Retromer forms low order oligomers on supported lipid bilayers

Retromer is a protein sorting device that orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long standing hypotheses regarding the Retromer sorting mechanism posit that oligomeric interactions between Retromer and associated accessory factors on the endosome membrane drives clustering of Retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers (∼18%), dimers (∼35%), trimers (∼24%) and tetramers (∼24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.


Introduction
Retromer is an evolutionarily conserved protein complex that orchestrates sorting and export of integral membrane proteins from the endosome. Loss of Retromer function, which is implicated in a variety of disease conditions, results in increased rates of turnover of plasma membrane proteins and retrograde cargo proteins in the lysosome, with broad consequences to cell and organism physiology (1)(2)(3).
Retromer is composed of three proteins VPS26, VPS29, and VPS35, that form a stable, soluble heterotrimer (4-7) that is recruited to the endosome by binding to sorting signals of integral membrane protein cargo and to other membrane-associated accessory proteins, including sorting nexins, RAB7 network components, and the WASH complex (2,8). Genetic and structural analyses of Retromer trimer complexed with different sorting nexins suggests that Retromer is a modular sorting device that associates distinctly with different sorting nexins (e.g., SNX-BARs, SNX3, SNX27) to establish cargo-specific sorting and trafficking pathways (2). Despite recent insights into Retromer structure, the molecular mechanisms of Retromer-mediated sorting remain poorly understood.
We discovered that the yeast (Saccharomyces cerevisiae) sorting nexin, SNX3/Grd19, functions as a cargo-selective Retromer adapter that associates with Retromer on the endosome membrane and aids in cargo recognition (9). Studies of cultured human cells and other model organisms confirmed the existence of a SNX3-Retromer sorting pathway in metazoans (10)(11)(12) where cargo sorting signal is recognized via the SNX3-Retromer interface (13). Using bulk biochemical reconstitution, we previously discovered that multivalent interactions between Retromer, SNX3, RAB7, and an integral membrane cargo confer recruitment of Retromer to the surface of small unilamellar vesicles (14). In this study we examined reconstituted components of the SNX3-Retromer sorting system on supported lipid bilayers by quantitative fluorescence microscopy. We find that when associated with a membrane, both in the presence or absence of cargo and accessory proteins, Retromer exists as monomers and lower order oligomers (dimer-to-tetramer). The results suggest that cargo is modestly concentrated by Retromer prior to export from the endosome by the SNX3-Retromer pathway, prompting a revision in long standing models of retromermediated cargo sorting.

Results and Discussion
As a coat protein of endosome-derived transport carriers that recognizes retrograde cargo sorting signals, Retromer has been proposed to concentrate integral membrane protein cargo prior to transport carrier formation (1,7,15,16). To test this, we first sought to determine the oligomeric state of Retromer when it is associated with a membrane.
Accordingly, a supported lipid bilayer (SLB) was constructed to mimic the relatively planar surface geometry of the vacuolar domain of the sorting endosome, where Retromer sorting domains are formed. The SLB contained physiological endosomal lipids, phosphatidylcholine (PC), phosphatidylserine (PS), and PtdIns3P, and non-physiological NiNTA-DGS, with a nickel ion-containing headgroup that is recognized by poly-histidine sequences, and trace amounts of rhodamine-phosphatidylethanolamine (Rh-PE) or NBDphosphatidylethanolamine (NBD-PE), used to assess lipid mobility and the quality of the bilayer ( Fig S1A). Fluorescence recovery after photobleaching (FRAP) analyses confirmed free diffusion of lipids within the supported bilayer; bilayers that were not fluid were exempt from analysis. Experiments also confirmed that association of a fluorescent His10-tagged peptide with the SLB is dependent upon the presence of NiNTA-DGS lipid and that the lipidassociated polypeptide is mobile ( Figure S1B, C).

