CROP2, a Retriever–PROPPIN complex mediating protein export from endosomes to the plasma membrane in human cells

Endosomal membrane traffic regulates the abundance and activity of many receptors and transporters at the plasma membrane, which renders it critical for the development and homeostasis of cells and tissues (McNally and Cullen, 2018). In line with this, perturbations in endosomal trafficking contribute to a wide range of diseases, including cancer and some of the most common neurological disorders (Carosi et al., 2023; Mellman and Yarden, 2013; Schreij et al., 2016). Many plasma membrane proteins cycle between the cell envelope, endosomes, and the Golgi. They can move on to lysosomes for degradation unless they are retrieved before endosomes fuse with lysosomes (Cullen and Steinberg, 2018). Endosomes thus serve as sorting stations at an intersection between several compartments. To collect cargo proteins, endosomes employ a variety of coats that form tubulo-vesicular carriers. Many of them are based on sorting nexins, which scaffold the membrane into narrow tubules. Sorting nexins can act on their own, such as in the ESCPE-1 complex (Kvainickas et al., 2017; Lopez-Robles et al., 2023; Simonetti et al., 2019; Simonetti et al., 2017), or together with complexes interconnecting them, such as Retromer or Retriever and the Commander complex, into which Retriever can be integrated (Bartuzi et al., 2016; Boesch et al., 2023; Chandra et al., 2024; Healy et al., 2023; Mallam and Marcotte, 2017; McNally et al., 2017; Phillips-Krawczak et al., 2015; Seaman, 2021). Further coats, such as clathrin adapter complexes, VINE, and ACAP1-clathrin can also act on endosomes (Dai et al., 2004; Hooy et al., 2022; Li et al., 2007; Pang et al., 2014; Ren et al., 2013; Sachse et al., 2002; Shortill et al., 2022). These coat components organize a variety of endosomal protein exit routes with partially overlapping cargo specificity (Bean et al., 2017; Kvainickas et al., 2017; McGough et al., 2014; Steinberg et al., 2013), which may reflect their capacity to form hybrid carriers integrating different classes of sorting nexins (Gopaldass et al., 2026).

Retromer is part of a protein coat mediating protein exit from endosomes in association with sorting nexins, such as Vps5, Vps17, or Snx3/Grd19 in yeast, and SNX27, ESCPE-1, or SNX3 in human cells (Seaman, 2021). Retromer consists of a VPS26–VPS29–VPS35 heterotrimer, which can form higher order complexes through VPS35 and VPS26 dimerization (Collins et al., 2008; Deatherage et al., 2020; Hierro et al., 2007; Kendall et al., 2022; Kendall et al., 2020; Kovtun et al., 2018; Leneva et al., 2021; Lucas et al., 2016; Purushothaman et al., 2017). While sorting nexins themselves change the spontaneous curvature of membranes and can tubulate them (Kovtun et al., 2018; Leneva et al., 2021; van Weering et al., 2012; Zhang et al., 2021), this activity can be enhanced by the association with Retromer, which may provide additional driving force for scaffolding the membrane (Gopaldass et al., 2023).

In human cells, numerous cargos recycle back to the cell surface in a Retromer-independent manner (Steinberg et al., 2013). Many of these depend on Retriever, an evolutionarily conserved complex of VPS26C/DSCR3, VPS29, and VPS35L/C16orf62, proteins that show distant homology to the Retromer subunits (McNally et al., 2017). Whereas the overall organization and shape of Retriever resemble those of Retromer, Retriever shows different cargo specificity and distinct features. Retriever forms part of a much larger complex, Commander, which integrates Retriever with the 12-subunit CCC complex and DENND10 and cooperates with the sorting nexin SNX17 to form coats (Boesch et al., 2023; Butkovič et al., 2024; Healy et al., 2023; Martín-González et al., 2025; Singla et al., 2024; Steinberg et al., 2012). Like Retromer, also Commander can interact with WASH and thereby link to actin (Derivery et al., 2009; Gomez and Billadeau, 2009; Guo et al., 2024; Harbour et al., 2012; Jia et al., 2012; Phillips-Krawczak et al., 2015; Seaman et al., 2009).

