A Mechanism for Retromer Endosomal Coat Complex Assembly with Cargo

A mechanism for retromer endosomal coat complex assembly with cargo

Megan S. Harrison, Chia-Sui Hung1, Ting-ting Liu1, Romain Christiano, Tobias C. Walther, and Christopher G. Burd2

Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520

Edited by Peter J. Novick, University of California, San Diego, La Jolla, CA, and approved November 20, 2013 (received for review September 3, 2013)

Retromer is an evolutionarily conserved protein complex com- are coordinately recognized by retromer and that these inter- posed of the VPS26, VPS29, and VPS35 proteins that selects and actions are sufﬁcient to recruit retromer to a membrane where packages cargo proteins into transport carriers that export cargo they poise retromer to capture integral membrane retrograde from the endosome. The mechanisms by which retromer is re- cargo. cruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bi- Results valent recognition of an effector of PI3K, SNX3, and the RAB7A The structure of yeast Snx3 shows that it is composed of short GTPase, by the VPS35 retromer subunit. These bivalent interac- unstructured regions at the amino and carboxy termini and a PX domain that, when bound to the endosome membrane, presents tions prime retromer to capture integral membrane cargo, which β A enhances membrane association of retromer and initiates cargo a prominent three-stranded -sheet to the cytoplasm (Fig. 1 ) sorting. The role of RAB7A is severely impaired by a mutation, (16) that we speculate serves as an interaction surface for binding other factors such as retromer and/or cargo proteins. Because K157N, that causes Charcot–Marie–Tooth neuropathy 2B. The both human and yeast SNX3 associate with retromer (12, 13, 17), results elucidate minimal requirements for retromer assembly on we implemented a functional assay in yeast to determine whether the endosome membrane and reveal how PI3K and RAB signaling residues on this surface are required for the function of yeast are coupled to initiate retromer-mediated cargo export. Snx3 in vivo, and then extended this information to human SNX3. Two or three codons encoding residues (depending on sorting nexin | mass spectrometry | biochemical reconstitution |