Retromer exists as monomers and low order oligomers on a membrane
Retromer was assembled in lysates of bacterial cells expressing individual Retromer proteins, as we have used previously (14). To directly visualize Retromer on the SLB by fluorescence microscopy, VPS26 was produced with N-terminal His10 and SNAP tags ( Figure 1), which facilitated binding to the SLB and labeling with a fluorescent dye (AlexaFluor488), respectively. Incorporation of His10-SNAP-VPS26 fusion protein into the Retromer trimer was indistinguishable to that of VPS26, indicating that Retromer structure is maintained in the tagged protein. After incubating labeled Retromer with the SLB for 2 hours, the SLB was washed to remove unbound proteins and total internal reflection fluorescence microscopy (TIRFM) was used to visualize SLB-associated proteins (17). The results show that Retromer is distributed homogenously on the SLB (Fig. S1C), suggesting that it does not self-organize into clusters over a range of concentrations (1 nM nominal Retromer concentration is shown in the figure).
Next we established a single molecule fluorescence microscopy assay to determine Retromer oligomeric state on the SLB. These experiments use lower protein concentrations in the medium (~75 pM), resulting in a lower protein density on the SLB, such that a minimum of 3-4 pixels (~0.75-1 micron) separation between fluorescence labeled particles is favored, facilitating single-particle analysis. We monitored intensity of AF488-labeled Retromer puncta continuously over time at high frame rate (17-18 ms exposure per frame) to capture single fluorophore bleaching steps until most puncta bleached away ( Fig. 2A).
Fluorescent particles photobleached in either single or multi-step bleaching profiles, indicative of either single or multiple fluorophores in the particle (Fig. 2B). We used the decrease in fluorescence intensity due to the last bleaching event in individual puncta to estimate the intensity of single AF488 fluorophores (Fig. 2B) and then to calculate Retromer copy number per particle. Importantly, in this procedure every single image stack provides its own single-molecule intensity calibration for the cluster copy number estimation, enhancing robustness of the approach against experiment-to-experiment variations.
Results for independent preparations of retromer were pooled together. The combined results of four independent Retromer preparations are plotted as a histogram showing the frequency distribution of Retromer complexes on the SLB (Fig. 2C). The predominant species of Retromer on the SLB are monomers (~18%), dimers (~35%), trimers (~24%) and tetramers (~13%). Also observed are pentamers and rare higher-order oligomers up to ten (~10%). This distribution of Retromer oligomers on SLBs is similar to that reported in a cryoelectron microscopy study where dimers and tetramers were also the prevalent Retromer species in vitrified ice (18). These results suggest that membrane association per se does not influence Retromer oligomeric sate.

Retromer accessory proteins do not influence oligomeric state
Retromer association with the endosome membrane is conferred by binding to integral membrane cargo proteins, sorting nexins, and RAB7 (14,(19)(20)(21), therefore we next sought to determine if these accessory factors influence Retromer oligomerization on a SLB. Histagged, AF488-labeled cargo protein was homogeneously dispersed on the SLB (Fig. S2A) and incubation with "untagged Retromer" (recombinant, not his-tagged or labeled) did not affect its appearance (not shown). However, the effect of Retromer may not be apparent due to the low affinity with which Retromer binds sorting signals. Accordingly, we added the sorting nexin, SNX3, which facilitates Retromer membrane recruitment and forms part of the DMT1-II cargo binding site (13), onto the SLB. After confirming that purified, labeled SNX3 associates with the SLB by binding to PtdIns3P (Fig. S1D), SNX3 and untagged Retromer were sequentially added in stochiometric excess of the nominal cargo concentration and the distribution of cargo fluorescence was monitored for any changes occurring with the addition of SNX3 and soluble Retromer (Fig. S2). At all times examined, cargo fluorescence was homogenously distributed on the surface of the SLB (Fig. S2B). Thus, at the protein concentrations accessible in our experimental system, a model Retromer cargo is not clustered by SNX3-Retromer (Fig. S2B, C). We next measured Retromer particle size at low protein density on the SLB using quantitative single-particle TIRFM. For these experiments we used a modified system because at the low densities of proteins on the SLB required for single-particle analysis, only a small proportion of cargo molecules will be bound by Retromer. Accordingly, we constructed a non-dissociable model of the Retromer-cargo complex, termed "Retromer- . This difference might be attributed to the different membrane models used (rigid SLB vs GUV), however, we note that non-quantitative methods were used to infer changes in relative protein abundances across the GUV membrane, and that clustering of yeast SNX3-Retromer depended on the presence of a second, non-physiological membrane binding site (a His6 tag) on ySNX3 (19).