Cargo carriers detach from endosomes by membrane fission (Gopaldass et al., 2024; Naslavsky and Caplan, 2023). Several factors have been implicated in this detachment, among them actin and the actin-controlling WASH complex (Derivery et al., 2009; Frisby et al., 2024; Gomez and Billadeau, 2009; Harbour et al., 2012; Jia et al., 2012; Seaman et al., 2009), dynamin-like GTPases and ATPases such as Vps1 and EHD1 (Arlt et al., 2015; Chi et al., 2014; Daumke et al., 2007; Deo et al., 2018; Gokool et al., 2007; Grant et al., 2001; Solinger et al., 2020), the ESCRT-III subunit IST1 (Clippinger et al., 2024) and the PROPPIN (β-propeller that binds phosphoinositides) WIPI1 and its yeast homolog Atg18 (De Leo et al., 2021; Gopaldass et al., 2024; Gopaldass et al., 2017). Coats and actin can exert forces on the underlying lipid bilayer, which not only allow the tubule to grow but may also produce friction between lipids and protein coats that contributes to fission (Johannes et al., 2014; Simunovic et al., 2018; Simunovic et al., 2017). In addition, dynamin-like proteins actively deform the bilayer to compress and destabilize it (Antonny et al., 2016). The lipid phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P₂) and/or phosphatidylinositol 5-phosphate (PI5P) is also required for fission (De Leo et al., 2021; Giridharan et al., 2022; Gopaldass et al., 2017; Rivero-Ríos and Weisman, 2022; Zieger and Mayer, 2012). PROPPINs are bona-fide effectors of PI(3,5)P₂ (Courtellemont et al., 2022; De Leo et al., 2021). They associate with membranes through two phosphoinositide-binding sites (Baskaran et al., 2012; Krick et al., 2012; Liang et al., 2019; Proikas-Cezanne et al., 2004; Vicinanza et al., 2015; Watanabe et al., 2012). Upon membrane binding, a hydrophobic loop, which is located between the two lipid binding sites, folds into an amphipathic α-helix that inserts into the bilayer. Thereby, PROPPINs can tubulate lipid bilayers and drive their fission (Courtellemont et al., 2022; De Leo et al., 2021; Gopaldass et al., 2017; Gopaldass and Mayer, 2024).

PROPPINs are a protein family that binds phosphatidylinositol 3-phosphate (PI3P), PI5P, and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) (Baskaran et al., 2012; Busse et al., 2015; Dove et al., 2004; Gopaldass and Mayer, 2024; Jeffries et al., 2004; Vicinanza et al., 2015). They are conserved from yeast (Atg18, Atg21, and Hsv2) to humans (WIPI1, WIPI2, WIPI3/WDR45B, and WIPI4/WDR45) and exist in most eukaryotes in several isoforms, which can be grouped into two evolutionary clades (Dove et al., 2004; Proikas-Cezanne et al., 2015). Atg18, Atg21, WIPI1, and WIPI2 belong to one clade, Hsv2, WIPI3, and WIPI4 to the second (Cong et al., 2021; Polson et al., 2010; Strong et al., 2021). WIPI1 and WIPI2 concentrate on phagophores when autophagy is induced and they are necessary for efficient formation of autophagosomes (Dooley et al., 2014; Polson et al., 2010; Proikas-Cezanne et al., 2004). WIPI2, which has a stronger impact on autophagy, interacts with key factors for autophagosome formation.