the length of the strand) with solvent-exposed side chains were CELL BIOLOGY proteoliposome changed to alanine (Fig. 1A), and then the mutant genes were used to replace the native SNX3 locus. Live cell fluorescence orting of cargo within the endosome determines whether it microscopy of GFP-tagged Snx3-β1 and Snx3-β3 mutant proteins Swill be retained and ultimately degraded via lysosome- shows that they localize to punctate endosomes, just as wild-type mediated turnover, or exported via a plasma membrane recycling Snx3-GFP (Fig. 1B). However, the Snx3-β2 mutant protein was fl or retrograde pathway that directs cargo to the TGN or recycling barely detectable by uorescence microscopy and immunoblotting, suggesting that it is structurally compromised and degraded endosome. Genetic dissection of endosomal retrograde pathways β β in budding yeast (Saccharomyces cerevisiae) led to the identifi- as a consequence. Because the Snx3- 1 and Snx3- 3 mutants cation of an endosome-associated protein complex termed ret- localize to endosomes, and appear to be as stable as wild-type Snx3, we focused further analysis on these mutants. romer, composed of a Vps5–Vps17 heterodimer and a trimeric complex of the Vps26, Vps29, and Vps35 proteins (1). The ret- “ ” Structural Requirements for Snx3 Function. To determine whether romer trimer, also called the cargo recognition complex, is the the β1andβ3 mutations affect Snx3 function in retrograde core functional unit of retromer, serving as a platform for recruiting many other factors to the endosome (2), and we shall Significance refer herein to the trimer as retromer. It is now appreciated that retromer constitutes an ancient, evolutionarily conserved protein sorting complex that operates in multiple endosomal cargo ex- The endosomal system is a network of organelles that play key port pathways (2, 3). Hence, elucidating the molecular mecha- roles in nutrient uptake, protein and lipid sorting, and signal nisms that underlie retromer function is key for understanding transduction. Integral membrane proteins are delivered to fi the endosomal system. endosomes via traf cking from the plasma membrane and the The formation of a vesicular transport carrier is typically ini- secretory pathway, and many of these proteins are then tiated by a GTPase module that elicits recruitment of a coat returned from the endosome for reuse. The selection and protein from the cytosol to a particular site on the membrane. packaging of many integral membrane proteins into transport Retromer is an effector of RAB7A [henceforth referred to as carriers that export cargo from the endosome requires a pro- RAB7 (human) or Ypt7 (yeast)], a GTPase regulator of endo- tein complex called “retromer,” whose function protects some dynamics and depletion of RAB7-GTP in cells results in organisms from metabolic defects, Charcot–Marie–Tooth neu- a substantial loss of endosome-associated retromer (4–9). In ropathy 2B, Parkinson’s disease, and Alzheimer’s disease. We addition to GTPase signaling modules, interactions of coat elucidate the minimal requirements for targeting of retromer proteins with membrane lipids, such as phosphoinositides, con- to the endosome membrane and show that this mechanism tribute to coat assembly by increasing the avidity of membrane facilitates retromer recognition of a cargo protein. binding. There is no evidence that retromer directly recognizes membrane lipids (10, 11). Instead, retromer membrane re- Author contributions: M.S.H., C.-S.H., T.-t.L., and C.G.B. designed research; M.S.H., C.-S.H., cruitment is attributed to its association with any of several dif- T.-t.L., and R.C. performed research; M.S.H., R.C., and T.C.W. contributed new reagents/ ferent sorting nexins (3), which are peripheral membrane proteins analytic tools; M.S.H., C.-S.H., T.-t.L., R.C., T.C.W., and C.G.B. analyzed data; and M.S.H. and C.G.B. wrote the paper. defined by the presence of a Phox homology (PX) domain that recognizes phosphatidylinositol 3-phosphate (PtdIns3P), a signa- The authors declare no conflict of interest. ture component of endosomal membranes. However, a formal This article is a PNAS Direct Submission. test of this hypothesis is lacking. Retromer binds sorting nexin 3 1C.-S.H. and T.-t.L. contributed equally to this work. [henceforth referred to as SNX3 (human) or Snx3 (yeast)] (12– 2To whom correspondence should be addressed. E-mail: [email protected]. 15), and in SNX3 knockdown cells, less retromer is associated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with endosomes (14). In this study, we show that SNX3 and RAB7 1073/pnas.1316482111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1316482111 PNAS | January 7, 2014 | vol. 111 | no. 1 | 267–272 Downloaded by guest on September 25, 2021 proportional to the amount of the GST-Ste13 fusion protein, and that both mutant forms of Snx3 were captured by the GST-Ste13 beads essentially as efficiently as wild-type Snx3. We therefore conclude that the β1 and β3 Snx3 mutant proteins are compromised in binding retromer but are not significantly compromised in recognition of cargo. To more directly address association of SNX3 with retromer, and to determine whether the structural requirements for the recognition of human SNX3 and retromer are conserved between the yeast and human proteins, we tested the ability of purified human retromer and human SNX3 to associate in vitro. Human retromer was assembled from proteins produced in Escherichia coli and immobilized on beads via an N-terminal GST moiety on GST-VPS35. As expected (13), purified recombinant human SNX3 was retained by these beads (Fig. 2B). Because the yeast Snx3-β1 mutant was the most severely functionally compromised