sorting out the cellular functions of sorting nexins

© 2002 Nature Publishing Group

Endocytic pathways function to internalize extra-cellular components, and they participate in diversesignal-transduction pathways. After their internaliza-tion by CLATHRIN-COATED VESICLES, cargo proteins aredelivered to peripheral EARLY ENDOSOMES or to sortingendosomes. Proteins in the early endosome compart-ment are sorted for delivery to one of at least threedestinations: the cell surface (for quick recycling);LYSOSOMES (through LATE ENDOSOMES); or the perinuclearendosomes, which include the TRANS-GOLGI NETWORK

(TGN)1–4 (FIG. 1).The SORTING NEXIN (SNX) family consists of a diverse

group of cytoplasmic and membrane-associated pro-teins that are involved in various aspects of endocyto-sis and protein trafficking through these membranouscompartments. Several putative sites of action for themammalian and yeast SNXs have been reported (FIG. 1).The hallmark of this protein family is the presence of aPX DOMAIN — a sequence of approximately 100–130amino acids that has recently been shown to bind vari-ous phosphatidylinositol phosphates (PtdInsPs),thereby potentially targeting these proteins to specificcellular membranes that are enriched in these phos-pholipids5–9 (FIG. 1). In addition to the PX domain,sorting nexins contain various protein–protein inter-action motifs that might participate in their subcellu-lar localization or their ability to form complexes at aparticular lipid-enriched membrane. So far, 25 humanSNXs have been identified, and recent studies haveuncovered roles for several of these proteins in the regulation of membrane trafficking.

This article describes the physical characteristics ofthe mammalian and yeast SNXs, such as the phospho-lipid-binding properties of their PX domains, their protein–protein interaction motifs and their cellularlocation, and, whenever possible, this information is correlated to the cellular function of the relevant protein.

The sorting nexin familyThe link between SNX proteins and the intracellulartrafficking of plasma-membrane receptors was postu-lated because of the homology that is shared betweenSNX1 and Mvp1, a yeast PX-domain-containing pro-tein that was isolated as a multicopy suppressor of vac-uolar sorting protein 1 (Vps1), which is a member ofthe dynamin GTPase family10. Vps1 mutant cells divertvacuole-bound membrane traffic to the plasma mem-brane11–13. Mvp1 interacts with Vps1 at a genetic level,colocalizes with Vps1 and is itself required for theefficient delivery of soluble proteins to the vacuole14.Given the role of Mvp1 in vacuolar sorting and thetwo-hybrid interaction that is formed between SNX1and the intracellular portion of the receptor tyrosinekinase epidermal growth factor receptor (EGFR),SNX1 was proposed to be involved in the targeting ofthe EGFR for lysosomal degradation through theendocytic pathway10. Other PX-domain-containingyeast proteins, Vps5 and Vps17, were subsequentlyshown to be involved in vacuolar protein sorting15. AsVps5 is thought to be the yeast orthologue of SNX1,this observation supported the postulate that SNXsfunction in membrane trafficking.

SORTING OUT THE CELLULARFUNCTIONS OF SORTING NEXINSCarolyn A. Worby and Jack E. Dixon

Sorting nexins are a diverse group of cellular trafficking proteins that are unified by thepresence of a phospholipid-binding motif — the PX domain. The ability of these proteins tobind specific phospholipids, as well as their propensity to form protein–protein complexes,points to a role for these proteins in membrane trafficking and protein sorting. It will beinteresting to determine whether the various sorting nexins have specialized or generalizedroles in protein trafficking.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | DECEMBER 2002 | 919

Department of BiologicalChemistry, University ofMichigan, Ann Arbor,Michigan 48109, USA.Correspondence to C.A.W.e-mail: [email protected]:10.1038/nrm974

CLATHRIN-COATED VESICLE

A vesicle that is coated withclathrin, which is a protein ableto self-assemble into triskelions(three-legged pinwheels) thatcoalesce with specific adaptorproteins (APs) to form apolyhedral lattice. Endocytosedvesicles are often coated with theclathrin–AP2 complex.

EARLY ENDOSOME

Irregularly shaped and mildlyacidic intracellular vesicle that isformed from an endocytosedclathrin-coated vesicle.

LYSOSOME

The cellular compartmentwhere the digestion of proteinsand other materials occurs.

R E V I E W S


LATE ENDOSOME

The acidic vesicle formed froman early endosome whereproteins are either sorted fortransport to the trans-Golginetwork or for digestion in thelysosome. They function as theprecursor vesicles for maturelysosomes.

TRANS-GOLGI NETWORK

(TGN). Membranouscompartment from whichvesicles bud to deliver proteinsand other materials to the cellsurface or to the late endosomesfor delivery to lysosomes.

SORTING NEXIN

(SNX). A hydrophilic protein of400–700 amino acids thatcontains a PX domain in themiddle or amino-terminal halfof the protein and regions ofpredicted coiled coils in thecarboxy-terminal half of theprotein. It functions in theendocytosis of plasma-membrane receptors and/or thetrafficking of proteins from onemembrane compartment toanother.

PX DOMAIN

(phox homology domain). Alipid- and protein-interactiondomain that consists of 100–130amino acids and is defined bysequences found in twocomponents of the phagocyteNADPH oxidase (phox)complex.

COILED-COIL DOMAIN

A bundle of α-helices that arewound into a superhelix. Coiledcoils were first described as themain structural element of alarge class of fibrous proteins,which includes keratin, myosinand fibrinogen.

SH3 DOMAIN

(Src homology 3 domain). Aprotein–protein interactiondomain of ~60 amino acids thatbinds to proline-rich (Pro-X-X-Pro) sequences.

TPR DOMAIN

(tetratricopeptide repeat). Thisdomain typically consists of 34amino acids and probablyfunctions as a protein–proteininteraction motif. It iscommonly found in proteinsthat aggregate in multi-proteincomplexes.

920 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

R E V I E W S

rion for inclusion in the SNX family is the presence of aPX domain, some proteins might be erroneouslyassigned to this family and might be involved in cellularfunctions that are unrelated to membrane trafficking.Only the systematic study of the cellular function ofthese individual proteins will be able to resolve thispotential miscategorization.

PX domainsThere are several, highly conserved phosphoinositol-binding motifs that have been described; these include theEPSIN AMINO (N)-TERMINAL HOMOLOGY (ENTH) domain, thePLEXTRIN HOMOLOGY (PH) domain, the FYVE (for Fab1/YOTB/Vac1/EEA1) domain and the PX domain6,26–29. TheENTH domain is found in several proteins that areinvolved in endocytosis and cytoskeletal organization,and the ENTH domains of epsin, adaptor protein 180(AP180) and clathrin assembly lymphoid myeloidleukaemia protein (CALM) have been shown to interactselectively with phosphatidylinositol-4,5-bisphosphate(PtdIns(4,5)P

2)28,30. The PH domain is present in various

proteins that are involved in cellular signalling (PKB/Akt,

A search for PX-domain-containing proteins usingthe Simple Modular Architecture Research Tool(SMART) program identified 25 putative human sort-ing nexins and the homologues for these proteins in arange of species from yeast to Arabidopsis16. The SNXfamily comprises proteins that contain very littlesequence homology except for the conserved sequencein the PX domain. On the basis of common domainstructures, this family can be divided into three sub-groups. SNX1, SNX2, SNX4, SNX5, SNX6, SNX7,SNX8, SNX15 and SNX16 all have long carboxy-termi-nal extensions that contain 1–3 COILED-COIL domains andcould be involved in homo- and/or hetero-oligomeriza-tion with other SNXs, as well as protein–protein interac-tions with non-SNX proteins17 (FIG. 2). SNX3, SNX10,SNX11, SNX12, SNX22, SNX23 and SNX24 can begrouped on the basis that they apparently contain onlya PX domain. The remaining sorting nexins containvarious protein–protein interaction sequences18–20(SH3,TPR), membrane targeting sequences21,22 (hydrophobicsequences, B41) or G-protein regulatory sequences23–25

(RGS, RA). It is important to note that as the main crite-

AP2

SNX1SNX2SNX4SNX6SNX15Mvp1Vps5

SNX3Grd19

SNX13

SNX9 (SH3PX1)

SNX13 (RGSPX1)

SNX17GTP

GDP

Gαs

Gαs

GPCR

Ligand

Ligand

Clathrin-coated pit

EEA1

PtdIns(3)P

PtdIns(3)P

Early endosome

Late endosome

Recycling endosome

4

3

2

2

1

PtdIns(3,4,5)P3PtdIns(4,5)P2

TFR TKR

TGNLysosome

βγ

Figure 1 | Protein and membrane trafficking. In this simplified scheme, binding of ligand to plasma-membrane receptors resultsin their internalization in clathrin-coated pits. Internalized clathrin-coated pits shed their clathrin coat and fuse with early endosomalvesicles that have various possible sorting pathways. The transferrin receptor (TFR) is often sorted into recycling endosomes for itsreturn to the plasma membrane (1). Tyrosine kinase receptors (TKRs) and seven-transmembrane G-protein-coupled receptors(GPCRs) are partly recycled through pathway 1, but they also accumulate in late endosomes (2) for transportation to the lysosomefor their subsequent degradation (2). The third pathway involves transportation to and from the trans-Golgi network (TGN) to the lateendosome and involves proteins such as hydrolases that are destined for the lysosome. Proteins are also sorted in the TGN fordelivery to the plasma membrane or for secretion from the cell (4). The positions where the sorting nexins (SNXs) are thought tofunction are indicated by the pink (mammalian) and purple (yeast) boxes. AP2, adaptor protein complex 2; EEA1, early endosomeautoantigen 1; PtdIns(3)P, phosphatidylinositol-3-phosphate. PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P3,phosphatidylinositol-3,4,5-triphosphate.