WIPI1 and Atg18 also support membrane fission and protein sorting on endosomes and lysosomes, but for these functions they rely on molecular features that are distinct from those important to autophagy (Courtellemont et al., 2022; De Leo et al., 2021; Gopaldass et al., 2017; Gopaldass and Mayer, 2024). WIPI1 localizes to endo-lysosomal compartments, where it affects the size of endosomes and the exit of proteins from endosomes to lysosomes (EGF receptor), to the Golgi (Shiga toxin), or to the cell surface (Transferrin receptor, GLUT1) (De Leo et al., 2021; Jeffries et al., 2004). Based on the differential effects of substitutions in the two phosphoinositide-binding sites of WIPI1 it was suggested that binding of PI3P allows WIPI1 to support the formation of endosomal transport carriers while PI(3,5)P₂ and lipid binding site two are needed for membrane fission at endosomes (De Leo et al., 2021). WIPI1 as well as its yeast homolog Atg18 associate with Retromer to form the CROP complex, which amplifies the fission activity of the PROPPIN and makes its binding to membrane PI(3,5)P₂ dependent (Courtellemont et al., 2022; Marquardt et al., 2023). This interaction depends on a conserved FSSS motif, which can be phosphorylated (Courtellemont et al., 2022; De Leo et al., 2021; Feng et al., 2015; Gubas et al., 2024; Strong et al., 2021). This phosphorylatable FSSS motif exists also in WIPI2, where it forms also part of the binding site for ATG16L1, a core component of the autophagic machinery (Gubas et al., 2024; Strong et al., 2021).

We have recently identified the Retromer- and WIPI1-containing CROP complex as an agent supporting the export of multiple cargos from endosomes, comprising both Retromer-dependent and Retromer-independent exit routes (Courtellemont et al., 2022; De Leo et al., 2021). Since not all cargo exit from endosomes depends on WIPI1 and CROP we analysed alternative PROPPINs that might be involved in WIPI1-independent exit from endosomes. This led us to uncover a novel role for a WIPI2–Retriever complex in trafficking of Integrin β1, which we present below.

WIPI1 promotes the exit of diverse cargos from endosomes, for example for transporting Transferrin to the plasma membrane, GLUT1 to the plasma membrane, EGF receptor (EGFR) to lysosomes, and Shiga toxin to the Golgi (De Leo et al., 2021). Integrin β1 provides an exception, its delivery to the cell surface being unaffected by knockout of WIPI1 (Courtellemont et al., 2022). We investigated whether the endosomal exit of Integrin β1 might depend on another PROPPIN. Since WIPI1 and WIPI2 belong to the same evolutionary clade (Bakula et al., 2013; Gopaldass and Mayer, 2024), share two predicted lipid binding sites as well as the potential to form an amphipathic helix on their CD loop 6 (Cong et al., 2021; Gopaldass et al., 2017; Polson et al., 2010), we focused our attention on WIPI2.

WIPI1 and WIPI2 are specific for distinct trafficking pathways

We tested the impact of WIPI2B knockdown (WIPI2^KD) on the afore-mentioned trafficking pathways (Figure 1). siRNA against WIPI2 reduced the protein in whole cell extracts by 94% when compared to cells transfected with control siRNA (Control) (Figure 1—figure supplement 1). We analysed trafficking of EGFR from early endosomes to degradative, LAMP1-containing compartments. After 24 hr of serum starvation, cells were stimulated with EGF and analysed for EGFR localization by immunofluorescence. Fifteen minutes after EGF addition, EGFR-positive structures colocalized with the early endosomal marker EEA1 to more than 75% in both WIPI2^KD and control cells, indicating that EGF had induced efficient internalization of EGFR in both cases (Figure 1A, B). After 30 and 60 min of EGF stimulation, EGFR increasingly shifted from EEA1- towards LAMP1-positive lysosomal structures in control as well as in WIPI2-silenced cells. EGFR signals declined after 60 min in the immunofluorescence images as well as in western blot analyses of the both cells (Figure 1C–E). Thus, WIPI2 knockdown does not impair EGFR trafficking from EEA1 endosomes to LAMP1-positive compartments and leaves lysosomal degradation of EGFR functional.

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To test the role of WIPI2 in Retromer-dependent trafficking we assayed the localization of the glucose importer GLUT1 because this protein accumulates on endosomes if its recycling to the plasma membrane through Retromer is impaired (Steinberg et al., 2013). To capture the surface fluorescence, we acquired z-stacks of non-permeabilized, immuno-stained cells, generated maximum intensity projections and integrated the fluorescence signal over the entire cell, normalized for its area as described in Materials and methods section. Non-permeabilized WIPI2^KD cells showed similar cell surface staining of GLUT1 as control cells (Figure 2A, B), suggesting that also Retromer-mediated recycling of GLUT1 occurs independently of WIPI2.