mutant, we prepared a recombinant form of human SNX3 in which the corresponding positions (F28, D30, E32) were Fig. 1. Mutations on the β1 and β3 strands ablate function but not endo- changed to alanine, and expressed and purified this protein from E. some localization of yeast Snx3. (A) Ribbon diagram of yeast Snx3. The lo- coli. Importantly, the solubility and purification of mutant SNX3-β1 cation of mutations on β1 (magenta E37A E39A H41A) and β3 (green R74A were identical to wild-type SNX3, suggesting that the mutations do K77A) are indicated, and the site of the Gly23Cys mutation used to de- not grossly perturb its structure. When assayed for binding to hu- rivatize Snx3 is indicated in red. The structure is based on coordinates pub- man retromer (Fig. 2B), a striking loss of binding is observed, in lished in Zhou et al (16). (B) In vivo localization of Snx3-GFP. Each mutant agreement with results for yeast SNX3-β1 mutant. These data show Snx3 gene replaced the wild-type locus and is expressed with a C-terminal that docking of both yeast and human SNX3 into retromer requires GFP tag. (C) The Snx3- and retromer-dependent cargo, Ste13, was expressed side-chain residues within the β1 strand of SNX3, and they formally as a GFP fusion in cells expressing the indicated form of Snx3 and imaged by establish that association of SNX3 with retromer is essential for deconvolution microscopy. For B and C, one image from the approximate SNX3 function in retromer-mediated cargo sorting. center of a Z series is shown. (Scale bar: 5 μm.) Recognition of SNX3 by Retromer. To gain insight into the retromer structural requirements for binding of SNX3, we first set out to sorting, the steady-state localization of Ste13, an integral mem- identify the retromer subunit(s) to which SNX3 binds. Retromer brane protease that is retrieved from the endosome in a Snx3- and functions as a trimer and the most highly conserved regions of retromer-dependent manner, was examined (18). In wild-type cells, the individual retromer proteins are those that mediate assembly GFP-Ste13 localizes to punctate Golgi and endosome compart- of the trimer, so we considered it important to address this ments, whereas in snx3Δ cells, it localizes to the vacuole mem- C question by using intact (i.e., full-length) proteins. Accordingly, brane and vacuole-associated endosomes (Fig. 1 ). In cells we devised a biochemical approach in which a chemical cross- β β expressing the Snx3 1or 3 mutant, GFP-Ste13 localizes prom- linking reagent incorporated into SNX3 was used to determine inently to the vacuole membrane and to punctate endosomes the “nearest neighbor” contacts in the reconstituted retromer– surrounding the vacuole, clearly demonstrating that mutations in SNX3 complex (Fig. 3A). β1 and β3 result in a loss of Snx3 function in vivo. We note that The mutagenesis data described above indicates that β1of the appearance of GFP-Ste13 in cells expressing the Snx3-β1 SNX3 is essential for binding retromer, so we targeted a residue mutant is indistinguishable to that of snx3Δ cells, whereas vac- (Gly23) that is near, but is not part of, this structural element uole localization in Snx3-β3–expressing cells is less prominent, (Fig. 1A) for conjugation to a chemical cross-linking biotin suggesting that the β1 mutations result in a severe impairment of transfer reagent, tetrafluorobenzoyl-biotinamidocaproyl-meth- Snx3 function, whereas the β3 mutant is markedly, but not com- anethiosulfonate (Mts-Atf-biotin). When incorporated into a “bait pletely, functionally compromised. Differential centrifugation ex- protein,” the tetrafluorophenyl azide (Atf) moiety can covalently periments confirm these findings (Fig. S1A). Two biochemical activities have been ascribed to yeast Snx3: binding to the cytoplasmic portions of the Ste13 and Ftr1 proteins that contain retrograde sorting signals (12, 18) and association with retromer (12, 13). In principle, loss of either function should result in a loss of Snx3-dependent retrograde sorting, so we examined each of these activities. In vivo association of Snx3 with retromer was monitored by coimmunoprecipitation after chemical cross-linking of intact cells (12). In this assay, the Vps29 retromer subunit contains a C-terminal HA epitope tag that permits the entire retromer complex, and associated Snx3, to be immunopurified from detergent solubilized cell lysates (12). By this assay, substantially less of the β1 and β3 mutant Snx3 proteins copurify with retromer (Fig. 2A). Notably, the amount of Snx3-β1 mutant protein that copurified was barely detectable, in accordance with the null phenotype of this mutant C with regard to Ste13 localization (Fig. 1 ). To monitor cargo β recognition, we used a pull-down assay that measures binding of Fig. 2. Mutations in 1 of yeast and human SNX3 perturb recognition by retromer cargo selective complex. (A) Coimmunoprecipitation of myc epi- Snx3 and the cytoplasmic segment of Ste13, which contains the tope-tagged Snx3 with Vps29-HA immunopurified from yeast cell lysates retrograde sorting signal. Snx3 was produced by coupled tran- 35 prepared in the presence of 5 mM DSP cross-linking reagent was visualized scription translation in the presence of [ S]methionine and in- by anti-myc blot (Lower). Vps29-HA levels were visualized by anti-HA blot cubated with beads coated with varying proportions of GST or (Upper). (B) Human retromer was immobilized and incubated with purified, GST-Ste13(1–117) (Fig. S1B). The results show that the amount recombinant SNX3 or SNX3-β1 mutant. Bound fractions were assayed by of wild-type and mutant Snx3 proteins captured by the beads is anti-SNX3 immunoblot (Upper) and staining with CBB (Lower).

