Recognising the signals for endosomal trafficking

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Recognising the signals for endosomal trafficking Saroja Weeratunga1, Blessy Paul1,2 and Brett M. Collins1 Abstract

The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised. Addresses 1 The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Queensland 4072, Australia 2 University of Texas Southwestern Medical Center, Department of Cell Biology, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA Corresponding author: Collins, Brett M ([email protected])

Current Opinion in Cell Biology 2020, 65:17–27 This review comes from a themed issue on Membrane Trafficking Edited by Frances M Brodsky and Jennifer L Stow For a complete overview see the Issue and the Editorial

https://doi.org/10.1016/j.ceb.2020.02.005 0955-0674/© 2020 Elsevier Ltd. All rights reserved.

Keywords Commander, Endosome, ESCRT, Retriever, Retromer, Sorting nexin, SNX.

Introduction Endosomes are dynamic and heterogeneous organelles that act as hubs for endocytic trafficking, recycling and degradation [[*1],[2]]. In a simplistic sense, transmembrane proteins that enter the endosome can undergo one of two fates; sorting to lysosomes for degradation, or retrieval to other compartments for reuse. Cargos that are typically recycled include cell surface receptors that are required for maintaining signalling and nutrient uptake, or receptors for

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lysosomal hydrolases that shuttle back and forth to the Golgi. Transmembrane proteins can be sorted into the early endosomal network via several different pathways, including endocytosis, phagocytosis, macropinocytosis, or via anterograde trafficking from the trans-Golgi network (Figure 1a). As the early endosome matures into a late endosome, intraluminal vesicles (ILVs) are formed by inward budding. This later compartment, often referred to as a multivesicular body, can then undergo fusion with the lysosome, delivering both transmembrane cargos for degradation, as well as fresh supplies of lysosomal hydrolase enzymes required for the turnover of proteins, lipids and other cellular components. In eukaryotes, the trafficking of integral membrane proteins between secretory and endocytic organelles occurs via incorporation into membrane vesicles or tubules. Tubulovesicular trafficking can occur either through bulk membrane flow or can involve the binding of soluble transport machineries to specific sorting signals in the cytoplasmic domains of the transmembrane proteins. Typically, a short peptide sequence or ‘motif ’ within the cytoplasmic domain of the transmembrane cargo is recognised by a specific adaptor protein, physically linking the cargo to the peripheral membrane coat during membrane vesicle formation. This selective sorting is perhaps best understood at the molecular level for clathrin-coated vesicle trafficking [3,4]. Motifs that are bound by clathrin adaptors such as AP1 and AP2 include tyrosine-based sequences (YxxF; where F is any hydrophobic amino acid [5]) [6] or acidic dileucine sequences ([DE]xxxL[LIM]) [7]. During the phase of early-to-late endosomal maturation, transmembrane cargos are sorted into separate membrane domains destined for either tubulovesicular recycling, or proteolytic destruction in the lysosome [[*1], [8,9]]. The separation of endosomes into retrieval and degradative domains is strikingly illustrated by the respective spatial segregation of Retromer and endosomal sorting complex required for transport (ESCRT) proteins on the large endosomes of the Caenorhabditis elegans coelomocyte [*10]. Whether particular cargos are sorted for endosomal degradation or recycling is primarily governed by a tug-of-war between the ESCRT protein machinery, which selects cargo for degradation Current Opinion in Cell Biology 2020, 65:17–27

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Figure 1

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Endosomal trafficking pathways for cargo retrieval and degradation. (a) Schematic diagram of the eukaryotic secretory and endocytic system. Endosomal sorting pathways are highlighted in blue. (b) Once transmembrane cargos enter the endosome, they encounter one of the two fates: sorting to ILVs by the ESCRT complex for degradation, or retrieval and recycling by a variety of different mechanisms. Sequence-dependent cargo recycling is often mediated by machineries that use different SNX family proteins as adaptors for cargo recognition. ILVs, intraluminal vesicles; ESCRT, endosomal sorting complex required for transport Current Opinion in Cell Biology 2020, 65:17–27

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Signals for endosomal trafficking Weeratunga et al.