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Next, we tested the requirement of WIPI2 for trafficking of Integrin β1. Surface accumulation of this Integrin subunit is unperturbed by loss of SNX27 or Retromer but depends on the SNX17–Retriever pathway (McNally et al., 2017; Steinberg et al., 2013). Knockdown of WIPI2 induced a loss of Integrin β1 immunofluorescence from the surface of HK2 cells (Figure 3A, B) and phenocopied the previously published knockdown of SNX17 (McNally et al., 2017; Steinberg et al., 2013). When the cells were detergent-permeabilized to partially remove the plasma membrane and improve accessibility of internal structures for immuno-staining, cells depleted of WIPI2 showed an accumulation of Integrin β1 on intracellular structures that colocalized with the early endosomal marker EEA1 and, to a lesser degree, with the late endosomal/lysosomal marker LAMP1 (Figure 3C–E, G). WIPI2 knockdown had no impact on the total EEA1 and LAMP1 fluorescence level per cell, suggesting that the abundance of these compartments remained the same (Figure 3F, H). In accord with previous observations (De Leo et al., 2021), Integrin β1 surface labelling in non-permeabilized cells was not affected by knockdown of WIPI1 (Figure 3—figure supplement 1). Thus, Integrin β1 recycling to the plasma membrane selectively requires WIPI2.

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The conserved amphipathic α-helical and FSSS motifs of WIPI2 promote Integrin β1 recycling

WIPI2 shares two critical features with other PROPPINs (Gopaldass and Mayer, 2024). These include an FSSS motif and the CD loop on blade 6 of the β-propeller structure. The CD loop promotes membrane fission activity of Atg18 during the fragmentation of yeast vacuoles (Gopaldass et al., 2017) and in the detachment of endosomal carriers (De Leo et al., 2021). Upon contact with the bilayer, the loop folds into an amphipathic α-helix that is conserved among PROPPINs and necessary for membrane fission activity. To test whether the amphipathic nature of this helix also contributes to the function of WIPI2 in Integrin β1 retrieval to the cell surface, we swapped two pairs of amino acids on opposite sides of this α-helix in WIPI2 (Figure 4A). This manipulation preserves the amino acid composition of the helix but abolishes its amphipathic character. The resulting ^EGFPWIPI2^SLoop (scrambled loop) variant and the wildtype version ^EGFPWIPI2^WT were expressed in control and WIPI2^KD cells at comparable levels, as shown by western blot (Figure 4—figure supplement 1). Whereas permeabilized WIPI2^KD cells displayed Integrin β1 concentrated on perinuclear endosomes, expression of ^EGFPWIPI2^WT rescued this phenotype (Figure 4B). ^EGFPWIPI2^SLoop failed to induce such recovery in WIPI2^KD cells. It even exerted a dominant-negative effect when expressed in control cells. (Figure 4C, D). Cells expressing ^EGFPWIPI2^SLoop also showed conspicuous long tubules, which were more readily detectable in live cell imaging (Figure 4—figure supplement 2) than in fixed cells (Figure 4).

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Alignments of Atg18 and WIPI1/2 orthologs have revealed a conserved FSS/TS motif in blade 2 (Courtellemont et al., 2022; Gopaldass et al., 2017; Gopaldass and Mayer, 2024; Strong et al., 2021). On the β-propeller structure of these PROPPINS, this motif is located at the membrane-distal side. It is a potential phosphorylation site and required for the interaction of Retromer with WIPI1 and Atg18 (Courtellemont et al., 2022; Feng et al., 2015). Since the overall structural arrangement of Retriever resembles that of Retromer (Bartuzi et al., 2016; Boesch et al., 2023; Healy et al., 2023; McNally et al., 2017), we tested whether WIPI2 requires its FSSS motif to bind Retriever. To this end, we generated WIPI2^S67E and WIPI2^S67A to substitute the central serine in the FSSS motif and mimic effects of potential phosphorylation at this site.