268 | www.pnas.org/cgi/doi/10.1073/pnas.1316482111 Harrison et al. Downloaded by guest on September 25, 2021 Fig. 3. SNX3 is recognized by VPS35 retromer subunit. (A) SNX3-Mts-Atf-biotin cross-linking strategy: Assembled SNX3*–retromer complex was exposed to UV light to activate the Atf (X) crosslinkage. Snx3* was subsequently released by reduction. An aliquot of the reaction was removed and probed by streptavidin-HRP blotting. The remainder was subjected to proteolysis, and derivatized peptides were identified by coupled liquid chromatography high resolution mass spectrometry (LC/MS). (B) CBB-stained gels of the indicated reactions (Left). Streptavidin-HRP blots of the same fractions (Right). Anti-SNX3 immunoblots of the blotted fractions are shown below the streptavidin-HRP blot. The asterisk indicates the position of free GST. (C) Summary of LC/MS analysis of Mts-Atf-biotin cross-linked products. Portions of the spectrum containing the major cross-linked peptides, which are each derived from VPS35, are shown. The corresponding mass data are shown in the table. The underlined Lys residues were modified with biotin. (D) Schematic diagram of VPS35 and location of SNX3–cross-linked peptides. VPS35 is proposed to be composed of 17 helical repeats (18). The approximate positions of the minimal regions of CELL BIOLOGY VPS35 that bind VPS26, VPS29, and RAB7 (red) and modified VPS35 peptides (yellow) are indicated.