R E V I E W S

PX domains were originally identified as a con-served motif in the p40phox and p47phox subunits ofthe neutrophil NADPH oxidase (phox) superoxide-generating complex6. Like the PH domains, PXdomains show limited sequence conservation andhave a wide range of PtdInsP-binding specificities(TABLE 1). In addition to the sorting nexins, PXmotifs are also found in proteins that are involvedin cell signal-transduction pathways, such as phos-pholipases D (PLD)1/2 and phosphatidylinositol 3-kinase (PI3K), and in yeast proteins that areinvolved in bud emergence and cell polarity, such asBem1 and Bem3 (REFS 35,36).

Teasdale et al.2 have proposed that the PXdomains of the SNXs represent a distinct subgroup ofthe PX superfamily, which has been designated as theSNX-PX DOMAIN. BLAST searches of the non-redundantprotein database using SNX-PX sequences do notretrieve other PX-domain-containing subfamilies2. Inaddition, a phylogenetic analysis of the PX domainsindicates that most of the SNX-PX domains mightcluster into distinct branches of the PX superfamily(FIG. 3). The PX domains of PI3K, Bem2 and p40phox

cluster together, as do the PX domains of the PLDsand Bem3 (FIG. 3). Only SNX27, the cellular functionof which is unknown, is not clustered with the otherSNXs. So, SNX-PX domains have probably evolvedindependently from other PX domains, and thereforemight have unique cellular functions.

On the basis of the phylogenetic analysis of theirPX domains, SNX1, SNX2, SNX5, SNX6, SNX7,SNX9 and SNX18 are grouped with Vps5, a yeast pro-tein that is involved in vacuolar-TGN trafficking37

(FIG. 3). SNX1 and SNX2 share extensive sequenceidentity throughout their amino-acid sequences andthey have recently been shown to be largely function-ally redundant38. SNX5 and SNX6 also share extensiveamino-acid identity. SNX9 and SNX18 are related toeach other, in that they both contain amino-terminalSH3 domains. The group that contains Grd19, SNX3,SNX11 and SNX12 (FIG. 3) has no recognizable motifsother than the PX domain. Grd19 is a yeast SNX thatis involved in protein sorting from the pre-vacuolarcompartment to the TGN39. It will be of interest todetermine if SNX3, SNX11 and SNX12 are involvedin similar sorting functions in mammalian cells. Theremaining SNXs are separated into four other groups.Little is known about the cellular functions of theseputative SNXs, but it is tempting to speculate that theSNXs that share conserved PX domains falling on thesame branch of the phylogenetic tree might sharecommon functions in the cell.

The PX domain structureThe structure of the PX domain of p40phox bound toPtdIns(3)P was solved recently and shown to adopt anovel fold that consists of three β-sheets packedagainst a helical subdomain, which consists of fourα-helices (α2, α3, α4 and α4′), a 3

10helix and a type II

polyproline helix (PPII)40 (FIG. 4). The amino-terminal

α-helix in this structure (α1) is not part of the PX

PDK1), cytoskeletal organization (spectrin, α-actinin),membrane trafficking (Vav, ARFP), as well as GTPases(dynamin)31. PH domains have limited sequence conser-vation, but are instead defined by a conserved tertiarystructure. In contrast to the ENTH domain, PH domainsshow a broad range of PtdInsP-binding specificities that include phosphatidylinositol-3,4,5-triphosphate(PtdIns(3,4,5)P

3), PtdIns(4,5)P

2and phosphatidyl-

inositol-3,4-bisphosphate (PtdIns(3,4)P2)32,33. Conversely,

the FYVE domain of the EARLY ENDOSOME AUTOANTIGEN 1

(EEA1) was originally defined as a sequence that isresponsible for binding exclusively to phosphatidyl-inositol-3-phosphate (PtdIns(3)P). So far, all FYVEdomains have maintained this binding specificity34.

B41 DOMAIN

(Band 4.1 homology domain,also known as anezrin/radixin/moesin (ERM)protein domain). This domain iscommonly found incytoskeletal-associated proteins,linking these proteins to theplasma membrane.

RGS DOMAIN

(regulator of G-protein-signalling domain). A sequenceof ~120 amino acids thatfunctions as a GTPase activatorthat stimulates the inactivationof heterotrimeric G proteins,thereby rapidly turning offG-protein-coupled-receptorsignalling pathways.

RA DOMAIN

(RasGTP effector domain).Stimulates the dissociation ofGDP from Ras-related proteins,thereby allowing the binding ofGTP and activation of theGTPases.

EPSIN AMINO (N)-TERMINALHOMOLOGY DOMAIN

(ENTH). A phospholipid-binding motif with high affinityfor PtdIns(4,5)P

2.

PLEXTRIN-HOMOLOGY DOMAIN

(PH). A phospholipid-bindingmotif that interacts with manydifferent phospholipidsincluding PtdIns(3,4,5)P

3,

PtdIns(4,5)P2

and PtdIns(3,4)P2.

FYVE DOMAIN

(Fab1/YOTB/Vac1/EEA1domain). A phospholipid-binding motif that interactsexclusively with PtdIns(3)P.

EARLY ENDOSOMAL AUTO-ANTIGEN 1

(EEA1). A FYVE-domain-containing protein that iscommonly used as a marker forearly endosomes. This protein isan autoantigen that is associatedwith subacute cutaneoussystemic lupus erythematosis.

SNX-PX DOMAIN

The classification for the PXdomains that are found insorting nexins and thatparticipate in lipid–proteinand/or protein–proteininteractions.

SNX1 (Vps5)SNX2

SNX9SNX18

SNX13SNX14

SNX17

SNX19

SNX21

SNX25

SNX27

SNX4SNX5SNX6

SNX8 (Mvp1)SNX7SNX15SNX16

SNX3 (Grd19)SNX10SNX11SNX12SNX22–24

PX CC CC CC

PX CC CC

PX

PX

PX

PX

PX

PX

CC

PX CC

CC

SH3

PXA RGS

PXRGS

PXPXA

B41

TPR

RA

Figure 2 | Human sorting nexins. The sorting nexins (SNXs)have been grouped in accordance with their domainstructures. Yeast homologues are in parentheses. Thedomains include the PX (phox homology), CC (coiled-coil), SH3(Src homology 3), PXA (PX-associated), RGS (regulator of G-protein signalling), B41 (or ezrin/radixin/moesin (ERM)), TPR(tetratricopeptide repeat) and RA (RasGTP effector) domains.The dark green bars represent the hydrophobic sequencesthat might indicate membrane association.



R E V I E W S

domain but was essential for the soluble expression ofthe isolated PX domain. The phosphoinositide ‘head-group’ occupies one corner of the pocket, the walls ofwhich are formed by the β3/α2-loop, the PP

II/α3-loop

and the amino-terminal halves of the β2-strand andα3-helix. The remainder of the pocket is filled withwell-ordered water molecules.

Conserved residues. Arginine (Arg) residue 58 (FIG. 4), aconserved residue in the β3/α2-loop, forms the mostextensive interactions with the D3 phosphate of thebound PtdIns(3)P. Mutation of this residue to gluta-mine (Gln) abolishes PtdIns(3)P binding in vitro andeliminates the punctate pattern that signifies the endo-somal localization of p40phox in vivo40. The presence of abasic amino-acid residue in this position is conservedin most PX domains (FIG. 5), and it is conserved in all PX domains that have been reported to bindPtdIns(3)P, including SNX3, SNX7, SNX16, Vam7,CISK and p47phox (REFS 5,7,9,41,42). PX domains that donot contain a basic residue at this position, such as thePX domain of PI3K-C2α, bind PtdIns(4,5)P

2 (REF. 43).

This indicates that a basic residue analogous to Arg58might be a signature for PtdIns(3)P binding. However,the PtdInsP-binding specificity of more PX domainswill have to be ascertained to test this hypothesis.

Another highly conserved basic residue in PXdomains, Arg105 (FIGS 4,5), forms hydrogen bondswith the 4- and 5-OH of the inositol moiety. Theseinteractions are also essential for PtdIns(3)P binding,as a mutation of Arg105 to alanine (Ala) abolishesphospholipid binding40. An interesting feature of thisstructure is that other interactions with the mem-brane outside the headgroup-binding pocket mightorientate the PX domain with respect to the surface ofthe membrane.