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Figure 2

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d

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Structural basis for cargo recognition by SNX adaptors for Retromer and Commander. (a) The Retromer and Commander endosomal retrieval complexes interact with specific SNX adaptors to mediate interactions with cognate peptide motifs in the cytoplasmic domains of cargos (green). Proteins in the complexes are outlined, with the specific SNX protein/domain required for cargo recognition indicated (blue). The sequence motifs are shown below. (b) Interaction of cargo with Retromer-SNX3 (PDB ID 5F0L; [**13]) . The cargo motif of DMT1-II (green) associates with a surface groove formed between the SNX PX domain (blue) and the VPS26 subunit of Retromer (cyan). The N-terminus of SNX3 interacts with the interface between VPS26 and VPS35 (wheat). (c) Interaction of cargo with Retromer-SNX27 (PDB IDs 5EMB and 4P2A; [**30,31]). As an example, the phosphorylated cargo motif of 5HT4(a)R (green) associates with the binding pocket of the SNX27 PDZ domain (blue). The phosphoserine group at position −5 binds to the conserved Arg58 of SNX27. The VPS26 subunit of Retromer (cyan) binds a unique b-hairpin structure of SNX27 and allosterically enhances the cargo binding affinity. (d) Interaction of cargo with Commander-SNX17 (PDB ID 4GXB; [28]). The cargo motif of P-selectin (green) binds in an extended conformation to the F3 subdomain of the SNX17 FERM domain (blue). SNX, sorting nexin; PX, phox homology

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and regulates the formation of ILVs, and a variety of opposing retrieval complexes (Figure 1b). Many of the known retrieval complexes include subunits from the sorting nexin (SNX) protein family [11], which can act as adaptors for hundreds of different cargos by association with specific sorting motifs in their cytoplasmic tails (Figure 2a; Table 1).

Retromer and SNX3 One of the most highly studied endosomal retrieval complexes is Retromer. Composed of three core subunits d VPS29, VPS26A/B and VPS35 d it can form dimeric arches [[12], [**13], [14]] that scaffold various regulatory factors such as Rabs, Rab GTP exchange factors and GTPase activating proteins, actinremodelling complexes and members of the SNX protein family [2,15e17]. A major part of Retromer’s function appears to be to couple the formation of dynamic actin-rich retrieval domains with transmembrane cargo selection, either directly, or more commonly through different SNX adaptor proteins. SNX3 is a highly conserved member of the SNX protein family, that interacts with the Retromer complex to recycle certain cargos from the endosome to the Golgi [[*18],[19e22]]. SNX3 belongs to the subfamily composed of only a core phox homology (PX) domain, which interacts with the endosomal lipid phosphatidylinositol-3-phosphate (PtdIns3P) [11,23]. Prominent cargos include the cation-independent mannose-6phosphate receptor (CI-MPR), and divalent metal transporter 1-II (DMT1-II). Sequences required for endosomal sorting were identified in the CI-MPR [24] and DMT1-II [25] cytoplasmic domains, 2370WLM2372

and 555YLL567 respectively, suggesting they contained a hydrophobic motif that could interact with Retromer. The subsequent crystal structure of Retromer and SNX3 in complex with the DMT1-II sequence confirmed this; however, it also showed that cargo recognition was mediated by both SNX3 and Retromer together rather than Retromer alone [**13] (Figure 2b). As indicated by previous cellular studies, cargo recognition by Retromer-SNX3 is based on a specific hydrophobic motif, UF[LMV] (U = aromatic amino acids; F = hydrophobic amino acids). While tethered to the endosomal membrane via its PX domain, the long, flexible N-terminal domain of SNX3 interacts with the interface between the VPS26A and VPS35 subunits of the Retromer complex. As a result of this interaction, the C-terminal b-sheet domain of VPS26A undergoes a small conformational change, exposing a hydrophobic ligand binding site for the recycling cargos. The overall cargo binding surface is formed by amino acid residues contributed by both VPS26A and SNX3, and involves a number of main-chain hydrogen bonds between the DMT1-II peptide and VPS26A. Because the DMT1-II Leu557 side chain makes the most extensive buried contact, this is referred to as position 0, with other side chains in the sequence numbered relative to this. The aliphatic side chains at positions 1 and 3 clamp Phe287 in VPS26A, whereas the Tyr555 side chain at position 2 inserts into a hydrophobic pocket at the interface between SNX3 and VPS26A and hydrogen bonds to SNX3 side chain His132. In addition to DMT1-II and CI-MPR, cargos including Sortilin, Wntless and pIgR are also thought to possess similar sorting signals, and hence are postulated to be recycled through the same mechanism [[**13], [25]].