cross-link to a “prey” protein within ∼11 Å upon activation with VPS35 containing the binding sites for VPS26, SNX3, and RAB7 UV light. Mts-Atf-biotin couples to cysteine residues in a bait comprise its most highly conserved region, underscoring the protein via a sulfhydryl reactive methanethiosulfonate (Mts) critical roles that, as we show below, SNX3 and RAB7 play in moiety, forming a reducible disulfide bond that upon reduction retromer recruitment to a membrane. elicits transfer of the biotin label to the prey protein. To con- The region of VPS35 containing the SNX3 binding site lies struct a SNX3 Mts-Atf-biotin bait protein, two mutations in immediately N-terminal to the Rab7 binding site (repeat 6) that SNX3 were engineered: Gly23Cys and Cys140Ser (which elimi- we identified (8) and overlaps the minimal VPS26 binding re- nates the single cysteine in native SNX3). The expression, solu- gion, raising the possibility that SNX3 may compete with VPS26 bility, and PtdIns3P binding properties of this mutant form of and/or RAB7 or, alternatively, that it forms a supercomplex with SNX3 were identical to that of wild-type SNX3, indicating that retromer and RAB7. To distinguish these possibilities, we these mutations did not structurally compromise SNX3. Mts-Atf- immobilized GST-RAB7 fusion proteins on beads and incubated biotin was coupled to C23, and varying amounts of modified them with purified recombinant retromer, SNX3, or retromer SNX3 (SNX3*) were incubated with retromer, which had been and SNX3 together, and then determined which proteins were immobilized on GSH beads via the GST moiety on GST-VPS35. captured (Fig. 4). All three retromer proteins and SNX3 are The bound SNX3*–retromer complexes were exposed to UV recovered on beads coated with the constitutively GTP-bound light, incubated with DTT, and the reaction components were form of GST-RAB7(Q67L) and, to a lesser extent, with native resolved by SDS/PAGE. Streptavidin-HRP blotting of the reaction GST-RAB7, but not by beads coated with the constitutively shows robust UV-induced biotin transfer to VPS35, whereas VPS26 GDP-bound form of GST-RAB7(T22N). Importantly, control is labeled to a small extent at the highest concentration of SNX3* experiments show that SNX3 is not captured by RAB7 beads in assayed (Fig. 3B). Neither VPS29, nor GST (derived from GST- the absence of retromer, indicating that retention of SNX3 is VPS35 fusion protein), were labeled above background. mediated by RAB7-bound retromer. This experiment demon- A mass spectrometry-based approach was implemented as an strates that retromer can bind SNX3 and RAB7 simultaneously. additional means to identify biotin-labeled retromer subunits and to map the site(s) of cross-linking. An aliquot of a cross- Requirements for Retromer Recruitment to a Membrane. The linked reaction (1 μM SNX3) was subjected to proteolysis with mechanism of retromer coat recruitment to the endosome is not LysC and Trypsin, and the resulting biotinylated peptides were known. RNAi-mediated knockdown of SNX3 and RAB7, and purified by using streptavidin-agarose and then directly analyzed expression dominant interfering RAB7 proteins in cells, results by liquid chromatography coupled online to high resolution mass in decreased endosome association of retromer (6, 13, 14, 19), spectrometry. In agreement with the streptavidin-HRP blotting implicating them in this process. We began investigating the results, analysis of purified biotinylated peptides identified two requirements for retromer membrane recruitment by assaying peptides with at least four spectral observations and the highest sedimentation of retromer with liposomes containing PtdIns3P, intensities (Fig. 3C). As expected, both peptides were derived to which SNX3 binds (22). A small amount of retromer sedi- from VPS35. One conjugated peptide, spanning amino acids ments with liposomes in the absence of SNX3, but this value 205–215, is contained within helical repeat 5 (Fig. 3D), and the varied (between 2 and 25%) with different preparations of ret- second peptide identified spans residues 25–44 (Fig. 3D) and is romer; we therefore considered this binding to be nonspecific contained within the minimal segment of VPS35 that assembles and subtracted it from subsequent measurements. Preincubation with VPS26 (19, 20). These results confirm and extend a recent of SNX3 with liposomes before the addition of retromer resulted yeast two-hybrid study that reported that SNX3 can interact with in nearly all SNX3 sedimenting with the liposomes; however, the N-terminal half of VPS35 (21). The N-terminal ∼300 aa of only a modest level (ranging from 8 to 22%) of retromer was