Table 1 | Properties of sorting nexins

SNX Lipid-binding preference‡ Protein–protein interactions/complexes Potential proteins sorted Located in cell

SNX1 PtdIns(3,4,5)P3>PtdIns(3,5)P2> HRS, VPS26, VPS29, VPS35, SNX1, SNX2, ALK4, EGFR*, IR, Leptin, Cytosol, endosomesPtdIns(3)P SNX4, SNX6, SNX15 PAS1*, PDGFR, TFR

SNX2 PtdIns(3)P>PtdIns(4)P>PtdIns(5)P FBP17, VPS26, VPS29, VPS35, SNX1, SNX2, ALK4, ALK5, EGFR, IR, Cytosol, endosomesSNX4, SNX6 leptin, PDGFR, TβRII KD

SNX3 PtdIns(3)P N/D N/D Cytosol, endosomes

SNX4 N/D SNX1, SNX2, SNX6 N/D Cytosol, endosomes

SNX5 N/D FANCA N/D Cytosol, endosomes

SNX6 N/D PIM1, SNX1, SNX2, SNX4 ALK1, ALK5, ALK6, EGFR, IR, Cytosol, endosomesleptin, PDGFR, TβRII KD

SNX9 N/D ACK2, AP-50, MDC9, MDC15, WASP EGFR* N/D

SNX13 PtdIns(3)P = PtdIns(5)P> N/D EGFR*, GPCR EndosomesPtdIns(3,5)P2 = PtdIns(4)P

SNX15 N/D N/D Furin*, PDGFR*, TGN38*, EndosomesTFR*

SNX17 N/D P-selectin LDLR* Cytosol, recyclingendosomes

*Those proteins determined experimentally to be sorted by the relevant sorting nexin. ‡Lipid-binding preference as determined by the protein–lipid overlay assay. ACK2, Cdc42-associated tyrosine kinase 2; ALK, type I receptor in the TGF-β family; AP-50, the Drosophila orthologue of the µ subunit of the AP2 protein complex; EGFR, epidermal growthfactor receptor; FANCA, Fanconi anaemia complementation group A protein; GPCR, G-protein-coupled receptor; HRS, hepatocyte growth factor (HGF)-regulated tyrosine-kinase substrate; IR, insulin receptor; LDLR, low-density lipoprotein receptor; MDC, metalloprotease, disintegrin, cysteine-rich proteins; N/D, not determined; SNX, sorting nexin;TFR, transferrin receptor; PDGFR, platelet-derived growth factor receptor; PtdIns(3)P, phosphatidylinositol-3-phosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate;PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-triphosphate; TβRII KD, TGF-β type II kinase dead receptor; TGN38, trans-Golgi network protein 38; Vps, vacuolar protein sortingreceptor; WASP Wiskott–Aldrich syndrome protein.

SNX8

Mvp1

SNX16

SNX19

SNX25SNX13

SNX22

SNX24

SNX14

SNX23 SNX15SNX21SNX4

SNX17

SNX10

SNX11

Grd19

SNX12

SNX3

SNX9

SNX6 SNX5SNX1 SNX2

SNX18

SNX27

SNX7

Vps5

Bem3

PLD2

PLD1

P13K

Bem2p40phox

Figure 3 | PX-domain phylogenetic tree. The amino-acid alignment for the PX domains wascreated using the CLUSTALW program (Open Gap penalty 10, Extend Gap penalty 0.05, Gapseparation distance 8) located at Expasy. The alignment preserved the secondary structures thathave been determined for the p40phox and Vam7 PX domains. The unrooted phylogenetic treewas created by Phylip using bootstrapping to generate multiple data sets90. The SNXs that arepresent on the branches that emanate from the base of the tree have been grouped in differentcoloured ellipses. The groups that contain the PX domains and are not part of the SNX-PXdomain subgroup are similarly coloured (purple).



R E V I E W S

residue is turned away from the PtdIns(3)P-bindingsite. Instead, it has a vital structural function becauseit forms a network of hydrogen bonds that stabilizesthe unique fold of the PX domain40. Mutation of theanalogous residue in p47phox results in chronic granu-lomatous disease (CGD) (BOX 1). The conserved lysine(Lys) residue that is immediately after the Pro-X-X-Pro motif (Pro, proline) and Arg105 present in theα3-helix participate in the binding pocket for thephosphate groups of PtdIns(3)P as described above.

Most of the SNXs also contain a Pro-rich sequencethat is located at the carboxyl terminus of the α2-helix (FIGS 4,5). This Hyd-Pro-X-X-Pro-X-(Lys,Arg)sequence is a potential SH3-domain-binding motif,although, so far, no SH3-binding partners have beenreported for any of the SNX-PX domains. However,SH3-domain interactions with Pro-rich sequences arethe principal protein–protein interactions betweenp40phox, p47phox, p67phox and NADPH oxidase subunits(for a review, see REF. 44). In the absence of extracellularstimuli, p47phox exists in an inactive conformation thatis attributable to an intramolecular interaction betweenthe Pro-X-X-Pro motif in its PX domain and its car-boxy-terminal SH3 domain. The NMR structure ofthis PX domain bound to the SH3 domain shows thatbinding alters the conformation of the Pro-X-X-Proloop and of the hydrophobic contacts between helicesin the structural core45. A high-resolution X-ray struc-ture for the p47phox domain has recently been reportedand shows that this PX domain contains two basicpockets that represent two potential phospholipid-binding sites46. One site is analogous to the p40phox

PtdIns(3)P-binding site and binds PtdIns(3,4)P2,

whereas the other site, which is unique to the PXdomains of p47phox and phospholipases D1 and D2,binds anionic phospholipids such as phosphatidic acidor phosphatidylserine. Karathanassis et al.46 haveshown that these two sites are strongly synergistic inmembrane binding and show that, in the full-lengthprotein, the SH3–Pro-X-X-Pro interaction affects theaccessibility of the PX-domain phosphoinositide-bind-ing pocket for the PtdInsP. At present, it is not knownwhether binding of the p40phox Pro-X-X-Pro motif toits SH3 ligand affects its ability to bind PtdIns(3)P.However, these scenarios raise the intriguing possibilitythat SH3-domain interactions could act as switchesthat regulate PX domain function.

Tertiary structure of the Vam7 PX domain. The three-dimensional solution-structure of the PX domain ofVam7, a SNARE protein that functions in vacuolarmembrane trafficking in yeast, was solved by NMRspectroscopy47. Vam7 consists of an amino-terminalPX domain and a carboxy-terminal coiled-coil target-membrane (t)-SNARE motif. It is localized specifi-cally to the vacuolar membrane as part of the vacuolarSNARE complex by the interactions between its PXdomain and PtdIns(3)P and by the interaction of itscoiled-coil domain with Vam3. This study showedthat PX domains share a structural core that lines thePtdInsP-binding pocket and that there are other

Tyrosine (Tyr) residue 94 (FIGS 4,5) interactshydrophobically with the glycerol moiety of thebound PtdIns(3)P. Hydrophobic residues in thisregion, which is known as the ‘membrane attachmentloop’, are conserved in many PX domains, which indi-cates that this might be a common feature with a rolein binding PtdInsPs in lipid bilayers (a discussion ofthe other conserved residues that are involved in stabi-lizing the binding of PtdIns(3)P (FIG. 5) can be foundin REF. 40).

Conserved structural features. An alignment of theSNX-PX domains and a schematic of the secondarystructure of p40phox can be seen in FIG. 5. Although fewindividual amino acids are conserved among SNX-PXdomains, analysis of the secondary structures, usingProteinPredict software, predicts that all of the SNX-PXdomains form secondary structures that are similar tothat of p40phox (REF. 2).

Throughout the PX domain are patches ofhydrophobic and hydrophilic conserved residues. TheArg-Arg-Hyd-Ser-(Asp,Glu)-Phe motif (Hyd, hydro-phobic; Ser, serine; Asp, aspartic acid; Glu, glutamicacid; Phe, phenylalanine) that is located in the β3α2-region is highly conserved and contains the basicresidue, Arg58, that forms part of the positivelycharged binding pocket in p40phox, which interactswith the negatively charged phosphate group ofPtdIns(3)P. The other highly conserved basic residuein this sequence, Arg57, was predicted to be a phos-phate ligand, but the structure showed that this

SNARE

(soluble N-ethylmaleimide-sensitive factor attachmentprotein receptor). SNAREs areproteins that are involved inmembrane-fusion events in thesecretory and endocyticpathways. SNAREs containcoiled-coil domains that formtight four-helix bundles, whichare proposed to drivemembranes into closeapposition ready for fusion.

N

C

α1

α2

α3

α4′

α4β2

β1

β3

310 PPII

R105

R58Y94

Figure 4 | Crystal structure of the p40phox domain. The PXdomain of p40phox bound to PtdIns(3)P (protein databank (PDB)ID: 1H6H) is depicted as a ribbon diagram91. The Pro-X-X-Pro(Pro, proline; X, any amino acid) loop that contains Tyr94 isshown in yellow. The side chains of Arg58 and Arg105 areblue. The PtdIns(3)P molecule is portrayed as a ball-and-stickmodel with carbon atoms in green, oxygen atoms in red andphosphorus atoms in magenta.