Table 1 Endosomal retrieval complexes and their sorting motifs. Retrieval complex

Cargo adaptor(s)

Retromer-SNX3

SNX3

Retromer-SNX27

SNX27

Commander-SNX17

SNX17

ESCPE-1

SNX5 SNX6

Other retrieval complex subunits Vps35 Vps26A/B Vps29 Vps35 Vps26A/B Vps29 Commd1-10 CCDC93 CCDC22 Vps35L Vps26C Vps29 SNX1 SNX2

Cargo motifsa

Key references

UF[LMV]0

[[**13], [25]]

[-][-]x[-][ST]xf0

[[*26], [**30]]

FxNxx[YF]0

[[28,43], [**45]]

FxU0xF[xn]F

[**50]

U = aromatic amino acids; F = hydrophobic amino acids; - = negatively charged Asp or Glu, or phosphorylated Ser or Thr [5]. The superscript ‘0’ indicates the residue designated as position zero for numbering of each motif sequence. SNX, sorting nexin; ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit-1

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Signals for endosomal trafficking Weeratunga et al.

Retromer and SNX27 Similar to RetromereSNX3, the RetromereSNX27 complex is also considered to be one of the major endosomal retrieval complexes, preventing lysosomal degradation of numerous cargos by recycling them to the plasma membrane [*26]. SNX27 is a distinctly different family member to SNX3, composed of an N-terminal PDZ (PSD95/Dlg/ZO1) domain, a central PX domain, and a C-terminal FERM (4.1/ezrin/radixin/moesin) domain [27] (Figure 2a). Similar to SNX3, the PX domain of SNX27 interacts with the endosomal lipid PtdIns3P for membrane recruitment. Although there is some evidence that the FERM domain of SNX27 can bind to transmembrane cargos [28], its primary role appears to be to scaffold other proteins including Rasrelated GTPases, actin remodelling complexes and other members of the SNX family [[*26], [27,29]]. The PDZ domain however, is well characterised as a major site of cargo binding and also interacts simultaneously with the Retromer subunit VPS26 to allosterically couple cargo capture to membrane sorting [[*26],[**30], [31]] (Figure 2c). Through the PDZ domain, Retromer-SNX27 interacts with transmembrane cargos possessing specific PDZ-binding motifs, which are always located at the C-terminus as binding invariably requires the free C-terminal carboxylate group [32]. PDZ domains are common in many scaffolding proteins, but the PDZ domain of SNX27 is unique in possessing an inserted b-hairpin structure, which is required for binding to the VPS26 subunit of Retromer. Many cargos are recycled by Retromer-SNX27 [[*26], [**30]]; and specific examples include the b2adrenergic receptor (b2AR) [33], 5-hydroxytryptamine receptor 4a (5-HT4(a)R) [[**30], [34]], parathyroid hormone receptor [35], glucose transporter 1 (GLUT1), and synaptic N-methyl-D-aspartate (NMDA) receptors [36]. The SNX27 PDZ domain binds type I PDZbinding motifs, with the core consensus sequence [ST] xF (where F is a hydrophobic residue at the C-terminus). Designated as position 0, the side chain of the F0 residue docks into a hydrophobic pocket, whereas its free carboxyl group hydrogen bonds with the backbone of a conserved loop in the PDZ domain (Figure 2c). In addition to the core sequence, SNX27 binding is enhanced by two distinct mechanisms. Constitutively high-affinity binding requires the presence of negatively charged side chains (Asp/Glu) at the 3 and 5 positions. These form hydrogen bonds and electrostatic contacts with Asn56 and Arg58 in the SNX27 binding pocket. High-affinity binding can also be promoted however, by the regulated phosphorylation of Ser/Thr residues in these positions, providing the necessary negative charges to coordinate with Arg58 [**30]. The binding affinity of cargo motifs for SNX27 is also allosterically enhanced by simultaneous binding to www.sciencedirect.com