Harrison et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 269 Downloaded by guest on September 25, 2021 with both SNX3 and RAB7(Q67L), nearly half of the added retromer cosedimented with the liposomes (43, 47, and 41% in the three independent experiments shown in Fig. 5 A–C). This level of binding required both active SNX3 and RAB7-GTP, and was not due to aggregation of the proteins, because the SNX3-β1 mutant and RAB7(T22N) are completely inactive in synergizing with RAB7(Q67L) or SNX3, respectively. Mutations in RAB7A are associated with Charcot–Marie– Tooth neuropathy 2B (23), and a previous report found that one of these, K157N, results in reduced coimmunopurification of GFP-RAB7A(K157N) with retromer and reduced retromer endosome localization (6). RAB7A(K157N) is predominantly GTP loaded in vivo (24), suggesting that it may not be recognized by retromer and native RAB7A. We addressed this hypothesis by biochemical reconstitution of RAB7-dependent retromer liposome association (Fig. 5C). These experiments confirm that the mutation of K157N, in the context of otherwise wild-type RAB7 or in RAB7(Q67L), is unable to induce signif- icant recruitment of retromer to liposomes individually, similar to RAB7(T22N) (Fig. 5C and Fig. S2B). Moreover, in combi- nation with SNX3, the RAB7(Q67L, K157N) is unable to elicit enhancement of retromer recruitment to liposomes (Fig. 5C). These findings are consistent with the K157N mutation impairing endosomal membrane recruitment of retromer and retromer- mediated sorting. Having reconstituted membrane targeting of retromer, we sought to test the hypothesis that these interactions prime cargo capture by retromer. We first confirmed that retromer binds directly to a cytoplasmic portion of human diivalent metal transporter 1 isoform II (DMT1-II), an iron transporter that bears a retromer sorting motif (25), and mapped this activity to the C- terminal portion of VPS35 in complex with VPS29 (Fig. S3). Next, we prepared proteoliposomes containing a peptide (250 pmol) Fig. 4. SNX3 and RAB7 bind retromer simultaneously. GST-RAB7 fusion proteins were immobilized on GSH beads: GST-RAB7 (native RAB7), RAB7- derived from DMT1-II containing the retromer sorting motif, or Q67L (GTP-bound mutant), and RAB7-T22N (GDP-bound mutant). A CBB stained a variant peptide bearing mutations in the retromer sorting motif gel (Top)anti-VPS26blot(Middle), and anti-SNX3 blot (Bottom) are shown. and nearby aromatic residues (which might also contribute to retromer recognition). Each peptide possesses a membrane spanning segment and a C-terminal cysteine residue to facilitate fl recruited to liposomes, even when SNX3 was present in substantial labeling of the peptide (with a uorescent dye) so that we could (16-fold) molar excess to retromer (Fig. 5 A–C). Importantly, determine the amounts of each peptide that was incorporated retromer recruitment was due to SNX3 recognition because into the proteoliposomes in the appropriate orientation. These the SNX3-β1 mutant protein was completely inactive in these proteoliposomes were ineffective in recruiting substantial assays (Fig. 5B). Given this modest level of recruitment, and amounts of retromer (Fig. 5D), indicating that retromer does not the low affinity of the SNX3–retromer interaction suggested avidly recognize cargo in this format. We next asked whether by this assay and the pull-down assays (Fig. 2) (13), we con- inclusion of SNX3 and RAB7 on the proteoliposomes influences clude that SNX3 recognition alone is insufficient to mediate retromer recruitment. To clearly distinguish any effect, the endosome loading of retromer. amounts of SNX3 (0.7 μM) and RAB7 (1 μM) were reduced to We next tested whether RAB7 is sufficient to mediate mem- the minimal amounts required to effectively recruit ∼40% of brane recruitment of retromer. To associate recombinant RAB7 added retromer. In the presence of SNX3 and RAB7, the native on the surface of liposomes, we constructed versions of RAB7 peptide had a striking effect, more than doubling the amount (to (Q67L) (GTP-bound conformation; “RAB7GTP” in Fig. 5) and >80%) of retromer that was recruited to the proteoliposomes RAB7(T22N) (GDP-bound conformation; “RAB7GDP”)inwhich (Fig. 5D). This enhancement is due to bona fide cargo recogni- its C-terminal prenylation site was replaced with a 6× histidine tion because the mutant peptide was entirely without effect. tag. Modified RAB7 proteins were incubated with liposomes containing 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl) Discussion iminodiacetic acid] succinyl} [DGS-NTA(Ni)], which contains The results presented here define the minimal requirements for a head group that is recognized by the 6×His tag. Although robust membrane recruitment of, and cargo capture by, retromer RAB7 proteins associated efficiently with these liposomes, only and establish it to function via a general paradigm of vesicle coat a modest level (ranging from 4 to 20%) of retromer sedimented assembly, whereby multivalent interactions involving lipid (e.g., with the liposomes, even with an eightfold molar excess of RAB7 phosphoinositide) recognition and a GTPase module initiate (Q67L) over retromer (Fig. 5 A and B). This recruitment was cargo sorting and the formation of a transport carrier. At the specific for the GTP-bound conformation of RAB7 [i.e., RAB7 steady state, SNX3 and RAB7 localize predominantly to early (Q67L)], as essentially no recruitment was elicited by RAB7 and late endosomes, respectively, indicating that the SNX3- and (T22N) (Fig. 5B) or RAB5(Q79L) (Fig. S2A). These results lead RAB7-dependent recruitment mechanism is restricted to ma- us to conclude that RAB7-GTP is insufficient to elicit recruit- turing sorting endosomes as they accrue RAB7 by Rab conver- ment of retromer to a membrane. The modest retromer recruit- sion (2, 13). From these endosomes, the mechanism defined here ment activity of SNX3 and RAB7 in these experiments, and the initiates sorting into retrograde pathways that direct cargo to the close proximity of the SNX3 and RAB7 binding sites on VPS35, TGN and the recycling endosome (13, 17, 25). In recent years it led us to consider the possibility that SNX3 and RAB7 might has become apparent that retromer sorts cargo at multiple exit cooperate to recruit retromer. The results of three experiments portals within the endosomal system, and our results suggest that support this hypothesis. First, when liposomes were preincubated multivalent interactions underlie localization of retromer at other