R E V I E W S

conserved residues in the Vam7 PX domain results inthe localization of Vam7 to the cytoplasm. This under-scores the importance of the PX domain for appropri-ate membrane targeting, however, localization ofVam7 to the vacuole membrane is also dependent onits coiled-coil domain interactions with Vam3. So, itcan be inferred that for some SNXs, protein–protein

membrane attachment sites in the hydrophobic loopthat connect the Pro-X-X-Pro motif with α3 (FIG. 5).Therefore, the membrane targeting ability of theVam7 PX domain might involve both a specific, basicPtdInsP-binding pocket and nonspecific hydrophobicinteractions between loop residues and the mem-brane. Cheever et al.7 showed that the mutation of

KK

IVVIIIIVIVHPLVVIDFLVLKAVATKFFL

Vps5SNX 1SNX 2SNX 5SNX 6SNX 7SNX 9SNX18Mvp1SNX 8Grd19SNX 3SNX10SNX11SNX12SNX13SNX14SNX15SNX16SNX4SNX17SNX19SNX21SNX22SNX23SNX24SNX25SNX27Vam7p40phox

K

LLLLFLLLLLLLLLLILLLLLLLILLLLLL

Q N N H WS E K H SA S K Y LI E T T DV E N E DE E A H PL V K F GA E K F PL K R Y PL H K F PI K E I SE R E S KQ S N A LQ R N A GE R D S KT E Q F ET E F H GA Y T H RK E M F PL V Y Y PR K E Y GE E K P DQ R Q F RK K L Y KK L K Y AK K C I KS E C V PK R E F AE R D V GE E R F G

K A V A F K V E V K D P V K V G E L T - - - - S I H V E Y T V I S E S S - - - - - - - - L L E L K Y A Q V S R R Y R D F R W L Y R Q G - - - - - - - - K VIIIIIPSMMNVQPVSPERVVIA-A-KTDT

D Q F D L T V G I T D P E K I G D G - - - - M N A Y V A Y K V T T Q T S L P - - - - - - L F R S K Q F A V K R R F S D F L G L Y E K Q N - - - - - - G FD I F D I E I G V S D P E K V G D G - - - - M N A Y M A Y R V T T K T S L S - - - - - - M F S K S E F S V K R R F S D F L G L H S K H V - - - - - - G YD P S L Q I D I P D A L S E R D - - - - - - - - K V K F T V H T K T T L P - - - - - - T F Q S P E F S V T R Q H E D F V W L H D T Y A - - - - - - G LD A A L Q V D I S D A L S E R D - - - - - - - - K V K F T V H T K S S L P - - - - - - N F K Q N E F S V V R Q H E E F I W L H D S Y A - - - - - - G Y

P D L K D L F I T V D E P E S H V T T - - - - I E T F I T Y R I I T K T S R G - - - - F D S S E F E V R R R Y Q D F L W L K G K - - - - - - - - T LT S T F D C V V A D P R K G S K M Y G - - - - L K S Y I E Y Q L T P - - - - - - - - -

-- E - T N T N R S V N H R Y K H F D W L Y E R S - - - - - - - A I

P Y P F Q C T I D D P T K Q T K F K G - - - - M K S Y I S Y K L V P - - - - - - - - - - - - - T H T Q V P V H R R Y K H F D W L Y A R - - - - - - - - V IR P L D A D I I I I E E I P E R E G L - - - - L F K H A N Y L V K H L I A L P - - - - - S T S P S E E R T V V R R Y S D F L W L R E I - - - - - - - - F RE L L A R D T V Q V E L I P E K K G L - - - - F L K H V E Y E V S - - - - - - - - - - - - - S Q R F K S S V Y R R Y N D F V V F Q E M - - - - - - - - Y RP E N F L E I E V H N P K T H I P N G M D S K G M F T D Y E I I C R T N L P - - - - - - S F H K R V S K V R R R Y S D F E F F R K C - - - - - - - - M L

P P S N F L E I D V S N P Q T V G V G - - - - R G R F T T Y E I R V K T N L P - - - - - - I F K L K E S T V R R R Y S D F E W L R S E - - - - - - - - V VE E F V S V W V R D P R I Q K E D - - - F W H S Y I D Y E I C I H T N S M - - - - - - C F T M K T S C V R R R Y R E F V W L R Q R - - - - - - - - L VE E V I T V R V Q D P R V Q N E G - - - S W N S Y V D Y K I F L H T N S K - - - - - - A F T A K T S C V R R R Y R E F V W L R K Q - - - - - - - - L VS N F L E I D I F N P Q T V G V G - - - - R A R F T T Y E V R M R T N L P - - - - - - I F K L K E S C V R R R Y S D F E W L K N E - - - - - - - - - I

A Y I S D T G V C N D H G - - - - K T Y A L Y A I T V H R R N L N - - - - - - - S E E M W K T Y R R Y S D F H D F H M R S - - - - - - - L SI S I P Y V D F F E D P S S E R K E K - - - K E R I P V F C I D V E R N D R R - - - A V G H E P E H W S V Y R R Y L E F Y V L E S K - - - - - - - - A FD F L R H Y T V S D P R T H P K G - - - - - - - Y T E Y K V T A Q F I S K - - - - K D P E D V K E V V V W K R Y S D F R K L H G D N L - - - F R R L ED R P S T P T I L G Y E V M E E R - - - - - A K F T V Y K I L V K K T P - - - - - - - - - - E E S W V V F R R Y T D F S R L N D K - - - - - - - - G F

K K I E I S V S E A E K R T G R N A M N - M Q E T Y T A Y L I E T R S V E H - - - - T D G Q S V L T D S L W R R Y S E F E L L R S Y - - - - - - - - H IM H F S I P E T E S R S G D S G - - - - G S A Y V A Y N I H V N - - - - - - - - - - - - - - G V L H C R V R Y S Q L L G L H E Q - - - - - - - - A N

P V I I Q N L R I T G T I T A R E H S G T G F H P Y T L Y T V K Y E T A L D G E N S S G L Q Q L A Y H T V N R R Y R E F L N L Q T R L R - - - - - - K FE V T S A N V V K D P P S - - - - - - K Y V L Y T L A V I G P G P - - - - - - - P D C Q P A Q I S R R Y S D F E R L H R N G - - - - - - - P M

V H I P S V G P E A E G P R Q S P E K - - - - - - S H M V F R V E V L C S - - - - - - - - - - - G R R H T V P R R Y S E F H A L H K R - - - - - - - - - -I K I S I P R Y V L C G Q G - - - - K D A H F E F E V K I T - - - - - - - - - - - V L D E T W T V F R R Y S R F R E M H K T - - - - - - - - E LI P S F R Y E E S D L E R - - - - - - G Y T V F K I E V L M N - - - - - - - - - - - G R K H F V E K R Y S E F H A L H K K - - - - - - - - - -

G M W K A S I T S G E V T E E N - - - - G E Q L P C Y F V M V S L Q E V G - - - G-- V E T K N W T V P R R L S E F Q N L H R K - - - - - - - - S LT E K Q A V P I S V P R Y K H V E Q N - - - - G E K F V V Y N V Y M A G - - - - - - - - - - - - - - R Q L C S K R Y R E F A I L H Q N N - - - - - - - - FN S V G K M S E K L R I K V D D V K I N - - - - P Y V L Y G V S T P - - - - - - - - - - - - - - - N K R L Y R Y S E F W L T R - - - - - S T I P YD D V A I S A N I A D I E E K R G - - - - - F T S H F V F V I E V K T K - - - - - - - - - - G G S K Y L I Y R Y R Q F H A L Q S K P D S K S S A L A C

Vps5SNX 1SNX 2SNX 5SNX 6SNX 7SNX 9SNX18Mvp1SNX 8Grd19SNX 3SNX10SNX11SNX12SNX13SNX14SNX15SNX16SNX4SNX17SNX19SNX21SNX22SNX23SNX24SNX25SNX27Vam7p40phox

P P P P E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q S V G S F K E N F I E N R R F Q M E S M L K K I C Q D P V L Q K - - - D K D F L L F L T S DP P P P E - - - - - - - - - - - - - - - - - - - S L I G M T K V K V G K E D S S S A E F L E K R R A A L E R Y L Q R I V N H P T M L Q - - - D P D V R E F L E K EP P A P E - - - - - - - - - - - - - - - - - - - S I V G M T K V K V G K E D S S S T E F V E K R R A A L E R Y L Q R T V K H P T L L Q - - - D P D L R Q F L E S SP P A P T P D F D G P R E K M Q K L G E G E G S M T K E E F A K M K Q E L E A E Y L A V F K K T V S S H E V F L Q R L S S H P V L S K - - - D R N F H V F L E Y DP P A P P P D F D A S R E K L Q K L G E G E G S M T K E E F T K M K Q E L E A E Y L A I F K K T V A M H E V F L C R V A A H P I L R R - - - D L N F H V F L E Y NP P L P E - - - - - - - - - - - - - - - - - - - - - - - - - F I V K G M V E R F N D D F I E T R R K A L H K F L N R I A D H P T L T F N - - - E D F K I F L T A QP S L P D -

K- - - - - - - - - - - - - - - - - - - - - - - - - - - Q V T G R F E E E F I K M R M E R L Q A W M T R M C R H P V I S E - - - S E V F Q Q F L N F R