The functionality of these WIPI2 variants was tested by rescuing WIPI2^KD cells through expression of siRNA-resistant variants of ^EGFPWIPI2. Both ^EGFPWIPI2^S67E and ^EGFPWIPI2^S67A were expressed to similar levels as an ^EGFPWIPI2 wildtype allele (Figure 4—figure supplement 1). Immunofluorescence staining of non-permeabilized WIPI2^KD cells showed that the expression of ^EGFPWIPI2^WT re-established the exposure of Integrin β1 at the cell surface, while ^EGFPWIPI2^S67A and ^EGFPWIPI2^S67E were ineffective (Figure 5A–C). Expression of the two variants also impaired surface exposure of Integrin β1 in control cells (Figure 5B–D). This dominant-negative effect is significant because it implies that the two variants compete with the endogenous protein. To be able to do so they should be folded, suggesting that the S67 substitutions do not grossly perturb WIPI2 structure. A conspicuous consequence of both FSSS substitutions was the accumulation of ^EGFPWIPI2^S67A and ^EGFPWIPI2^S67E in long tubules. These tubules were very extensive in live cell imaging and easily decayed upon fixation. Co-expression of ^RFPRAB4 or ^mCherryRab5 revealed that these tubules often connected larger dot-like structures, which were positive for ^mCherryRab5 and/or ^mRFPRAB4 (Figure 4—figure supplement 2A). The tubules themselves preferentially accumulated ^RFPRAB4 (70% colocalization) and only little ^mCherryRab5 (21%) (Figure 4—figure supplement 2B). These results are consistent with the view that WIPI2-associated tubules originate mainly from early and recycling endosomes and that WIPI2 contributes fission activity to Retriever-mediated trafficking through its amphipathic helix and its FSSS motif.

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WIPI2 interacts with Retriever, Commander, and SNX17

Next, we tested whether WIPI2 and WIPI1 interact with Retriever or Retromer and could become part of their respective coats. In that case, WIPI2 should interact not only with Retriever itself, but also with the sorting nexin SNX17, which binds Retriever, and perhaps the CCC subunits, which integrate Retriever into the Commander complex (Butkovič et al., 2024; Martín-González et al., 2025; Singla et al., 2024). We assayed this by co-immunoadsorption. An HA-tagged allele (WIPI2^HA) was expressed in HK2 cells using lentiviruses. Extracted proteins were adsorbed to anti-HA beads and analysed by western blotting (Figure 6). Whereas the Commander subunit CCDC93 and the Retriever subunit VPS26C co-adsorbed to the beads in extracts from cells expressing WIPI2^HA (Figure 6A, C), these signals were absent from cells expressing only non-tagged WIPI2. SNX17 also co-adsorbed with WIPI2^HA. This interaction was abolished by knockdown of the Retriever subunit VPS26C (Figure 6B), suggesting that Retriever links WIPI2 to SNX17. WIPI2 differentiated strongly between Retromer and Retriever. Although VPS26C is quite similar to the Retromer subunit VPS26, only VPS26C co-adsorbed with WIPI2^HA whereas VPS26 was not detectable (Figure 6C, D).

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The situation was inversed when the HA tag was attached to WIPI1. In this case, VPS26 was recovered on the beads but VPS26C was not (Figure 7A, B). SNX27, a sorting nexin that interacts with Retromer (Simonetti et al., 2022; Temkin et al., 2011), was also co-adsorbed with WIPI1^HA (Figure 7C). The immunoprecipitation results were supported by parallel immunofluorescence studies. WIPI2 colocalized preferentially with Retriever and SNX17 (Figure 7—figure supplement 1) while WIPI1 colocalized preferentially with SNX27 and Retromer (Figure 7—figure supplement 2). Moreover, WIPI2 colocalized with Integrin-β1 on early endosomal compartments positive also for SNX17, but not on late endosomal LAMP1 containing compartments (Figure 7—figure supplement 3). These results suggest that Retriever bridges WIPI2 and SNX17 and that Retromer bridges WIPI1 with SNX27, integrating these PROPPINs into their cognate Retriever or Retromer coats.