270 | www.pnas.org/cgi/doi/10.1073/pnas.1316482111 Harrison et al. Downloaded by guest on September 25, 2021 CELL BIOLOGY

Fig. 5. Dual recognition of SNX3 and RAB7 recruits retromer to a membrane. (A) Liposomes were preincubated with purified SNX3, SNX3-β1, and/or RAB7- Q67L-His, and then purified retromer was added. Liposomes were collected by centrifugation, and the supernatant (S) and pellet (P) fractions (equal amounts) were loaded onto gels. A CBB-stained gel (Upper) and anti-VPS26 blot (Lower) are shown. The proportions of VPS26 in the pellet fractions, determined from this blot, are indicated below it. (B) Liposomes were incubated with RAB7-Q67L-His (“RAB7GTP”) or RAB7-T22N-His (“RAB7GDP”) with and without wild-type SNX3. Cosedimentation of retromer was monitored as described above. (C) Liposomes were preincubated with RAB7GTP or RAB7GTP K157N, with or without SNX3, and purified retromer was added. Cosedimentation of retromer was monitored as described above. (D) Retromer sedimentation was assayed with proteoliposomes alone or preincubated with SNX3 and RAB7. The proteoliposomes contained a retromer sorting motif (Tyr-Leu-Met; Center) or a peptide with each residue of this sequence, and surrounding aromatic residues, substituted by alanine.

exit sites. A candidate component of a mechanism to recruit ret- likely underlies the substantially increased association of retro- romer to the early endosome is SNX27, which is recognized by the mer with the membrane. Cargo binding activity has also been VPS26 retromer subunit (26–29), where it functions in a reported for the VPS26 subunit of retromer, which binds to the plasma membrane recycling pathway that is regulated by Rab4. opposite end of VPS35 (31), raising the possibility that recognition Multivalent interactions allow diverse retromer recruitment of different cargos may drive distinct retromer coat assembly mechanisms and can explain how retromer functions in organ- pathways. isms, such as Arabidopsis thaliana, which do not possess a SNX3 An outstanding question regards the relationship between ortholog. SNX3 and SNX-BAR proteins in retromer recruitment. In yeast, In solution, retromer is a ∼210-Å-long flexible rod (19), and the Vps5–Vps17 SNX-BAR heterodimer, which was originally our data demonstrate that it becomes initially anchored to the defined as stoichiometric components of retromer, is required endosome membrane by interactions involving SNX3 and RAB7 for retromer endosome localization, but Snx3 is not (8, 12). The at the N-terminal proximal region of VPS35. Mutations in yeast stable pentameric form of yeast SNX-BAR retromer, however, is Vps35 that result in a cargo-specific sorting defect of Vps10 map distinguished from metazoan retromer, which does not associate to the C-terminal region of Vps35 (30), and our data show that avidly with SNX-BAR proteins. Although depletion of retromer this region in human retromer is responsible for recognition of SNX-BAR proteins in cultured human cells is reported to in- DMT1-II (Fig. S3). The location of these interactions poise ret- crease the cytosolic pool of retromer (32), multivalent recogni- romer on the endosome membrane to survey and capture cargo tion of SNX3, RAB7, and cargo by metazoan retromer could via a binding site that localizes to the opposite end of VPS35. serve to recruit retromer on the relatively flat vacuolar mem- Upon cargo recognition, retromer is stabilized on the membrane, brane domain, juxtaposing retromer near a high local concen- effectively capturing cargo and initiating sorting. Thus, recognition tration of SNX-BARs that coat tubules. The incorporation of of cargo by this region of retromer would anchor the two ends of retromer into the SNX-BAR–coated tubular endosomal network the retromer complex to the membrane, and this arrangement may be coordinated with termination of RAB7 signaling by the