P H L P E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q A T - R F E E D F I S K R R K G L I W W M N H M A S H P V L A Q - - - C D V F Q H F L T C PP E L P P - - - - - - - - - - - - - - - - - - - - - - - - - - - - R I G S Q N A D Q L F L K K R R I G L S R F I N L V M K H P K L S N - - - D D L V L T F L T V RP A L P P - - - - - - - - - - - - - - - - - - - - - - - - - - - - R M - - L G A D R E F I E A R R R A L K R F V N L V A R H P L F S E - - - D V V L K L F L S F SP K V M V P - - - - - - - - - - - - - - - - - - - - - - - H L P G K I L L S N R F S N E V I E E R R Q G L N T W M Q S V A G H P L L Q S G - - S K V L V R F I E A EP L P G K A - - - - - - - - - - - - - - - - - - - - - F L R Q L P F R G D D G I F D D N F I E E R K Q G L E Q F I N K V A G H P L A Q N - - - E R C L H M F L Q D EP E L P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - K N L F F N M N N R Q H V D Q R R Q G L E D F L R K V L Q N A L L L S - - - D S S L H L F L Q S HP E L P G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K S T F F G T S D E F I E K R R Q G L Q H F L E K V L Q S V V L L S - - - D S Q L H L F L Q S QP P L P G K - - - - - - - - - - - - - - - - - - - L K R K L P F R G D E G I F E E S F I E E R R Q G L E Q F I N K I A G H P L A Q N - - - E R C L H M F L Q E EL K L P G K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K T F N N M D R D F L E K R K K D L N A Y L Q L L L A P E M M K A S - - P A L A H Y V Y D F LA Q L P S K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R I I G P K N Y E F L K S K R E E F Q E Y L Q K L L Q H P E L S N - - - S Q L L A D F L S P NP A F P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R A Q V F G R F E A S V I E E R R K G A E D L L R F T V H I P A L N N - - - S P Q L K E F F R G GA L P P K R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - W F K D N Y N A D F L E D R Q L G L Q A F L Q N L V A H K D I A N - - - C L A V R E F L C L DP P L P E K - - - - - - - - - - - - - - - - - - - - - R A E F V W H K L S A D N M D P D F V E R R R I G L E N F L L R I A S H P I L C R - - - D K I F Y L F L T Q EP A F P P K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K L F S L T P A E V E Q R R E Q L E K Y M Q A V R Q D P L L G S - - - S E T F N S F L R R AN V K G P K - - - - - - - - - - - - - - - - - - - - - K-- K L F P D L P F G N M D S D R V E A R K S L L E S F L K Q L C A I P E I A N - - - S E E V Q E F L A L NI S F P R K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R L R R N F T A E T I A R R S R A F E Q F L G H L Q A V P E L R H - - - A P D L Q D F F V L PP D F P S K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R L P N W R T R G L E Q R R Q G L E A Y I Q G I L Y L N Q E V P - - - - K E L L E F L R L RL E F P P K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K L F G N K D E R V I A E R R S H L E K Y L R D F F S V M L Q S A T - - S P L H I NP E I P S K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - H V R N W V P K V L E Q R R Q G L E T Y L Q A V I L E N E E L P - - - - K L F L D F L N V RV Q L P S L S- - - - - - - - - - - - - - - - - - - - - - - - - -- K L P F K S I D Q K F M E K S K N Q L N K F L Q N L L S D E R L C Q - - - S E A L Y A F L S P SP R L P G K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - W P F S L S E Q Q L D A R R R G L E E Y L E K V C S I R V I G E - - - S D I M Q E F L S E SP E K P - - - - - - - - - - - - - - - - - - - - - - - - - G V L D R R W Q R R Y D D P E M I D E I G L E R F L N E L Y N D R F D S R W R D T K I A Q D F L Q L SP T L P A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V Y V G V K Q E I A E M I P A L N A Y M K S L L S L P V W V L M - - D E D V R I F F Y S

P P XX X K

β1 β2 β3 α2

α3 α4

E--

KR

R RRK

KKKK

K

RK

KK

K

A-

2771411382829272472731257032248

1427563507

8105561

527134

4715

65418

390268265169170147357382243178158

124129149673626126214184105659242108174103322172121136

148

206

*

Figure 5 | SNX-PX domain alignment. The CLUSTALW parameters for this alignment are described in the legend to FIG. 3.Hydrophobic residues are highlighted by green boxes, whereas hydrophilic residues are highlighted by blue (basic) or red (acidic)boxes. The Pro-X-X-Pro motif (Pro, proline; X, any amino acid) is designated by yellow boxes. The amino acids that are known to beinvolved in binding the phospholipid for the p40phox and Vam7 PX domains are highlighted by orange boxes, and the secondarystructure determined for these PX domains is indicated by arrows (β-sheets) and ovals (α-helices). The lipid-micelle-interactingelement in Vam7 is boxed. The arginine residue that is mutated in chronic granulomatous disease patients is denoted by an asterisk.Arg58, Arg105 and Y94 are indicated by pink triangles. The Genbank accession numbers of the sorting nexins (SNXs) that wereused for this aligment are as follows: SNX1, Q13596; SNX2, O60749; SNX3, O60493; SNX4, O95219; SNX5, Q9Y5X3; SNX6,Q9UNH7; SNX7, Q9UNH6; SNX8, Q9Y5X2; SNX9, Q9Y5X1; SNX10, Q9Y5X0; SNX11, Q9Y5W9; SNX12, Q9YMU4; SNX13,Q9Y5W8; SNX14, Q9Y5W7; SNX15, Q9NRS6; SNX16, P57768; SNX17, Q15036; SNX18, Q96RF0; SNX19, Q92543; SNX21,Q969T3; SNX22, Q96L94; SNX23, Q96L93; SNX24, Q9Y343; SNX25, Q9H3E2; SNX27, Q96L92.



R E V I E W S

for the retrieval of Kex2 and A-ALP (two Golgi-resident proteins), but not Vps10, and therefore doesnot have a role in sorting soluble enzymes to the vacuole39.

Mvp1 is the yeast homologue for SNX8 (REF. 14).Mvp1 was isolated as a multicopy suppressor ofVps1 mutants that are deficient in trafficking of thecarboxypeptidase Y receptor. Mvp1, in conjunctionwith Vps1, is involved in sorting proteins in the lateGolgi for delivery to the vacuole. It might also functionin the retrieval of proteins from the pre-vacuolarendosomes to the late Golgi14.

Functions of sorting nexins in humansSNX1. SNX1 was the first mammalian SNX to becharacterized10. It was isolated from a two-hybridscreen by using the kinase domain and the LYSOSOMAL

TARGETING SEQUENCE (tyrosine- or di-leucine-basedmotifs) of the EGFR as a bait. The portion of SNX1that was isolated in this screen (the carboxy-terminalcoiled-coil sequences) interacted specifically with thelysosomal targeting motif, Tyr-Leu-Val-Ile.Importantly, overexpression of full-length SNX1 inAfrican green monkey kidney (CV1) cells markedlydownregulated the amount of activated EGFR onthe cell surface. This effect was highly specific asoverexpression of SNX1 did not alter ERBB2 orplatelet-derived growth factor receptor (PDGFR)levels in these cells. Furthermore, overexpression ofthe amino terminus of SNX1 (which includes the PXdomain) did not affect the rate of ligand-inducedendocytosis of the EGFR, which indicates that SNX1does not affect the AP2 clathrin-coat complex butmore probably directs vesicular trafficking and pro-tein sorting in the endocytic compartment. The fateof endocytosed membrane proteins, such as theEGFR, is determined in the sorting endosomes.Therefore, SNX1 is postulated to be a candidateENDOSOMAL RETENTION protein.

SNX1 is both membrane associated and cytosolic,where it probably exists as a tetramer in large proteincomplexes49 (TABLE 1). Similar to its yeast homologueVps5, SNX1 associates with the proteins that make upthe retromer complex — VPS26, VPS29 and VPS35(REF. 50). So far, there is no known human homologuefor Vps17, which is part of the retromer complex inyeast. It is probable that the SNX1 protein complexcan associate reversibly with membranes of the endo-somal compartment, thereby coating these vesicles51.It is also probable that SNX1 forms complexes with SNX2, as these proteins have been shown to co-immunoprecipitate50. Co-transfection analysesand the subsequent subcellular localization of SNX1and SNX1, which is detected by immunofluorescence,indicate that SNX1 and SNX2 colocalize in a punctatestaining pattern that partially overlaps with the early-endosome marker EEA1 (REFs 49,51,52). SNX1 hasalso been shown to interact with SNX4, SNX6 andSNX15, but no functional significance has been attrib-uted to these interactions. However, the interaction ofSNX1 with hepatocyte growth factor (HGF)-regulated

interaction motifs might help the PX domain to targetthe protein to its appropriate cellular membrane.

Functions of sorting nexins in yeastPtdInsPs have long been known to have roles in cargosorting and vesicle formation, and PX-domain-containing proteins — in conjunction with other pro-teins that contain PtdInsP-binding modules — probablyfunction as adaptors and/or cargo sorters. Althoughthis area of research is in its infancy, as compared withmammalian SNXs, several yeast PX-domain-containingproteins are known to have well-characterized roles inprotein sorting. In particular, the yeast proteins Mvp1,Grd19, Vps5/Grd2 and Vps17 have all been shown tofunction in protein trafficking.

Vps5, the yeast homologue of SNX1 and SNX2,was identified as a protein that is responsible for vac-uolar trafficking37,48. A defective Vps5 results in thesecretion of the hydrolase carboxypeptidase Y, and theincorrect delivery of the hydrolase receptor Vps10 tothe vacuole. Under normal conditions, Vps10 bindscarboxypeptidase Y in the TGN and carries it to pre-vacuolar endosomes where the hydrolase dissociates.The hydrolase is delivered to the vacuole, whereasVps10 is recycled to the TGN for further rounds ofhydrolase transportation. In addition to this recyclingstep, Vps5 is also required for the retention ofenzymes such as dipeptidyl amino peptidase (DPAP)and Kex2 in the late Golgi — it does this by retrievingmisplaced molecules from the pre-vacuolar endo-somes37,48. Vps5 carries out its biological functions byassembling into the ‘retromer complex’, which isdescribed in BOX 2.

