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SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking

['Yangli Tian', 'Key Laboratory Of Molecular Biophysics Of The Ministry Of Education', 'College Of Life Science', 'Technology', 'Huazhong University Of Science', 'Wuhan', 'Hubei', 'Qiaoju Kang', 'Xuemeng Shi', 'Yuan Wang']

Date: None

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.

Trafficking of internalized materials through the endolysosomal system is essential for the maintenance of homeostasis and signaling regulation in all eukaryotic cells. Early endosomes are the sorting hub on the endocytic pathway. After internalization, the plasma membrane lipid, proteins, and invading pathogens are delivered to early endosomes for further degradation in lysosomes or for retrieval to the plasma membrane or the trans-Golgi network for reuse. However, when, where and by what mechanism various cargo proteins are sorted from each other and into the different pathways largely remain to be explored. Here, we identified SNX-3, a PX-domain only sorting nexin family member, as a novel regulator for the tubular endosomes underlying recycling of a subset of CIE cargoes. Compared with EEA-1, the superior recruitment of SNX-3 at the CIE-derived subpopulation of endosomes is critical for preventing these endosomes from converging to the classical sorting endosomes and subsequently into the multivesicular endosomal pathway. We speculate that through a spatio-temporal interplay with the retromer, SNX-3 is involved in different recycling transport carriers. Our finding of SNX-3’s role in modulating the formation of tubular endosomes provides insight into the sorting and trafficking of CIE pathways.

Funding: This work was supported by the Major Research Plan of the National Natural Science Foundation of China (91954107) and the National Natural Science Foundation of China (31571468) to RZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The present work explores the mechanisms underlying the sorting and trafficking of CIE cargoes within the endo-lysosomal system. We performed an imaging-based family-wide screening of SNX proteins and identified candidates whose dysfunction disrupted hTAC-containing tubular endosomes in the C. elegans intestine. SNX-3 was identified as a novel regulator of the tubular endosomes that mediates specific ARF-6-associated CIE recycling independent of the retromer complex. The loss of SNX-3 abolishes the ARF-6-associated CIE recycling endosomal tubules and results in misrouting of hTAC into the lysosome for degradation. Furthermore, we demonstrated a competitive relationship between SNX-3 and EEA-1 for association with PtdIns(3)P-enriched EEs. Accordingly, the loss of SNX-3 results in the merge of hTAC-laden endosomal intermediates with EEA-1-positive endosomes and ultimately entry into lysosomes. The identification of the role of SNX-3 in modulating the formation of the tubular endosomes and recycling of the ARF-6-associated CIE cargoes provides insight into the sorting and trafficking of CIE pathways.

The entry and subsequent intracellular itinerary followed by various ARF6-associated CIE cargo proteins have been investigated using the cultured mammalian cells [ 3 ]. Usually the internalized CIE proteins such as the major histocompatibility complex class I (MHCI), the GPI-anchored protein CD59, and GLUT1 join the classical SEs containing the CDE cargo transferrin receptor (TfR) and the early endosomal antigen 1 (EEA1) soon after internalization, where they are sorted for recycling or degradation [ 7 ]. However, a subset of CIE proteins, like CD44, CD98, and CD147, enter cells with MHCI and directly join the recycling tubules, by-passing the merge with classical EEA1-positive SEs [ 4 ]. The existence of these divergent itineraries suggests that the CIE cargoes are sorted at different points along the endo-lysosomal pathway. Consequently, it remains controversial as to how many independent types of recycling transport carriers are formed from endosomes and what their relative relationship is. The α-chain of the human interleukin-2 receptor (IL-2Rα, also termed hTAC) is also a classical marker of ARF6-associated CIE pathway [ 17 ]. Using the Caenorhabditis elegans (C. elegans) intestinal integrated hTAC, Grant and his colleagues have identified a set of CIE specific recycling regulators, including RAB-10, EHBP-1 and ALX-1 [ 18 – 20 ]. We previously found that a component of the exocyst complex SEC-10, in concert with RAB-10 and the microtubule cytoskeleton, plays an important role in the formation of the interconnected endosomal tubules required for efficient recycling of hTAC in the C. elegans intestine [ 21 ]. However, there is still a far way to go to fully understand the mechanisms involved in remodeling/generation of the CIE-relevant tubular endosome membrane and how this process is coordinated with selective capture of recycling cargoes.

Several protein complexes are involved in the sorting of transmembrane cargoes into the recycling/retrograde transport pathway or the degradative multivesicular endosomal pathway (MVE). Endosomal complexes required for transport (ESCRTs) are implicated in the formation and budding of intraluminal vesicles (ILVs) to recognize and sort ubiquitinated membrane proteins that arrive via the endocytic pathway [ 8 ]. The sorting nexin family of proteins (SNXs) are implicated in receptor recycling to the PM or retrieval to TGN and are characterized by the presence of a conserved phox (PX) domain, which mediates interactions with endosomal phosphoinositides (mainly phosphatidylinositol 3-phosphate, PtdIns(3)P) [ 9 , 10 ]. The evolutionarily conserved retromer, comprising a vacuolar protein-sorting trimer of VPS26-VPS29-VPS35 that is proposed to regulate cargo selection, can form alternative protein-sorting complexes with different SNX members [ 9 ]. Recent studies have documented that the SNX-BAR dimer and retromer complex function in endosome-to-TGN retrieval of CI-MPR [ 11 , 12 ]. By contrast, SNX27-retromer is implicated to recycle a variety of cell surface proteins back to the PM, including GLUT1 and many important neurotransmitter receptors, such as AMPA receptor, serotonin-4a receptor, and the β2 adrenergic receptor [ 13 ]. Interestingly, although SNX3 consists only of a PX-domain, SNX3-retromer complex is formed and involved in the retrograde transport of cargo receptors including the Wnt sorting receptor Wntless, the divalent metal ion transporter Dmt1-II, and even CI-MPR [ 14 – 16 ]. Nevertheless, when, where and by what mechanism the CIE/CDE cargo proteins are sorted from each other and into the different pathways of degradation and recycling is poorly understood.

