alexa-488 intensity β Surface PDGFR β STEAP3 STEAP3 N-cadherin MCT1 PDGFR 120 120 120 120 GLUT1 100 100 100 100 80 * 80 80 80 * 60 60 60 60 * VPS35 40 * * 40 40 40 * of control 20 20 20 20 SNX27 in percentage 0 0 0 0 Contr.27 35 Contr.27 35 Contr.27 35 Contr.27 35 Lysate Actin Surface abundance Figure 1 Interactome analysis combined with a quantitative analysis of the Odyssey scanner. SNX27 and VPS35 were suppressed with two individual surface proteome of SNX27- and VPS35-depleted HeLa cells uncovers oligonucleotides and surface fractions were analysed for the abundance transmembrane cargo for SNX27 and retromer. (a) HeLa cells were of the indicated proteins. The quantifications show the mean of at least grown in three different SILAC media, SNX27 and VPS35 expression three independent experiments (n D 3 for EGFR and CSPG4; n D 4 for was suppressed with siRNA and the biotinylated surface proteome was N-cadherin and MCT1; n D 5 for GLUT1; n D 6 for PDGFRβ; ∗ P < 0:05; quantified compared with control cells. Proteins in the yellow circle were unpaired t -test; error bars, s.e.m.; individual data points shown in statistics lost at least 1.4-fold from SNX27-suppressed cells; the red circle contains source data; uncropped blots in Supplementary Fig. S5). (c) ATP7A surface proteins lost at least 1.4-fold from the surface of VPS35-deficient cells. levels were analysed under copper-induced (200 µM CuCl2) conditions The blue circle contains proteins that were at least threefold enriched in SNX control and SNX27-depleted cells over a time course of 7 h in a SILAC-based SNX27 interactome analysis. Hence, proteins in the (graph represents the mean of three independent experiments; ∗P < 0:05; lower overlap region of the three circles interact with SNX27 and are lost unpaired t -test; error bars, s.e.m.; individual data points are shown in from the surface of SNX27- and VPS35-depleted cells, which makes them Supplementary Table S7 titled Statistics Source Data). (d) Flow cytometric high-confidence new cargo for the SNX27 and retromer. (b) Validation of analysis of surface-resident CD97, PTPRJ and TRAILR1 in SNX27- and selected hits by fluorescence-based quantitative western blotting on an VPS35-deficient cells. of control, SNX27- and retromer- (VPS35) suppressed cells through the highly efficient GFP-trap method12. Besides robustly detecting the triple SILAC analysis to uncover cargoes that depend on these known interaction partners DGKZ (ref. 13), β-Pix, GIT1 and GIT2 proteins for endosomal recycling and surface abundance. To obtain (ref. 14) (Supplementary Table S1), our screens identified 77 integral the interactome, GFP–SNX27 and GFP were lentivirally expressed in membrane proteins that were at least threefold enriched over control SILAC-labelled human RPE1 and HeLa cells and precipitated with (Supplementary Table S2). Many of these SNX27 interactors harbour 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION © 2013 Macmillan Publishers Limited. All rights reserved. ARTICLES a class I PDZ ligand at their C terminus (S-TX'), with an apparent that became more obvious when plasma membrane translocation preference for a negative charge (E or D) at the −3 position and, as was induced by high-copper conditions16 (Fig. 1c). Furthermore, published previously, a lack of a positive charge at the −5 position15 flow cytometric analysis confirmed that the surface receptors CD97, (also see text in Supplementary Table S2).