Grd19 is the yeast homologue of the group ofSNXs, including SNX3, that contain only a PXdomain. It is a small hydrophilic protein that is pre-dominantly localized in the cytosol. However, in ayeast Vps mutant that accumulates an enlarged pre-vacuolar compartment, Grd19 accumulates in thatcompartment39. Grd19 has been shown to be a com-ponent of the retrieval machinery that functions byinteracting directly with the cytosolic tails of certainresident TGN proteins during the sorting process atthe pre-vacuolar compartment. Grd19 is important

LYSOSOMAL TARGETING

SEQUENCE

There are two types of lysosomaltargeting sequences: thetyrosine-based sequence, whichconsists of a tyrosine residuefollowed by a hydrophobic(Hyd) residue three amino acidslater (Tyr-X-X-Hyd), and the di-leucine repeat (LL). Usuallymore than one of these targetingsequences is found in proteinsthat are destined for thelysosome.

AP2

This adaptor protein complex isa component of clathrin-coatedvesicles that are associated withthe plasma membrane. Thecomplex is heterotetramericwith two large subunits, α andβ2, a medium-sized µ-subunitand a small σ-subunit. AP-50 isthe Drosophila equivalent of theµ-subunit that has beenimplicated in cargo selection.

ENDOSOMAL RETENTION

A mechanism that traps specificproteins inside the endosomalcompartment, therebypreventing recycling of theprotein to the cell surface.

Box 1 | PX domains and disease

The PX domain was first characterized by a comparison of protein domains in thephagocytic NADPH oxidase (phox) complex, which has a crucial role in host defenceagainst microbial infection by generating microbicidal oxidants83. Chronicgranulomatous disease (CGD) is an inherited disorder in which these oxidants are notgenerated, thereby resulting in recurrent and life-threatening infections. The activeoxidase complex contains cytochrome b558 (the catalytic core), the small GTPase Rac,p67phox and the PX-domain-containing protein p47phox. Another PX-domain protein,p40phox, is crucial for activating the complex that is normally dormant in resting cells.Naturally occurring mutations in the catalytic core, p67phox or p47phox have been found inCGD patients84. An arginine to glutamine mutation in p47phox (the analogous residue inp40phox is marked by an asterisk in FIG. 5) has been identified in a subset of these patients.This mutation, which is located in the PX domain, eliminates phosphoinositide binding,thereby causally linking PX-domain function to the manifestation of CGD.



R E V I E W S

protease-activated receptor-1 (PARS1) is regulated bySNX1. Overexpression of the carboxyl terminus ofSNX1 results in the accumulation of PARS1 in theearly-endosomal compartment instead of the lyso-some. This implies that SNX1 is involved in sortingPAR1 from early endosomes to lysosomes. The chal-lenge in the case of all of the aforementioned recep-tors is to determine the functional role of SNX1 intheir cellular trafficking.

The association of SNX1 with endosomal mem-branes requires PI3K activity, which indicates that theD3 phosphate is essential for PtdInsP recognition55. AsPtdIns(3)P is a well-established lipid component ofendosomes and as all yeast PX domains recognizePtdIns(3)P (REF. 56), the observation that the SNX1 PXdomain specifically recognizes PtdIns(3,4,5)P

3is

unusual, because this lipid is localized principally tothe plasma membrane where it assembles PH-domain-containing proteins57,58. A recent study hasconfirmed that, in protein–lipid overlay assays, SNX1does bind PtdIns(3,4,5)P

3. However, these researchers

also showed that SNX1 binds PtdIns(3)P andPtdIns(3,5)P

2— but not PtdIns(3,4,5)P

3— in lipo-

some-based assays59. To give credibility to the latterresult, the subcellular localization of SNX1 was deter-mined under conditions in which plasma-membranePtdIns(3,4,5)P

3levels were significantly elevated59.

Under those conditions, SNX1 failed to associate withthe plasma membrane, whereas a control PH-domain-containing protein became strongly associ-ated with the plasma membrane. Although the reasonfor this lipid-binding discrepancy remains obscure,these results indicate that the lipid-binding specifici-ties of the SNXs should be verified by alternativeexperimental procedures.

In addition to its lipid-binding abilities, the PXdomain might participate in the homo- and hetero-oligomerization of SNX1 and SNX2. However, the roleof PX domains in protein–protein interactions remainsto be confirmed. Yu and Lemmon proposed that ‘low-affinity’ PX domains provide specificity in localization,but that other targeting domains are needed to drivemembrane association56. Indeed, most PX-domain-containing proteins are associated with large proteincomplexes. In the case of SNX1, mutation of either thePX domain or the coiled-coil carboxyl terminus abol-ishes vesicle localization, which leads to the conclusionthat both the PX and coiled-coil domains are necessaryfor correct cellular localization. As noted previously,Vam7 has similar requirements for its appropriatemembrane targeting. The Vam7 PX domain alone tar-gets the protein to endosomal and vacuolar membranes.The coiled-coil domain of Vam7 is necessary to restrictthe protein to its normal vacuolar localization.

Recently, mice that lack Snx1 and/or Snx2 havebeen generated38. Snx1−/− and Snx2−/− mice are viableand fertile, but the development of Snx1−/−/Snx2−/−

embryos is arrested at midgestation, which indicatesthat Snx1 and Snx2 are functionally redundant andprovide a necessary function in embryogenesis. Thephenotype of the double-knockout mice is similar to

tyrosine-kinase substrate (HRS) — a FYVE-domain-containing protein that is localized to early endosomesand is a prominent target for phosphorylation by vari-ous receptors, including the EGFR — does have func-tional consequences53. HRS and SNX1 compete forthe same binding site on the EGFR, and overexpres-sion of HRS inhibits ligand-induced degradation ofthe EGFR, which indicates that HRS might modulatelysosomal trafficking of the receptor by sequesteringSNX1 (REF. 54).

SNX1 has also been shown to interact with severalother members of the receptor tyrosine kinase family,including the PDGFR and the insulin receptor (IR). Inaddition, SNX1 co-immunoprecipitates with the longform of the leptin receptor — a cytokine receptor thatsignals through the activation of Janus tyrosinekinases — and transferrin receptors (TFR), which arereceptors that internalize and recycle constitutively51

(TABLE 1). Finally, downregulation of G-protein-coupled

HRS

A FYVE-domain-containingprotein that is localized to earlyendosomes. HRS contains aclathrin-binding motif as well asa ubiquitin-interaction motifand can therefore affect thesorting of ubiquitylated proteinsinto the clathrin-coatedmicrodomains of earlyendosomes.

Box 2 | Complex formation by sorting nexins

The PX-domain-containing proteins Vps5and Vps17 are part of thewell-characterizedretromer complex inSaccharomyces cerevisiae.This complex functions inthe endosome-to-Golgiretrieval of the vacuolarprotein sorting receptorVps10, as well as theGolgi-resident membraneprotein A-ALP and the endopeptidases Kex2 and dipeptidyl amino peptidase(DPAP)48,85. The other members of this complex that have been identified so far areVps35,Vps29 and Vps26 (REF. 86). The protein subcomplexes that are involved in cargoselection and vesicle formation are bracketed (see figure). Although each of theretromer-complex proteins cycles between a cytoplasmic and a membrane localization(as indicated by the arrows),Vps35 is mostly membrane associated and its interactionwith Vps29 is responsible for bringing this protein to the membrane48.Vps35 acts as a‘receptor protein’ for cargo proteins, and recruits them into retrograde vesicles by directinteraction with (Y/F)XXΦ motifs (where Φ is hydrophobic) that are present in theircarboxyl termini85,87.Vps29 might assist in this process by enhancing the association ofVps35 with the membrane and/or the other complex members.Vps26 assists bypromoting the interaction between the cargo-selective component (Vps35) and thestructural components (Vps5 and Vps17) of the retromer complex88.Vps5 and Vps17dimerize strongly through the coiled-coil motifs that are present in their carboxyltermini89. Even though Vps5 and Vps17 both contain PX domains, only the Vps17 PXdomain has been shown to be essential for their membrane localization, whereas theamino terminus of Vps5 is responsible for interacting with the Vps35–Vps29–Vps26complex89. The PX domains of Vps5 and Vps17 bind phosphatidylinositol-3-phosphate(PtdIns(3)P), but the function of this interaction is unknown. Although Vps17 does notinteract directly with Vps35–Vps29–Vps26, it clearly facilitates the interaction betweenVps5 and this subcomplex89. In addition to assembling to form the retromer complex,Vps5 has been shown to have intrinsic self-assembly activity in vitro, by forming large15–20-nm particles of a uniform shape and size48. As SNX1 also has self-assemblyactivity, as well as the propensity to heterodimerize with other SNXs, thismultimerization property is hypothesized to be important in the cellular roles of theseproteins49,50. This could be especially important in driving vesicle formation, and,indeed,Vps5 is found clustered on pre-vacuolar endosomes at sites where vesicles seemto be budding from the membrane48.