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Retromer subunit VPS26 [[**30],[31]]. Thus, although the mechanisms are different, both SNX3 and SNX27 binding to cargos is directly coupled to their association with the core Retromer complex. Finally, an intriguing new study showed that the soluble enzyme OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) has an exceptionally high affinity for the SNX27 PDZ domain [37]. OTULIN specifically removes Met1linked ubiquitin chains conjugated by linear ubiquitin chain assembly complex and was identified as a SNX27 interactor by proteomics. The interaction does not affect OTULIN activity but does block the association of SNX27 with transmembrane cargos and Retromer. Structural studies showed that OTULIN binds simultaneously to both the PDZbm-pocket, and to the bhairpin that associates with VPS26, suggesting a negative regulatory role in Retromer-SNX27emediated trafficking in the cell.

Commander and SNX17 The SNX17 protein (and the bladder-specific homologue SNX31) shares similarities with SNX27, in that it also has a PtdIns3P-binding PX domain, coupled to a Cterminal FERM domain [27] (Figure 2a). However, SNX17 and SNX31 lack a PDZ domain for cargo interaction. Instead they have long been known to interact via the FERM domain with cargos that have FxNxx[YF] motifs, such as the amyloid precursor protein [38], members of the lipoprotein receptor family including LDLR and LRP1 [39e41], and integrins [42e44] for endosomal recycling to the plasma membrane. It was recently shown that SNX17 is associated with the Commander complex, an assembly composed of at least fifteen proteins including two subcomplexes named Retriever and the CCC (CCDC92/CCDC93/COMMD) complex. Retriever consists of three proteins VPS35L, VPS26C and VPS29 that are structurally similar to Retromer [[15],[**45]] (Figure 2a). Knockdown or knockout of either Commander or SNX17 have similar effects on integrin recycling [**45], and it is proposed that together these proteins represent a novel machinery for endosomal recycling, with some similarities to the distantly related Retromer assembly. The specific mechanism by which SNX17 recognises FxNxx[YF] motifs was demonstrated by the cocrystal structure of the SNX17 FERM domain bound to the sequence 758FTNAAY763 from the cytoplasmic domain of the P-selectin cargo protein [28,46] (Figure 2d). FERM domains possess three sub-domains F1eF3, and the peptide motif binds to a highly conserved pocket in the F3 region. The two most important side chains providing specificity are the aromatic [YF] residue at position 0, and the Asn side chain at position 3. The two side-chains dock into complementary pockets, with the [YF]0 residue stacking with SNX17 Arg319, and the N 3 residue forming a network of hydrogen bonds with Current Opinion in Cell Biology 2020, 65:17–27

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both the SNX17 and peptide backbones. A small sidechain at position 1 allows for the peptide to make a tight-turn required for binding, and this is typically facilitated by a Pro residue at position 2, although this is not strictly required. Finally, a hydrophobic side chain at the 5 position further contributes to the overall interface, together with a series of main-chain hydrogen bonding interactions between peptide and protein.