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Since the FSSS motif of WIPI2 is necessary for its transport functions, we tested whether this motif affects the interactions of WIPI2 with Retriever. To this end, wildtype and the two FSSS variants of WIPI2^HA were stably expressed and cell extracts were probed by the same co-adsorption approach as above (Figure 8). Whereas the Retriever subunit VPS26C was efficiently co-adsorbed with WIPI2^WT-HA, WIPI2^S67A-HA, and WIPI2^S67E-HA labilized this interaction. Cells carrying only the non-tagged, endogenous WIPI2 yielded no VPS26C signal. Thus, the FSSS motif is required for the interaction between WIPI2 and Retriever. These results were also confirmed by immunofluorescence analysis (Figure 8—figure supplement 1), where VPS26C colocalized less with WIPI2^S67A and WIPI2^S67E (16%) than with the wildtype form (56%).

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The interaction of WIPI2 and Retriever is necessary for their localization on recycling endosomes

To test for the in vivo consequences of the WIPI2–Retriever interaction, we analysed the effect of the FSSS motif on the colocalization of the respective WIPI2^EGFP variants with different endosomal RAB-GTPases, which were N-terminally tagged with mCherry. In line with previous studies (Polson et al., 2010; Puri et al., 2018) ^EGFPWIPI2^WT mostly co-localized with ^DsRedRAB11 (58%) and ^mCherryRAB5 (44%), and much less with ^mCherryRAB7 (6%) (Figure 9, Figure 9—figure supplements 1–3). By contrast, ^EGFPWIPI2^S67A and ^EGFPWIPI2^S67E co-localized mainly with ^mCherryRAB5 (60–63%) and ^mCherryRAB7 (36–38%), and much less with ^DsRedRAB11 (12–14%). The Retriever subunit VPS35L was detected by immuno-staining. In cells expressing WIPI2^WT, it mainly co-localized with ^DsRedRAB11 (42%) and ^mCherryRAB5 (43%) and less with ^mCherryRAB7 (23%). In ^EGFPWIPI2^S67E and ^EGFPWIPI2^S67A expressing cells, VPS35L distribution shifted, similarly as for ^EGFPWIPI2, indicating reduced presence on ^DsRedRAB11 compartments (12–13%), whereas co-localization with ^mCherryRAB5 (42–44%) was maintained and that with ^mCherryRAB7 (26–27%) slightly increased. Collectively, these results suggest that the FSSS motif in blade 2 of WIPI2 allows this PROPPIN to associate with Retriever, and that this association is necessary for localizing WIPI2 and Retriever on Rab11-containing recycling endosomes.

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Our experiments reveal pathway specificity for WIPI1 and WIPI2 (Figure 10). The two proteins support distinct cargoes and transport routes and interact with different partners. This isoform selectivity contrasts with the roles of these proteins in their second functional domain, autophagy, which strongly depends on WIPI2 but is also significantly reduced by ablation of WIPI1 (De Leo et al., 2021; Dooley et al., 2014; Guan et al., 2001; Obara et al., 2008; Polson et al., 2010; Proikas-Cezanne et al., 2015; Proikas-Cezanne et al., 2004). Furthermore, the autophagic function of the PROPPINs also exploits other molecular features than endosomal protein exit. For autophagy, WIPI1 and its yeast orthologue Atg18 do not require the amphipathic helix in CD loop 6, which is essential for endosomal transport. It was hence proposed that WIPI1 and Atg18 perform distinct functions in autophagy and in endosomal protein exit (Courtellemont et al., 2022; De Leo et al., 2021; Gopaldass et al., 2017).

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In endosomal transport, WIPI1 and WIPI2 display selectivity in their partners (Figure 10). WIPI1 interacts with Retromer and WIPI2 with Retriever, mirroring the impact of the two PROPPINs on the respective transport routes and cargos. WIPI1 and WIPI2 utilize the same molecular features to support these transport routes. Their FSSS motif is necessary for both isoforms to integrate with Retromer and Retriever, respectively. This, together with the similarity in the overall organization of Retromer and Retriever, suggest that they might form complexes in a similar manner. In analogy to the WIPI1–Retromer complex, which was termed CROP (Courtellemont et al., 2022), we hence refer to the WIPI2–Retriever complex as CROP2.

