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

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.

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A presynaptic endosomal trafficking pathway controls synaptic growth signaling

The Journal of Cell Biology ◽

10.1083/jcb.201009052 ◽

2011 ◽

Vol 193 (1) ◽

pp. 201-217 ◽

Cited By ~ 57

Author(s):

Avital A. Rodal ◽

Aline D. Blunk ◽

Yulia Akbergenova ◽

Ramon A. Jorquera ◽

Lauren K. Buhl ◽

...

Keyword(s):

Endocytic Pathway ◽

Physical Interaction ◽

Endosomal Trafficking ◽

Synaptic Growth ◽

Recycling Endosomes ◽

Sorting Nexin ◽

Trafficking Pathway ◽

Early Endosomes ◽

Protein Receptors ◽

Escrt Complex

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)–mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.

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Retromer-driven membrane tubulation separates endosomal recycling from Rab7/Ypt7-dependent fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e16-08-0582 ◽

2017 ◽

Vol 28 (6) ◽

pp. 783-791 ◽

Cited By ~ 17

Author(s):

Latha Kallur Purushothaman ◽

Henning Arlt ◽

Anne Kuhlee ◽

Stefan Raunser ◽

Christian Ungermann

Keyword(s):

Protein Sorting ◽

Endocytic Pathway ◽

Dual Function ◽

Major Protein ◽

Tubule Formation ◽

Sorting Nexin ◽

Retromer Complex ◽

Endosomal Recycling ◽

Membrane Tubulation ◽

Reconstituted System

Endosomes are the major protein-sorting hubs of the endocytic pathway. They sort proteins destined for degradation into internal vesicles while in parallel recycling receptors via tubular carriers back to the Golgi. Tubule formation depends on the Rab7/Ypt7-interacting retromer complex, consisting of the sorting nexin dimer (SNX-BAR) and the trimeric cargo selection complex (CSC). Fusion of mature endosomes with the lysosome-like vacuole also requires Rab7/Ypt7. Here we solve a major problem in understanding this dual function of endosomal Rab7/Ypt7, using a fully reconstituted system, including purified, full-length yeast SNX-BAR and CSC, whose overall structure we present. We reveal that the membrane-active SNX-BAR complex displaces Ypt7 from cargo-bound CSC during formation of recycling tubules. This explains how a single Rab can coordinate recycling and fusion on endosomes.

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Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

Journal of Experimental Medicine ◽

10.1084/jem.177.3.583 ◽

1993 ◽

Vol 177 (3) ◽

pp. 583-596 ◽

Cited By ~ 95

Author(s):

P Romagnoli ◽

C Layet ◽

J Yewdell ◽

O Bakke ◽

R N Germain

Keyword(s):

Major Histocompatibility Complex ◽

Mhc Class Ii ◽

Class Ii ◽

Endocytic Pathway ◽

Invariant Chain ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Late Endosomes ◽

Early Endosomes ◽

Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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Abstract 18060: TRAIL Promotes Angiogenesis and Ischemia-Induced Neovascularisation via NOX4, H2O2 and Nitric Oxide-Dependent Mechanisms

Circulation ◽

10.1161/circ.130.suppl_2.18060 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Belinda A Di Bartolo ◽

Sian P Cartland ◽

Leonel Prado-Lourenco ◽

Nor Saadah M Azahri ◽

Thuan Thai ◽

...

Keyword(s):

Nitric Oxide ◽

Angiogenic Factor ◽

Tubule Formation ◽

Nitric Oxide Synthase Inhibitor ◽

Therapeutic Implications ◽

Synthase Inhibitor ◽

Oxide Synthase ◽

First Time

Background: Angiogenesis and neovascularization are essential processes that follow ischemia insults. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) not only induces endothelial cell (EC) death and inhibits angiogenesis, but also promotes EC migration, invasion and proliferation in vitro . These seemingly opposite effects make its role in angiogenesis in vivo unclear. Using TRAIL -/- and wild-type mice, we sought to determine the role of TRAIL in angiogenesis and neovascularisation. We also sought mechanisms in vitro . Methods and Results: Reduced vascularisation assessed by real-time in vivo 3D Vevo ultrasound imaging and CD31 staining was observed in TRAIL -/- mice 28 d after hindlimb ischemia. Moreover, reduced capillary formation and increased apoptosis was evident in TRAIL -/- muscles even at 3 d after ischemic surgery. We have previously shown that fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, regulates TRAIL gene expression in vascular smooth muscle cells. Indeed, FGF-2 also regulates TRAIL expression in ECs, and FGF-2-inducible proliferation, migration and tubule formation was inhibited with siRNA targeting TRAIL. Notably, both FGF-2 and TRAIL significantly increased NOX4 expression. TRAIL-inducible angiogenic activity in ECs was inhibited with siRNAs targeting NOX4, and consistent with these, NOX4 mRNA was reduced in 3 d ischemic hindlimbs of TRAIL -/- mice. TRAIL stimulated intracellular H 2 O 2 levels in ECs, and TRAIL-inducible proliferation, migration and tubule formation was inhibited with not only PEG-catalase, a H 2 O 2 scavenger, but also blocked with L-NAME, a nitric oxide synthase inhibitor. Conclusions: This is the first demonstration showing that TRAIL promotes angiogenesis in vivo . We show for the first time that the TRAIL stimulates NOX4 expression to mediate nitric oxide-dependent angiogenic effects. This has significant therapeutic implications such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with CVD and diabetes.