PX

CC

CC

PX

CC

CC

Vps5 Vps17

Cargo selection

CargoVps10(Y/F)XXΦ

Vesicle budding

PtdIns(3)P

Lipid bilayerMembrane

Vps29

Vps26

Vps35



R E V I E W S

Also, analogous to SNX1, both SNX2 and SNX4 inter-act to varying degrees with several receptors, includingthe EGFR, PDGFR, IR and the long form of the leptinreceptor. Unlike SNX1, however, SNX2 and SNX4 donot interact with the TFR, which indicates differingbinding specificities (TABLE 1). Despite the extensiveidentity that is shared between their PX domains(70%), the SNX2 PX domain binds preferentiallyPtdIns(3)P, but not PtdIns(3,4,5)P

3(REF. 55).

embryos that lack Hβ58, the mouse homologue ofVps26, which indicates that the mammalian retromercomplex is structurally conserved with respect to itsyeast counterpart.

SNX2. SNX2 and SNX1 share 63% sequence identityand colocalize in cells. Like SNX1, SNX2 can oligomer-ize with itself and/or form heteromeric complexes withSNX1, SNX4 and proteins of the retromer complex.

AP2Early endosome

TGN

TGN

Lysosome

VPS29

VPS26

VPS35

VPS29

VPS26

VPS35

Retromercomplex

Cargo Vps5/Vps17

VacuolePtdIns(3)P

Recyclingendosome

Lipid bilayer

EGFR

PAR1

LDLRTFR

PDGFR

Mammals

SNX1/2 HRS Furin TGN38

Yeast

Vps5/Vps17 Vam7 A-ALP Vps10 DPAP Kex2

SNX1/2SNX9SNX13

Grd19

Mvp1

PtdIns(3)P

SNX1/2SNX17

SNX15

SNX1/2

SNX3

Figure 6 | The functions of the sorting nexins. This schematic depicts the known functions of mammalian and yeast sortingnexins (SNXs). In mammals, most of the functions of SNXs revolve around their interaction with plasma-membrane receptors andtheir ability to affect the turnover of these receptors. SNX1, SNX2, SNX9 and SNX13 have been shown to be involved in the sortingof the tyrosine kinase receptor epidermal growth factor receptor (EGFR). SNX1 and SNX2 have also been shown to sort the G-protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1). Similarly, endocytosis of the low-density lipoproteinreceptor (LDLR) is affected by SNX17, whereas endocytosis of the transferrin receptor (TFR) and platelet-derived growth factorreceptor (PDGFR) are affected by SNX15. SNX15 also affects the cellular location of furin and TGN38. SNX1 and SNX2 are part ofthe mammalian retromer complex, which in yeast has been shown to be important for recycling proteins from pre-vacuolarendosomes back to the Golgi. In yeast, most of the research on SNXs has revolved around the retromer complex and sorting fromthe Golgi to pre-vacuolar endosomes and then back to the Golgi. Vps5, the yeast orthologue of SNX1/2, is present in the retromercomplex with another PX-domain-containing protein, Vps17. Two other PX-domain-containing proteins, Mvp1 and Grd19, areinvolved in this process but are not known to be members of the retromer complex. Vam7 is a SNARE protein that is involved invesicle fusion with the vacuole. A-ALP, model late-Golgi membrane protein in which the cytosolic domain of DPAP is fused to thetransmembrane and lumenal domains of alkaline phosphatase; AP2, adaptor protein complex 2; DPAP, dipeptidyl amino peptidase;HRS, hepatocyte growth factor (HGF)-regulated tyrosine-kinase substrate; PDGFR, platelet-derived growth factor receptor;PtdIns(3)P, phosphatidylinositol-3-phosphate; TFR, transferrin receptor; TGN, trans-Golgi network; TGN38, trans-Golgi networkprotein 38; Vps, vacuolar protein sorting.



R E V I E W S

weakly associates with ALK4 (type I receptor) andSNX2 associates moderately with ALK4 and ALK5,but strongly with TβRII KD receptors. SNX4 associ-ates weakly with ALK5 and ALK6, and strongly withTβRII KD receptors. SNX6 was also able to interactwith receptor tyrosine kinases, namely the EGFR,PDGFR, IR and the long form of the leptin receptor(TABLE 1). At a functional level, overexpression of SNX6inhibits TGF-β and activin signalling in HepG2 cells,as measured by the activity of 3TP-LUX , a reporterfor TGF-β family receptors. SNX6, which is alsoknown as TFAF2 (TRAF4-associated factor 2), hasbeen shown to interact with PIM1, which is a proto-oncogene that has serine/threonine kinase activity64.SNX6 interacts with PIM1 using its amino-terminalsequence, which includes the PX domain. This is thesame region that was shown to interact with the TGF-βfamily of receptors that were mentioned above. In thiscase, the coiled-coil sequences do not participate sig-nificantly in these protein–protein interactions.Association of PIM1 with SNX6 results in the localiza-tion of SNX6 to the nucleus. In addition, PIM1 canphosphorylate SNX6. However, the functional signifi-cance of SNX6 phosphorylation is unclear, as a kinase-inactive PIM1 mutant can still translocate SNX6 tothe nucleus.

SNX9. SNX9, which is also known as SH3PX1, wasoriginally isolated as a two-hybrid positive for MDC9and 15 (MDC, metalloprotease, disintegrin, cysteine-rich)65. SNX9 binds preferentially to the precursor, butnot the processed forms, of these metalloproteasesthrough the interaction of its SH3 domain with Pro-rich sequences that are present in the cytoplasmic tailsof the metalloproteases. Although localized predomi-nantly in the cytoplasm of cells, SNX9 interacts withproteins that are associated with clathrin-coated pitssuch as the Cdc42-associated tyrosine kinase 2(ACK2), which has been shown to associate withclathrin and the µ-subunit of the AP2 clathrin-coatprotein complex (known as AP-50 in Drosophilamelanogaster)66–68. In Drosophila, SNX9 was isolated asa member of a protein complex that was purified fromS2 cells by using the SH2 domain of Dock, theDrosophila orthologue of the mammalian SH3/SH2adaptor protein NCK in pull-down experiments.Drosophila Ack was also isolated in this complex, andwas subsequently shown to co-immunoprecipitatewith and phosphorylate SNX9 (REF. 69). Taken together,these results indicate that SNX9 might function withthe clathrin-coat adaptor proteins in the endocyticprocess. Indeed, in mammalian cells, SNX9 has beenshown to regulate the endocytosis of EGFR in con-junction with ACK2, and ligand-induced stimulationof EGFR results in the tyrosine phosphorylation ofSNX9 by ACK2 (REF. 70). In Drosophila, Ack phosphory-lates SNX9 on a Tyr residue in the SH3 domain69.Phosphorylation of this Tyr residue diminishes theinteraction of SNX9 with Wiskott–Aldrich syndromeprotein (WASP), while enabling it to associate withDock69. Members of the WASP family are multidomain

SNX3. SNX3 and related SNXs are small hydrophilicproteins that consist of little sequence other than thePX domain. Perhaps owing to the lack of coiled-coildomains, SNX3 does not form heteromeric complexeswith other SNXs51. Lipid-overlay analysis shows thatthe SNX3 PX domain almost exclusively interacts withPtdIns(3)P (REF. 41). This supports its cellular distribu-tion in early and perinuclear endosomes, where it colocalizes with EEA1 and the TFR, respectively.Overexpression of SNX3 leads to an expansion of thetubular-vesicular structures that are characteristic ofearly, recycling and late endosomes. Concomitantwith this, SNX3 overexpression results in the delay orinhibition of EGFR transport to the lysosome,whereas the inhibition of SNX3 function by microin-jecting antibodies directed against SNX3 prevents thetransport of internalized TFR antibody from the earlyto the RECYCLING ENDOSOMES41. Taken together, theseresults indicate that SNX3 has a function in mem-brane trafficking from the early endosome to the recy-cling endosome. The inability of SNX3 to interactwith the other SNXs raises the intriguing possibilitythat SNX3 could restrict the functions of the otherSNXs by binding to PtdIns(3)P at crucial membranesites, thereby preventing the binding of the otherSNXs and their accompanying protein partners tothese sites.

SNX5. SNX5 has been reported to bind the Fanconianaemia complementation group A protein(FANCA)60. Fanconi anaemia (FA) is an autosomal-recessive genetic syndrome that is characterized byprogressive bone-marrow failure, several develop-mental abnormalities and a predisposition to malig-nancy61. FA is genetically heterogeneous — it consistsof at least eight complementation groups (from FA-Ato FA-H) — and FANCA is the gene that is mutated inFA-A62. In a two-hybrid screen, the FANCA proteinwas shown to interact with SNX5 and was subse-quently shown to co-immunoprecipitate with SNX5in cells that overexpress both proteins60. The overex-pression of SNX5 in cells also results in an increase ofFANCA, but the significance of this is unknown.SNX5 shares 66% amino-acid identity with SNX6 andboth contain an insert in their PX domains (FIG. 5),which indicates that these two SNXs might share cellular functions.