Figure 3

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The endosomal SNX-BAR sorting complex for promoting exit-1 complex In yeast, the Retromer complex associates with Vps5 and Vps17, two SNX family proteins possessing bin/ amphiphysin/rvs (BAR) domains, for retrieval of vacuolar cargos [12,47e49]. In higher eukaryotes however, the respective SNX-BAR homologues SNX1/SNX2 and SNX5/SNX6, are only loosely associated with Retromer if at all [15]. Recent proteomics studies however show that these proteins are critical for the endosomal retrieval of a large range of different transmembrane proteins in a Retromer-independent manner [**50]. Simonetti et al. named these complexes of SNX5, SNX6, SNX1 and SNX2, the ESCPE-1 (Figure 3a).

b

Mechanistically, ESCPE-1 operates through the sequence-dependant recognition of the endosomal transmembrane cargos by the PX domains of either of the closely related homologues SNX5 or SNX6. All ESCPE-1 subunits possess a PX domain followed by a BAR domain, and form heterodimers of SNX5 or SNX6 with SNX1 or SNX2 through the C-terminal BAR domain structure. The BAR domain is a banana-shaped dimer with positively charged surface residues that associate with negatively charged membrane phospholipids to promote membrane curvature and tubulation [51]. Although the PX domains of SNX1 and SNX2 have a typical structure and preferentially recognise late endosomal PtdIns(3,5)P2 lipids [52], the PX domains of SNX5 and SNX6 have a unique extended helical structure and no membrane-binding capacity. Instead this helical extension provides a docking site for cargosorting peptide motifs (Figure 3b).

Current Opinion in Cell Biology

Structural basis for cargo recognition by the ESCPE-1 complex. (a) The ESCPE-1 complex is composed of heterodimers of SNX5 or SNX6 combined with either SNX1 or SNX2 via their BAR domains, and interacts with cognate peptide motifs in the cytoplasmic domains of cargos (green) via the PX domain of SNX5 or SNX6 (blue). (b) Interaction of cargo peptides (green) with the PX domain of SNX5 (blue). The CI-MPR can bind SNX5 with two very similar b-hairpin structures, differing only in the hydrophobic residue in the second b-strand that inserts into a hydrophobic pocket on SNX5 (Form 1, Leu2370, PDB ID 6N5X; Form 2, Met2371, PDB ID 6N5Y; [**50]). The cargo motif of SEMA4C binds in a similar b-hairpin structure, but has a much shorter loop between b-strands 1 and 2. SNX, sorting nexin; ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit-1. Current Opinion in Cell Biology 2020, 65:17–27

Cargo interactions with SNX5 and SNX6 were first hinted at through studies of their interactions with a secreted bacterial effector protein, IncE from Chlamydia trachomatis [53,54]. A C-terminal peptide of IncE forms a b-hairpin structure bound to SNX5 and SNX6 (and the neuronal specific homologue SNX32) and interacts with a very highly conserved surface groove. Although the sequences are unrelated to IncE, it was subsequently shown that the natural cargo proteins CI-MPR and Semaphorin 4C (SEMA4C) bind SNX5 using the same surface and a similar b-hairpin structure (Figure 3b). Structures, mutagenesis and cellular validation have defined a core motif in endosomal cargos that forms the first b-strand FxUxF. The central aromatic side chain, www.sciencedirect.com

Signals for endosomal trafficking Weeratunga et al.

defined here as U0, inserts into a hydrophobic pocket to stack with the SNX5 Tyr132 side chain, a hydrophobic residue at F2 (often aromatic) packs against SNX5 Phe136, and a hydrophobic residue at the F 2 position also associates with a complementary hydrophobic pocket. The interaction is further stabilised by a second b-strand formed by the cargo cytoplasmic sequence, so that overall the peptide generates an extended b-sheet with SNX5 that packs against the extended helical structure. The second b-strand shows little sequence specificity, and is connected to the first b-strand via a highly variable loop region; the only key characteristic is a hydrophobic side chain that packs against Tyr132, Leu133 and Phe136 side chains of SNX5. Indeed, two separate structures of the same sequence of the CIMPR cargo showed that different hydrophobic residues could be used in this position (Figure 3b) [**50]. The current working model for the recycling of transmembrane cargos is that the SNX1/SNX2 subunits of ESCPE-1 interact with PtdIns(3,5)P2 for the endosomal recruitment, whereas SNX5/SNX6 interacts with the cargos in a sequence-dependant manner. The SNX5/ SNX6 and SNX1/SNX2 BAR domains induce membrane curvature and membrane remodelling to generate membrane tubules to coordinate the packing of cargoes to produce cargo-enriched tubulovesicular transport carriers (Figure 3a).