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A Role for Sorting Nexin 2 in Epidermal Growth Factor Receptor Down-regulation: Evidence for Distinct Functions of Sorting Nexin 1 and 2 in Protein Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0711 ◽

2004 ◽

Vol 15 (5) ◽

pp. 2143-2155 ◽

Cited By ~ 74

Author(s):

Anuradha Gullapalli ◽

Tiana A. Garrett ◽

May M. Paing ◽

Courtney T. Griffin ◽

Yonghua Yang ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Protein Trafficking ◽

Growth Factor Receptor ◽

Down Regulation ◽

Sorting Nexin ◽

Px Domain ◽

Early Endosomes ◽

Epidermal Growth

Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps)5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.

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Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Molecular Biology of the Cell ◽

10.1091/mbc.10.9.2891 ◽

1999 ◽

Vol 10 (9) ◽

pp. 2891-2904 ◽

Cited By ~ 35

Author(s):

Valérie Brachet ◽

Gérard Péhau-Arnaudet ◽

Catherine Desaymard ◽

Graça Raposo ◽

Sebastian Amigorena

Keyword(s):

Mhc Class Ii ◽

Class Ii ◽

Endocytic Pathway ◽

Cross Linking ◽

Histocompatibility Complex ◽

Early Endosomes ◽

Peptide Loading ◽

Golgi Network ◽

Mhc Class Ii Molecules

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

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The Alzheimer's gene SORL1 is a key regulator of endosomal recycling in human neurons

10.1101/2021.07.26.453861 ◽

2021 ◽

Author(s):

Swati Mishra ◽

Allison Knupp ◽

Marcell Szabo ◽

Chizuru Kinoshita ◽

Dale W. Hailey ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cell Surface ◽

Induced Pluripotent Stem Cell ◽

Endosomal Trafficking ◽

Altered Expression ◽

Therapeutic Implications ◽

Endosomal Recycling ◽

Human Neurons ◽

Early Endosomes

Background: Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer's disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD's cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1). Methods: We used isogenic hiPSCs engineered to have SORL1 depleted or to have enhanced SORL1 expression. We differentiated neurons from these cell lines and mapped the trafficking of APP, TRKB and GLUA1 within the endosomal network using confocal microscopy. We also performed cell surface recycling and lysosomal degradation assays to assess the functionality of the endosomal network. Finally, we analyzed alterations in gene expression in SORL1 depleted neurons using RNA-sequencing. Results: We find that as with APP, endosomal trafficking of GLUA1 and TRKB is impaired by loss of SORL1. Conversely, increased SORL1 expression enhances endosomal recycling for APP and GLUA1. Our unbiased transcriptomic data further support SORL1's role in endosomal recycling. We observe altered expression networks that regulate cell surface trafficking and neurotrophic signaling Conclusion: Collectively, and together with other recent observations, these findings suggest that SORL1 is a key and broad regulator of retromer-dependent endosomal recycling in neurons, a conclusion that has both pathogenic and therapeutic implications for Alzheimer's disease.

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation

10.1101/2021.10.03.21264281 ◽

2021 ◽

Author(s):

Franziska Paul ◽

Calista Ng ◽

Shahriar Nafissi ◽

Yalda Nilipoor ◽

Ali Reza Tavasoli ◽

...

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Muscle Weakness ◽

Germline Mutations ◽

Lysosomal Degradation ◽

Mendelian Disorder ◽

Progressive Muscle Weakness ◽

Endosomal Recycling ◽

Early Endosomes ◽

Recycling Pathway

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively-acting germline alleles p.Arg180Gly and p.Gly183Arg which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate PI3P and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts exhibit accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

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Codon usage and protein sequence pattern dependency in different organisms: A Bioinformatics approach

Journal of Bioinformatics and Computational Biology ◽

10.1142/s021972001550002x ◽

2015 ◽

Vol 13 (02) ◽

pp. 1550002

Author(s):

Mohammad-Hadi Foroughmand-Araabi ◽

Bahram Goliaei ◽

Kasra Alishahi ◽

Mehdi Sadeghi ◽

Sama Goliaei

Keyword(s):

Gene Regulation ◽

Codon Usage ◽

Protein Sequence ◽

Multinomial Logistic Regression ◽

Translation Efficiency ◽

Translation Rate ◽

Protein Levels ◽

Synonymous Codons ◽

First Time

Although it is known that synonymous codons are not chosen randomly, the role of the codon usage in gene regulation is not clearly understood, yet. Researchers have investigated the relation between the codon usage and various properties, such as gene regulation, translation rate, translation efficiency, mRNA stability, splicing, and protein domains. Recently, a universal codon usage based mechanism for gene regulation is proposed. We studied the role of protein sequence patterns on the codons usage by related genes. Considering a subsequence of a protein that matches to a pattern or motif, we showed that, parts of the genes, which are translated to this subsequence, use specific ratios of synonymous codons. Also, we built a multinomial logistic regression statistical model for codon usage, which considers the effect of patterns on codon usage. This model justifies the observed codon usage preference better than the classic organism dependent codon usage. Our results showed that the codon usage plays a role in controlling protein levels, for genes that participate in a specific biological function. This is the first time that this phenomenon is reported.

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