WIPI2-dependent transport of Integrin β1 depends on the amphipathic character of the α-helix in CD loop 6. In Atg18 and WIPI1, this helix is necessary to convey membrane fission activity, which is used both for division of the lysosome-like vacuoles of yeast and for detachment of endosomal carriers (De Leo et al., 2021; Gopaldass et al., 2017; Zieger and Mayer, 2012). This fission activity is potentiated by integrating Atg18 and WIPI1 with Retromer into the CROP complex (Courtellemont et al., 2022). Our observations suggest that WIPI2 contributes pathway-specific membrane fission activity by integrating with Retriever. In line with this, ablating the amphipathic character of its helix on CD loop 6 disrupts Integrin β1 recycling to the cell surface and leads to its accumulation in endosomal compartments. Disrupting the interaction of WIPI2 with Retriever by substituting the FSSS motif has the same effect. It also leads to the accumulation of long tubular structures, which we presume to be exaggerated endosomal carriers that continue to grow but fail to detach. This phenotype is consistent with a block in the membrane fission step that is necessary to allow the endosomal carrier to depart.

Retriever couples to other proteins that are required for endosomal protein transport. These include the CCC complex, which associates with Retriever to yield the very large Commander complex (Boesch et al., 2024; Healy et al., 2023; Laulumaa et al., 2024). How Commander is placed on the membrane is currently unknown, but the interaction between SNX17 and Retriever contributes to its recruitment (Butkovič et al., 2024; Healy et al., 2022; Singla et al., 2024; Steinberg et al., 2012) and presumably to the formation of a coat. Our pull-downs of WIPI2 detected not only the Retriever subunit VPS26C as an interactor, but also SNX17 and the CCC subunit CCDC93. This co-fractionation could be due to a direct interaction of WIPI2 with Retriever or reflect an interaction via another component of Commander. Our immunoprecipitation assays cannot distinguish and more detailed structural and interaction studies with pure compounds will be necessary to elucidate the nature of this interaction. Since Retriever is structurally similar to Retromer, WIPI2 is structurally similar to WIPI1 and Atg18, and a Retromer–Atg18 interaction could be reconstituted from pure proteins (Courtellemont et al., 2022), we consider a direct WIPI2–Retriever interaction as the more likely scenario. But in both cases our observations suggest that WIPI2 integrates into the Retriever coat and might thus bring membrane fission activity to the place where it is required for carrier formation. Here, it could synergize with other factors that support membrane fission in endosomal transport, such as EHD1, which interacts with SNX17, and the actin-regulating WASH complex, which binds to Commander (Bartuzi et al., 2016; Dhawan et al., 2022; Dhawan et al., 2020; Phillips-Krawczak et al., 2015).

The requirement of two distinct WIPI proteins, using the same molecular features, in Retromer- and Retriever-dependent transport uncovers a novel conserved element between these two pathways. In combination with the similar overall organization of Retromer and Retriever and their common interactions with EHD proteins (Arlt et al., 2015; Chi et al., 2014; Daumke et al., 2007; Deo et al., 2018; Gokool et al., 2007; Grant et al., 2001; Solinger et al., 2020) and WASH (Derivery et al., 2009; Gomez and Billadeau, 2009; Guo et al., 2024; Harbour et al., 2012; Jia et al., 2012; Phillips-Krawczak et al., 2015; Seaman et al., 2009), this supports the notion that these two membrane coats share similar mechanistic principles for their formation and/or detachment.

Source data files are provided. All cell lines and constructs produced for this study are available from the corresponding author upon request.

1. 1. Antonny B
   2. Burd C
   3. De Camilli P
   4. Chen E
   5. Daumke O
   6. Faelber K
   7. Ford M
   8. Frolov VA
   9. Frost A
   10. Hinshaw JE
   11. Kirchhausen T
   12. Kozlov MM
   13. Lenz M
   14. Low HH
   15. McMahon H
   16. Merrifield C
   17. Pollard TD
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Author details

1. Maria Giovanna De Leo

   Department of Immunobiology, University of Lausanne, Lausanne, Switzerland

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1523-9121

2. Andreas Mayer

   Department of Immunobiology, University of Lausanne, Lausanne, Switzerland

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   andreas.mayer@unil.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6131-313X

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