Endocytosis and post-endocytic trafficking through the endolysosomal system are essential for the maintenance of homeostasis and signaling regulation in eukaryotic cells. After internalization, the plasma membrane (PM) lipids and proteins, as well as nutrients, are delivered to the early endosomes (EEs) or sorting endosomes (SEs). Here, cargo proteins are sorted for degradation in lysosomes or alternatively retrieved and recycled to the PM or the trans-Golgi network (TGN) for reuse [ 1 , 2 ]. There are two general types of endocytic pathways, the clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE) [ 3 ]. As increasing PM proteins are found to enter cells by CIE pathways [ 4 ], CIE mechanisms are recognized to be fundamental for many physiological processes, such as immune surveillance, cell signaling, cell migration, and metastasis [ 5 ]. However, compared to the well-established CDE pathway, our understanding of the details of CIE and post-endocytic trafficking is particularly limited [ 6 , 7 ].

Results

hTAC traffics via the ESCRT pathway for lysosomal degradation in snx-3 mutants Two opposing cargo sorting systems are located at the dynamic SEs. The ESCRT complex serves to recognize and sort ubiquitinated endosomal proteins for degradation. It is also involved in deforming the endosomal-limiting membrane inward to generate MVBs [36,37]. By contrast, the retromer complex associates with alternative SNXs and mediates the sorting and transport of various transmembrane proteins destined either to the Golgi or directly to the PM [15,38]. To clarify how the shunted cargo molecule is delivered to lysosomes, we used transgenes expressing fluorescently tagged HGRS-1/Hrs to mark the degradative ESCRT domain [8,37] and examined whether hTAC or DAF-4 proteins pass through ESCRT-positive structures to lysosomes. The Manders’ coefficients for the cytoplasmic hTAC-GFP and RFP-HGRS-1 revealed an increased colocalization of them in snx-3(tm1595) mutants; specifically, M1 increased from 0.20 in WT to 0.44 in snx-3(tm1595) mutants and M2 increased from 0.22 to 0.39 (Fig 4A and 4B). Similarly, M1 and M2 Manders’ coefficients for DAF-4-GFP and RFP-HGRS-1 increased significantly in snx-3(tm1595) mutants compared to those of WT animals (S5C and S5D Fig). The results indicated that the shunted CIE cargoes enter the lysosome via the ESCRT pathway. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. hTAC is captured by the ESCRT complex in snx-3(tm1595) mutants. (A) Confocal images showing the overlap of RFP-HGRS-1 with hTAC-GFP is increased in snx-3(tm1595) mutants. (B) Manders’ colocalization coefficients for hTAC-GFP and RFP-HGRS-1 as depicted in A were calculated, error bars are mean ± 95% CI (WT: [ROI] = 13, n = 9; snx-3(tm1595): [ROI] = 12, n = 7). ****P<0.0001 (Student’s t test). M1: green pixels overlapping red; M2: red pixels overlapping green. (C) Confocal images showing the distribution of hTAC-GFP in intestines of snx-3(tm1595) mutant, vps-4 RNAi-treated and snx-3(tm1595); vps-4(RNAi) double mutant animals. hTAC-GFP accumulated into clusters of vacuoles in the intestine of snx-3(tm1595); vps-4(RNAi) double mutants. (D) The average FIs of hTAC-GFP depicted as Z-projection images in C were calculated. Error bars are mean ± 95% CI ([ROI] = 12, n = 12). ns, not significant; ***P<0.001, **P<0.01 (Kruskal-Wallis test with Dunn’s post hoc test for multiple comparison). In A and C, the arrowheads indicate positive overlap. Asterisks depict the intestine lumen. Scale bars: 5 μm. Quantitative data are available in S1 File. https://doi.org/10.1371/journal.pgen.1009607.g004 VPS4 is a component of the AAA ATPase and is involved in the final step of MVB vesicle formation by dissociating or driving sequential polymerization of ESCRT-III [39,40]. To verify that the shunted cargo molecule is captured by the ESCRT complex for lysosomal degradation, the subcellular distribution of hTAC-GFP in snx-3(tm1595); vps-4(RNAi) double mutants was examined and compared with the individual single mutant (Fig 4C and 4D). After treatment with RNAi against vps-4, hTAC-GFP still mainly localized beneath the basolateral plasmalemma in tubular-vesicles with relative less puncta within the cytoplasma, as observed in control animals. Double depletion of SNX-3 and VPS-4, however, produced prominent clustering of hTAC-GFP-containing vacuoles in the cytoplasm (Fig 4C). In contrast to the snx-3(tm1595) mutants, the average total intensity of hTAC-GFP in the snx-3(tm1595); vps-4(RNAi) double mutants was comparable to those in WT or vps-4-RNAi treated animals (Fig 4D). These data suggest that even though the loss of SNX-3 causes hTAC to deviate from the recycling pathway, the depletion of VPS-4 further leads to the blockage of downhill flow of hTAC into the lysosome, resulting in clustered hTAC-containing structures. Taken together, these results demonstrated that the shunted CIE recycling cargo is mediated by ESCRT into the lysosome for degradation in snx-3(tm1595) mutants.

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009607

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