ESCRT-mediated degradation of ubiquitinated cargos As described previously, retrieval of cargos often involves interactions that are mutually exclusive with one another. For example, the motifs in the CI-MPR cargo recognised by either SNX3-Retromer or ESCPE-1 are overlapping, and cargo interaction with RetromerSNX27 uses the same binding surface as OTULIN. This implies that the interactions must be dynamic to allow exchange between different complexes and will also be context dependent, that is, they will be allosterically coupled to other signals such as membrane lipids or regulatory proteins. A prime example of how dynamic exchange between different complexes can promote sequential cargo capture, sequestration and packaging is the sorting of ubiquitinated proteins by ESCRT. ESCRT is composed of four subcomplexes, ESCRT-0 to ESCRT-III, that mediate cargo sorting and formation of ILVs in a series of interactions at the endosomal limiting membrane (Figure 4a) [55e58]. The primary mechanism of cargo sorting into the ESCRT-mediated degradation pathway is via the post-translational addition of Lys63-linked chains of polyubiquitin. At each stage of ESCRT assembly, ubiquitin-tagged cargos are sequestered by different ESCRT-subunits using distinct mechanisms [57]. www.sciencedirect.com

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Structures have now been solved of ubiquitin bound to almost all of the interacting domains of ESCRT (Figure 4a and b). Both subunits of the ESCRT0 heterodimer of HRS/STAM can bind to ubiquitin; in each instance using both an N-terminal VHS domain and central ubiquitin-interacting motif or di-ubiquitine interacting motif. The VHS domain is an a-helical solenoid structure [59,60], whereas the ubiquitin-interacting motif and di-ubiquitineinteracting motif are short sequences that form a single a-helix [61]. In ESCRT-I ubiquitin is bound by domains from both VPS23 and UBAP1/ multivesicular body 12 subunits. VPS23 uses a small globular ubiquitin-conjugating enzyme E2 variant domain [62], whereas UBAP1 possesses a solenoid of overlapping UBAs domain [63]. The solenoid of overlapping UBAs domain is composed of three overlapping a-helical UBA structures and is predicted to bind up to three ubiquitin molecules in tandem (Figure 4b). Finally, ESCRT-II subunit VPS36 interacts with ubiquitin through its GRAM-like ubiquitin binding in EAP45 (GLUE) domain [64]. The sequential interactions of ubiquitinated cargos with ESCRT-0, eI and II is able to sequester them into endosomal domains where they are de-ubiquitinated and sorted into ILVs, formed by the filamentous assembly of the ESCRT-III complex [58]. Although each ESCRT component binds to ubiquitin using a different protein structure, they all associate with the same surface of ubiquitin, encompassing the key Ile44 side chain. In other words, no single ubiquitin monomer can bind more than one ESCRTsubunit at any given moment. The sequestration of ubiquitinated cargo into ILVs therefore depends on a number of factors [55,65,66]. First, cargos are often modified by polyubiquitin chains or multiple sites of monoubiquitination. Secondly, ubiquitin-binding affinities are typically modest (in the micromolar Kd range), allowing for on-and-off binding and exchange with neighbouring ESCRT molecules in the two-dimensional membrane environment. Finally, reversible ubiquitination by ubiquitin ligases and deubiquitinases provides additional spatial and temporal cues for efficient cargo sorting.