WASHC2C disordered segment does not influence Retromer oligomeric state
In metazoans, Retromer recruits the WASH protein complex from the cytosol to the endosome membrane where it promotes ARP2/3-dependent actin polymerization and Retromer-dependent sorting (8,(22)(23)(24)(25). Binding of WASH to Retromer is conferred by an approximately 1100 amino acid long unstructured segment of the WASHC2C/FAM21C subunit containing 21 "Leucine-Phenylalanine-acidic motifs ("LFa": L-F-[D/E]3-10-L-F) shown to constitute Retromer-binding sites by solution-phase binding assays (24,26). On the basis of the multi-valency of WASHC2C-Retromer interaction, Jia et al. (24) speculated that recruitment of WASH to the endosome membrane would result in clustering of membrane associated Retromer-cargo complexes (24). We tested this hypothesis using our single particle analysis platform.
We first determined if SLB-associated Retromer can recruit WASHC2C-21 ( Fig. 1 Finally, we asked if Retromer, cargo, SNX3, RAB7, and WASHC2C act synergistically to influence Retromer clustering at low protein densities (Fig. 5). As before, Retromer-RRS was used to enforce cargo occupancy. In the absence of any other factors, the distribution of Retromer-RRS oligomers (in the presence of SNX3 and RAB7 GTP ) is similar to the distribution of Retromer oligomers (Fig. 2). Next, WASHC2C-21 was added to the reactions to examine the effect of sub-stoichiometric (50:1) and stoichiometric (1:1) WASHC2C-21.
Single particle analyses revealed small increases in the proportions of Retromer-RRS monomer at both Retromer-RRS:WASHC2C-21 ratios, similar to the effect of sub-stoichiometric amounts of WASHC2C-5 (Fig. 4). These results indicate that binding of Retromer and WASHC2C on the SLB is (or is close to) stoichiometric, and further suggest that WASHC2C does not exert an effect (e.g., allosteric) to promote Retromer oligomerization. Consistent with these interpretations, Jia and colleagues reported that only the last two of the 21 LFa motifs (LFa20-21), which are those bound by VPS35 with the highest affinity, are essential for WASH-dependent sorting of integral membrane cargo (24).
These findings suggest that WASH does not exert its role in the Retromer pathway by clustering Retromer or Retromer-cargo complexes.

