Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors

ABSTRACT The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.

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Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The Journal of Cell Biology ◽

10.1083/jcb.200804048 ◽

2008 ◽

Vol 183 (3) ◽

pp. 513-526 ◽

Cited By ~ 262

Author(s):

Raul Rojas ◽

Thijs van Vlijmen ◽

Gonzalo A. Mardones ◽

Yogikala Prabhu ◽

Adriana L. Rojas ◽

...

Keyword(s):

Cathepsin D ◽

Retrograde Transport ◽

Phosphatidylinositol 3 Kinase ◽

Sorting Nexin ◽

Retromer Complex ◽

Endosomal Membranes ◽

Guanosine Triphosphatase ◽

Vacuolar Protein ◽

Golgi Network ◽

Phosphatidylinositol 3

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

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Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

The Journal of Cell Biology ◽

10.1083/jcb.201702137 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3677-3693 ◽

Cited By ~ 60

Author(s):

Arunas Kvainickas ◽

Ana Jimenez-Orgaz ◽

Heike Nägele ◽

Zehan Hu ◽

Jörn Dengjel ◽

...

Keyword(s):

Quantitative Proteomics ◽

Retrograde Transport ◽

Dependent Manner ◽

Sorting Nexin ◽

Trans Golgi Network ◽

The Core ◽

Retromer Complex ◽

Mannose 6 Phosphate ◽

Golgi Network

The retromer complex, which recycles the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), is thought to consist of a cargo-selective VPS26–VPS29–VPS35 trimer and a membrane-deforming subunit of sorting nexin (SNX)–Bin, Amphyphysin, and Rvs (BAR; SNX-BAR) proteins. In this study, we demonstrate that heterodimers of the SNX-BAR proteins, SNX1, SNX2, SNX5, and SNX6, are the cargo-selective elements that mediate the retrograde transport of CI-MPR from endosomes to the TGN independently of the core retromer trimer. Using quantitative proteomics, we also identify the IGF1R, among more potential cargo, as another SNX5 and SNX6 binding receptor that recycles through SNX-BAR heterodimers, but not via the retromer trimer, in a ligand- and activation-dependent manner. Overall, our data redefine the mechanics of retromer-based sorting and call into question whether retromer indeed functions as a complex of SNX-BAR proteins and the VPS26–VPS29–VPS35 trimer.

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The retromer complex regulates C. elegans development and mammalian ciliogenesis

10.1101/2021.09.24.461716 ◽

2021 ◽

Author(s):

Shuwei Xie ◽

Ellie Smith ◽

Carter Dierlam ◽

Danita Mathew ◽

Angelina Davis ◽

...

Keyword(s):

Potential Function ◽

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Vulval Development ◽

Sorting Nexin ◽

Capping Protein ◽

C Elegans ◽

Retromer Complex ◽

Mother Centriole

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

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A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking

Journal of Virology ◽

10.1128/jvi.01499-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10145-10155 ◽

Cited By ~ 36

Author(s):

David Pim ◽

Justyna Broniarczyk ◽

Martina Bergant ◽

Martin P. Playford ◽

Lawrence Banks

Keyword(s):

Pdz Domain ◽

Endosomal Trafficking ◽

Sorting Nexins ◽

Human Papillomavirus 16 ◽

Sorting Nexin ◽

Papillomavirus Infection ◽

Infection Dynamics ◽

And Function ◽

Golgi Network ◽

Interfering Rna

ABSTRACTPrevious studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.IMPORTANCEThe trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.

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Retromer revisited: Evolving roles for retromer in endosomal sorting

The Journal of Cell Biology ◽

10.1083/jcb.201708111 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3433-3436 ◽

Cited By ~ 7

Author(s):

John P. Chamberland ◽

Brigitte Ritter

Keyword(s):

Mammalian Cells ◽

Endosomal Sorting ◽

Trans Golgi Network ◽

Retromer Complex ◽

Cell Biol ◽

The Individual ◽

Golgi Network

The highly conserved retromer complex has been linked to cargo retrieval from endosomes to the trans-Golgi network. In this issue, Kvainickas et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201702137) and Simonetti et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201703015) fundamentally question the current retromer model and demonstrate that in mammalian cells, the individual retromer subcomplexes have functionally diverged to organize multiple distinct sorting pathways.

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GGA proteins: new players in the sorting game

Journal of Cell Science ◽

10.1242/jcs.114.19.3413 ◽

2001 ◽

Vol 114 (19) ◽

pp. 3413-3418 ◽

Cited By ~ 1

Author(s):

Annette L. Boman

Keyword(s):

Membrane Trafficking ◽

Sequence Homology ◽

Mammalian Cells ◽

Gene Knockout ◽

Protein Domains ◽

Vesicle Formation ◽

Trans Golgi Network ◽

And Function ◽

Golgi Network ◽

Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

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Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

Journal of Cell Science ◽

10.1242/jcs.113.11.1993 ◽

2000 ◽

Vol 113 (11) ◽

pp. 1993-2002 ◽

Cited By ~ 5

Author(s):

H. Cao ◽

H.M. Thompson ◽

E.W. Krueger ◽

M.A. McNiven

Keyword(s):

Mammalian Cells ◽

Living Cells ◽

Dominant Negative ◽

Coated Vesicle ◽

Large Numbers ◽

Golgi Structure ◽

And Function ◽

Golgi Network ◽

Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

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Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

Scientific Reports ◽

10.1038/srep03362 ◽

2013 ◽

Vol 3 (1) ◽

Cited By ~ 17

Author(s):

Divyanshu Mahajan ◽

Boon Kim Boh ◽

Yan Zhou ◽

Li Chen ◽

Tobias Carl Cornvik ◽

...

Keyword(s):

Mammalian Cells ◽

Active Form ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Vitro Assay ◽

Guanine Nucleotide Exchange ◽

And Function ◽

Golgi Network

Abstract Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

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Nradd Acts as a Negative Feedback Regulator of Wnt/β-Catenin Signaling and Promotes Apoptosis

Biomolecules ◽

10.3390/biom11010100 ◽

2021 ◽

Vol 11 (1) ◽

pp. 100

Author(s):

Ozgun Ozalp ◽

Ozge Cark ◽

Yagmur Azbazdar ◽

Betul Haykir ◽

Gokhan Cucun ◽

...

Keyword(s):

Wnt Signaling ◽

Mammalian Cells ◽

Genetic Disorders ◽

Negative Regulator ◽

Neurotrophin Receptor ◽

P75 Neurotrophin Receptor ◽

Death Domain ◽

C Terminus ◽

And Function ◽

Functional Analyses

Wnt/β-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/β-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/β-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/β-catenin signaling during development.

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Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies

PLoS ONE ◽

10.1371/journal.pone.0236538 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0236538

Author(s):

David J. Vance ◽

Amanda Y. Poon ◽

Nicholas J. Mantis

Keyword(s):

Monoclonal Antibodies ◽

Mammalian Cells ◽

Type I ◽

Dependent Manner ◽

Type Ii ◽

Single Chain ◽

Carbohydrate Recognition Domains ◽

And Function ◽

Golgi Network ◽

Dose Dependent

Ricin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.

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