SNX6. Initially, studies of SNX6 defined the associa-tions of SNXs with the receptor serine/threoninekinases that make up the TRANSFORMING GROWTH FACTOR

(TGF)-β FAMILY OF RECEPTORS63. SNX6, which was originallyisolated as a two-hybrid protein interaction partnerfor SMAD1, interacts strongly with ALK5, moderatelywith ALK6 (ALK5 and ALK6 are type I receptors) andstrongly with TβRII kinase dead (KD) receptors (type IIreceptors) (TABLE 1). These studies localized SNX6 inthe cytoplasm where it is thought to target proteins tothe TGN63. As SNX6 hetero-oligomerizes with SNX1,SNX2 and SNX4, the associations of these SNXs withthe TGF-β family of receptors were also tested. SNX1

TGF-β RECEPTOR SUPERFAMILY

Receptors in this superfamily areactivated by numerous peptideligands including TGF-β,actinin/inhibin, bonemorphogenetic proteins, growthand differentiation factors, glial-derived neurotrophic factor andMullerian inhibitory substance.The receptors for thissuperfamily fall into twocategories: type II (Ser/Thrkinases), which bind ligand, andtype I, which, followingrecruitment and subsequentphosphorylation by the type IIreceptor, transduces the signal to the cell.

RECYCLING ENDOSOMES

Vesicles that are derived fromearly endosomes destined forfusion with the plasmamembrane.



R E V I E W S

cycles continually from the TGN to the plasma mem-brane, and returns to the TGN though sorting/recycling or late endosomes77,78. The overexpression ofSNX15 alters the morphology of several endosomalcompartments, thereby altering the steady-state local-ization of furin, which results in the delayed process-ing of several furin substrates75. Taken together, theseresults indicate that SNX15 has a role in the proteintrafficking processes that involve the endocytic pathway and the TGN.

SNX17. SNX17 was isolated on the basis of its abilityto interact with P-selectin in a two-hybrid screen79.P-selectin is a cell-adhesion molecule that is found inplatelets and endothelial cells that mediate the bindingof leukocytes80. It is stored in secretory granules and istransported to the plasma membrane after cell activa-tion. It is also internalized rapidly and then either re-cycled or degraded. SNX17 binding to P-selectin doesnot require its PX domain, but the sequences that areinvolved in this binding have yet to be elucidated. Likeother SNXs, SNX17 has a mainly cytoplasmic localiza-tion with some localization to punctate membranestructures. The function of SNX17 in P-selectin traf-ficking is unknown. Recently, SNX17 was reported tobind to the intracellular domain of some members ofthe low-density lipoprotein receptor (LDLR) familyincluding LDLR, VLDLR, APOER2 and LDLR-relatedprotein81. In this case, functional studies showed that overexpression of SNX17 enhances the rate ofendocytosis of the LDLR.

Summary and future investigationsIn summary, SNXs are hydrophilic molecules that arelocalized in the cytoplasm and have the potential formembrane association either through their lipid-binding PX domains or through protein–proteininteractions with membrane-associated protein com-plexes. Indeed, several of the SNXs require several tar-geting motifs for their appropriate cellular localization.In almost every case studied, mammalian SNXs canbe shown to have a role in protein sorting (FIG. 6), withthe most commonly used experimental model beingplasma-membrane receptor endocytosis and sortingthrough the endosomal pathway. However, it isequally probable that SNXs sort vesicles that are notderived from the plasma membrane, and have a func-tion in the accurate targeting of these vesicles andtheir cargo. This scenario has been studied extensivelyin the case of the yeast SNXs (FIG. 6). It will be of inter-est to ‘sort out’ the various functions of the SNXs incells and to determine if they have specialized or gen-eralized roles in protein trafficking. Much workremains to be done in this area as the specific lipid-and protein-binding abilities of the SNXs have yet tobe characterized. Indeed, whether lipid and proteinbinding are mutually exclusive is not known.

The cellular regulation of the SNXs has not yet beenaddressed. In the case of p47phox, serine and threoninephosphorylation is important for this protein to be ableto form an active NADPH oxidase complex82. So far,

proteins that function as scaffolds, which bringtogether components of signal-transduction pathwayswith the cellular machinery that promotes actin poly-merization and microfilament reorganization71.Therefore, SNX9 might be involved in linking changesin the actin cytoskeleton to these endocytic processesthat involve clathrin-coated vesicles.

SNX13. SNX13 was identified in a database search forcandidate proteins that contain RGS domains24 andwas named RGS-PX1. RGS domains affect the cellularsignal transduction that is triggered by seven-transmembrane G-PROTEIN-COUPLED RECEPTORS (GPCRs).GPCRs signal by the nucleotide exchange of guanineon the α-subunit of heterotrimeric G proteins, whichresults in the conversion of the inactive guanosinediphosphate (GDP)-bound α-subunit to the activatedGTP-bound form72. RGS domains facilitate the con-version of the active protein back to the inactive GDP-bound form by potentiating an intrinsic GTPaseactivity that is present in the α-subunit72. SNX13 is thefirst RGS that was shown to promote GTP hydrolysison Gαs

, thereby modulating Gαsactivity in cells. The

β2-adrenergic receptor (β2AR) signals through Gαs,

which results in the activation of adenylate cyclase,thereby increasing the cAMP levels in the cell.Treatment of HEK293 cells with the β2AR agonist iso-proterenol increases their cellular cAMP level, whereasexpression of the RGS domain of SNX13 reduces thisincrease by ~70%. The PX domain of SNX13 bindsPtdIns(3)P and PtdIns(5)P preferentially, whereas itassociates weakly with PtdIns(3,5)P

2and PtdIns(4)P

(TABLE 1). Cellular localization studies using a greenfluorescent protein (GFP) fusion construct showedthat RGS-PX1 colocalizes with EEA1 to early endosomesin COS-7 cells. Targeting of SNX13 to endosomesindicates that it might function as a SNX. Indeed,overexpression of SNX13 inhibits degradation of theEGF receptor, the opposite effect to that seen for over-expression of SNX1 (REF. 24). Perhaps more physiologi-cally relevant, expression of SNX13 in the endosomescan regulate the ability of the β2AR to signal from thiscompartment24,73. Activated β2AR cycles throughendosomes, so the presence of SNX13 in endosomesmight be important for attenuating the activity of thereceptor in this cellular compartment.

SNX15. SNX15 was isolated as a result of a databasesearch using a PX-domain consensus sequence that wasobtained from SNX1, SNX2, SNX3 and SNX4 (REF. 74).The PX domain of SNX15 interacts with the PDGFreceptor, and this interaction is independent of theactivation state of the receptor74,75. So far, the molecu-lar details of this interaction have not been defined.Overexpression of SNX15 negatively affects the inter-nalization and degradation of the PDGF and transferrinreceptors, and impairs the post-translational process-ing of the IR and hepatocyte growth factor (HGF)receptor precursors. Both the IR and HGF receptorprecursors are substrates of the endoprotease, furin76.Furin is localized predominantly in the TGN, but

G-PROTEIN-COUPLED

RECEPTORS

(GPCR). Receptors that span theseven-transmembrane domainand that, on ligand stimulation,activate small G proteins byfacilitating the exchange of GDPfor GTP.



R E V I E W S

might have important functional consequences69,70. Thecellular regulation of the SNXs and their importance inthe convergence of membrane trafficking and signaltransduction are exciting areas for future studies.

there have been no reports on the significance of serine/threonine phosphorylation in SNX function. Although,SNX9 tyrosine phosphorylation by ACK, which resultsin a switch in its protein–protein interaction partners,

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AcknowledgementsMany thanks go to R. Holz and N. Leff for their critical reading of this manuscript, and to M. Wishart for his critical reading andhelp with the phylogeny study. J. Stuckey’s help with the p40phox

structure was much appreciated.

Online links

DATABASESThe following terms in this article are linked online to:Swiss-Prot: http://www.expasy.ch/Grd19 | Mvp1 | p40phox | p47phox | SNX1 | SNX2 | SNX3 | SNX4 |SNX5 | SNX6 | SNX9 | SNX13 | SNX15 | SNX17 | Vam7 | Vps5

FURTHER INFORMATIONJack E. Dixon’s laboratory:http://dixonlab.biochem.med.umich.edu/index.htmlExpasy: http://ca.expasy.org/SMART: http://smart.embl-heidelberg.deAccess to this interactive links box is free online


CORRECTION

SORTING OUT THE CELLULAR FUNCTIONS OF SORTING NEXINSCarolyn A. Worby and Jack E. Dixon

Nature Rev. Mol. Cell Biol. 3, 919–931 (2002)

The authors would like to apologize for the following omissions:

The following reference should have been included in the reference list:92. Wang, Y., Zhou, Y., Szabo, K., Haft, C. R. & Trejo, J. Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1.

Mol. Biol. Cell 13, 1965–1976 (2002).

Reference 92 should have been cited on page 926, right column, 2nd line, at the end of the sentence: ‘Finally, downregulation ofG-protein-coupled protease-activated receptor-1 (PAR1) is regulated by SNX1 (REF. 92).’

On page 926, right column, 1st line, the abbreviation ‘PARS1’ should have read ‘PAR1’

On page 922, Table 1, 4th column, 1st row,‘PAS1*’ should have read ‘PAR1*’, and a definition of the acronym should have appeared inthe table’s footnote: PAR1, protease-activated receptor-1.

The online versions of the article have been corrected.

sorting out the cellular functions of sorting nexins

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