Implications for Retromer sorting mechanism
When associated with a membrane, human Retromer has an intrinsic propensity to form low order clusters (<5 monomers) and neither membrane association, nor the membrane- where Retromer monomer and dimer were the most prevalent species (13,18,27). In our study, Retromer dimers were generally the most abundant species observed on SLBs and this likely reflects two different Retromer-to-Retromer binding modes, where one is the 2fold symmetric dimer observed in solution phase described by Kendall and colleagues (18), and the second mode is mediated by the VPS35-VPS35 dimerization interface observed in the Retromer-SNX-BAR coat (28) and in solution (13). A distinct binding mode(s) is needed to explain the small proportion of higher order oligomers observed in our study, which might consist of chains Retromer complexes in solution-phase observed by cryo-electron microscopy (18).
Retromer has been proposed to constitute a coat protein complex for endosome-derived transport carriers that, by analogy to better characterized conventional vesicle coats (e.g., clathrin) that polymerize on the membrane to enrich nascent carriers in particular integral membrane cargo (7,16). A key feature of the conventional paradigm is the small area of coated membrane that gives rise to small (<100 nm diameter) transport carriers. In contrast, sorting of retrograde and recycling integral membrane in the endosomal system is weakly concentrative, relying instead on the large surface area of endosome-derived carriers to mediate bulk export of proteins and lipid from the endosome (29). Results presented here suggest that Retromer concentrates cargo minimally, though this study was necessarily limited so it is possible that conditions or factors not examined, such as different membrane topologies, rigidities, and/or WASH-mediated actin polymerization, influence Retromer oligomerization. We note, however, that the structure of yeast VPS5-Retromer coated membrane tubules that show the coat to be highly heterogenous with limited long range order, which is consistent with a minimally concentrative sorting mechanism (2,28).
Collectively, data do not support longstanding hypotheses of Retromer sorting that invoke oligomerization as a driving force for sorting of integral membrane proteins in the endosomal system. Rather, low order oligomerization of Retromer and associated factors is likely an adaptation of bulk membrane trafficking pathways characteristic of the endosomal system that ensure membrane homeostasis of the plasma membrane and endo-lysosome organelles. Retromer, RAB7, and SNX3 were prepared as described in Harrison et al. (14).
Proteins were prepared fresh on the first or second day of each three-day experiment.
Proteins were expressed by auto-induction (30)  After thoroughly rinsing with Mili-Q-filtered water and drying the slide, the clean wells are sealed with PCR sealing foil sheets (ThermoScientific). To form an SLB, an individual well was opened and cleaned with two 1-hour incubations at 50°C of freshly prepared, sterile filtered 5M NaOH. The well was rinsed twice with 0.5 ml Mili-Q water and twice with Buffer A. The rinsed well was then filled with 0.2 ml of Buffer A and 10 l of the liposomes were added. After incubation for one hour at 37°C, the well was washed three times with buffer A to remove any non-adhered liposomes. The SLB was then blocked with 0.1% casein in Buffer A for 20 minutes at 37°C. We checked the mobility of the SLB by FRAP or visual inspection (described below) for each experiment; immobile membranes were discarded.
In one three channel co-localization experiment (Fig. 3), the fluorescence signal was too weak for visualization on the SLB alone because of the low affinity between Retromer and WASHC2C. In this experiment, we looked at the bright spots where more lipid and thus protein was concentrated, and signal-to-noise was high enough to detect co-localization.

Protein attachment to supported bilayers
His10-tagged proteins were bound to NiNTA-DGS lipids in the SLB. Proteins were added to the well containing 0.2ml of Buffer A with 0.1% casein and 1 mM TCEP. The protein and the bilayer were incubated in the well in the dark for two hours at 30°C to get a secure attachment to the bilayer. After protein addition, the bilayer was washed 3X with 0.1% casein Buffer A. Proteins not binding through His10 tags (SNX3, Retromer, and WASHC2C), were incubated with the SLB for 30 minutes in the dark at room temperature and then washed 3X with 0.1% casein Buffer A. Wells with immobile proteins were not used.

Calculation of Retromer copies per cluster
Calibration of single fluorophore intensities. Fluorescently labeled proteins bound to a SLB were imaged using TIRF microscopy using continuous stream acquisition until nearly all spots bleached. Imaging started in a virgin region that was never exposed to excitation light.  Multiple bleaching movies were collected as described above and changes to the Retromer particle distribution were quantified. After analysis of the puncta of fluorescence in the micrographs (Fig. 4A), results were plotted as histograms (Fig. 4.A, B), with the order of the oligomer (i.e. 1 (monomers), 2 (dimers), 3 (tetramers), etc.) on the horizontal axis and relative frequency of the oligomer on the vertical axis. Statistical differences between the distributions were tested as described above.  Retromer complexes in individual puncta. Note that the data in the top panel is also presented in Figure 2D.