Harrison et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 271 Downloaded by guest on September 25, 2021 RAB7 GTPase activating protein, TBC1D5, that binds retromer incubated with RAB7 and/or SNX3 at room temperature for 60 min, then (6). In further support of this model, we recently found that in with retromer at 4 °C for 60 min. Liposomes were collected by centrifugation yeast cells lacking Ypt7-GTP (the ortholog of RAB7), retromer at 200,000 × g for 20 min at 4 °C, and the supernatants pellet fractions were cargo accumulates in the endosome (8) and, in cultured human harvested. More detailed information, including the sequences of the pep- cells, acquisition of RAB7 on the endosome by Rab conversion is tides used to produce the proteoliposomes, is provided in SI Materials temporally correlated with the appearance of retromer-coated and Methods. tubules that bud from the endosome (33). The biochemical reconstitution system established here provides an avenue to de- Cross-Linking Assay. Purified SNX3 (G23C C140S) was conjugated with Mts- finitively address these questions. Atf-biotin reagent (Pierce Chemical) according to manufacturer’s instruc- tions. Conjugated SNX3 (SNX3*) was incubated with GSH bead-immobilized Materials and Methods retromer in binding buffer (without DTT) in the dark, beads were washed, 20% were removed for the “before UV” control, and the remainder was Cell Culture and Molecular Biology. All yeast strains were constructed in the exposed to UV light. Samples were resolved by SDS/PAGE for CBB staining or BY4742 background (MATα his3Δ 1, leu2Δ 0, lys2Δ 0, ura3Δ 0) and propa- immunoblotting with antibodies against SNX3 or Streptavidin-HRP, or bi- gated by standard methods (34). Additional information is provided in SI otin-labeled proteins were enriched on streptavidin agarose and subjected Materials and Methods. to proteolysis and mass spectrometry. Additional information is provided in SI Materials and Methods. Recombinant Protein Expression, Purification, and Binding Assays. The purification, processing procedures, and conditions used for protein binding assays are provided in SI Materials and Methods. Mass Spectrometry. The methods are described in SI Materials and Methods.

Liposome Binding Experiments. Liposomes were produced from pure synthetic ACKNOWLEDGMENTS. We thank members of the Reinisch laboratory (Yale School of Medicine) for assistance with recombinant protein production, Drs. lipids (Avanti Polar Lipids) by mixing dioleoyl-phosphatidylcholine, dioleoyl- μ Da Jia and Mike Rosen (The University of Texas Southwestern Medical phosphatidylserine, nickel salt (DOGS-NTA-Ni), and extrusion through 1- m Center) for retromer expression vectors, Dr. Lei Shi for technical assistance, ﬁ pore-size lters by using a miniextruder (Avanti Polar Lipids). Proteolipo- and members of our laboratories for helpful discussions. This work was sup- somes were produced in a similar manner with peptides added to the lipid ported by National Institute of Health Grants GM060221 (to C.G.B.) and mixture before extrusion. For binding assays, liposomes (50 nmol lipid) were GM095982 (to T.C.W.).

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