Concluding perspectives For simplicity, we have purposely focused on the dichotomy between lysosomal destruction and the broader process of endosomal retrieval. However, there are many potential destinations to which different cargos can be retrieved, and a major outstanding question is how this downstream targeting is specified. There are likely a number of contributing factors. Targeting of cargo carriers and positioning of the endosomes themselves will depend on the particular cytoskeletal motors and adaptors that are recruited [67,68], and will require the involvement of specific SNAREs (which will need to be Current Opinion in Cell Biology 2020, 65:17–27

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Figure 4

a

b

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Structural basis for ubiquitin-modified cargo recognition by ESCRT. (a) Ubiquitinated receptors destined for degradation are recognised by sequential ESCRT complexes for sorting into ILVs. ESCRT-0, –I, –II, –III complexes are indicated schematically, with domains known to bind ubiquitin indicated in blue. Grey dashed lines indicate known interactions between the different ESCRT subunits. (b) Known structures of the ESCRT domains (blue) in complex with ubiquitin (orange). For spatial reference, the ubiquitin K63 side-chain involved in covalent ubiquitin chain formation, and the conserved I44 side-chain important for ligand recognition are indicated in spheres. The structures with PDB IDs are as follows: (1) ubiquitin-STAM1 VHS domain (3LDZ) [59], (2) ubiquitin-STAM2 VHS domain (2LOT) [60], (3) ubiquitin-HRS DIUM (2D3G) [61], (4) ubiquitin-VPS23 UEV domain (1UZX) [62], (5) model of the ubiquitin-UBAP1 SOUBA domain (4AE4 combined with 2MJ5) [63], and (6) ubiquitin-VPS36 GLUE domain (2HTH) [64]. ILVs, intraluminal vesicles; PX, phox homology; UEV, ubiquitin-conjugating enzyme E2 variant; SOUBAs, solenoid of overlapping UBAs.

co-packaged with appropriate cargos), and membrane tethers for docking of the transport tubules and vesicles [69,70]. For example, different membrane tethers of the Golgin family can mediate the Golgi recruitment of carriers for the CI-MPR cargo generated by either Retromer-SNX3 or the ESCPE-1 complex [*18]. The Current Opinion in Cell Biology 2020, 65:17–27

docking of endosome-derived carriers with the plasma membrane is controlled by a different tethering complex called Exocyst. The turnover of endosomal phosphoinositide lipids is essential for Exocyst-mediated fusion, as disease-causing mutations or depletion of myotubularin-1 (MTM1) leads to accumulation of 3www.sciencedirect.com

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phosphoinositide lipids on endosomes decorated by several members of the SNX family, including SNX1, SNX3, SNX17 and SNX27 [71]. One of the most remarkable studies regarding the destination of endosome-derived carriers was the analysis of the Wntless and b2AR cargos by Varandas and colleagues [*72]. Wntless has a Retromer-SNX3 binding UF [LMV] motif required for trans-Golgi network delivery, whereas b2AR interacts with Retromer-SNX27 via its Cterminal PDZbm for recycling to the plasma membrane. It was found that both proteins could enter the same Retromer-positive endosomal carrier domains, although the kinetics of b2AR was slower. Despite this, the steady state localisation of b2AR and Wntless are at the plasma membrane and Golgi, respectively. Intriguingly, if the sorting motifs are switched between the two cargos, their kinetics of entering the Retromer-positive domains are reversed but their final destinations remain the same as the wild-type proteins. This implies that while they can enter the same Retromer carriers (albeit with different enrichment) their final destinations are mediated by as yet unknown downstream pathways. Although we now understand how many cargos are selected within the endosomal system, there are almost certainly other machineries and sorting motifs that contribute to the retrieval process that remain to be identified. Also, it will be important to understand how cargo interactions are regulated by other contextdependent signals. Most peptide motifs bind with only moderate affinity, so that cargo interactions with the correct peripheral membrane trafficking complexes invariably requires coincidence detection [73e75]. Probing cargo association in the presence of factors including lipids, small GTPases and conformational alterations will provide a deeper understanding of how avidity and cooperativity contribute to cargo capture into specific endosomal membrane domains.

Conflict of interest statement Nothing declared.

Acknowledgements BMC is supported by a National Health and Medical Research Council Senior Research Fellowship (APP1136021). Work in the laboratory is supported by funding from the NHMRC (APP1156493) and the BrightFocus Foundation (A2018627S).

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