scholarly journals Regulation of the endosomal SNX27-retromer by OTULIN

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Aurelia Stangl ◽  
Paul R. Elliott ◽  
Adan Pinto-Fernandez ◽  
Sarah Bonham ◽  
Luke Harrison ◽  
...  
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Pdz Domain ◽  
Binding Motif ◽  
Ubiquitin Chain ◽  
Sorting Nexin ◽  
Catalytic Function ◽  
Retromer Complex ◽  
Mass Spectrometric Identification ◽  
Affinity Interaction

Abstract OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

scholarly journals An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking

2009 ◽  
Vol 418 (2) ◽  
pp. 345-367 ◽  
Author(s):  
Heather L. Wieman ◽  
Sarah R. Horn ◽  
Sarah R. Jacobs ◽  
Brian J. Altman ◽  
Sally Kornbluth ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Pdz Domain ◽  
Binding Motif ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
Growth Factor Regulation

Cell surface localization of the Glut (glucose transporter), Glut1, is a cytokine-controlled process essential to support the metabolism and survival of haemopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. In the present study, we show that a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ-domain protein, GIPC (Gα-interacting protein-interacting protein, C-terminus), bound to Glut1 in part via the Glut1 C-terminal PDZ-binding motif, and we found that GIPC deficiency decreased Glut1 surface levels and glucose uptake. Unlike the Glut1 degradation observed on mutation of the Glut1 PDZ-binding domain, however, GIPC deficiency resulted in accumulation of intracellular Glut1 in a pool distinct from the recycling pathway of the TfR (transferrin receptor). Blockade of Glut1 lysosomal targeting after growth factor withdrawal also led to intracellular accumulation of Glut1, a portion of which could be rapidly restored to the cell surface after growth factor stimulation. These results indicate that the C-terminal PDZ-binding motif of Glut1 plays a key role in growth factor regulation of glucose uptake by both allowing GIPC to promote Glut1 trafficking to the cell surface and protecting intracellular Glut1 from lysosomal degradation after growth factor withdrawal, thus allowing the potential for a rapid return of intracellular Glut1 to the cell surface on restimulation.

scholarly journals SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry

2022 ◽  
Vol 119 (4) ◽  
pp. e2117576119
Author(s):  
Bo Yang ◽  
Yuanyuan Jia ◽  
Yumin Meng ◽  
Ying Xue ◽  
Kefang Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Viral Entry ◽  
Complex Structure ◽  
Pdz Domain ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Binding Motif ◽  
Type I

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27–retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

scholarly journals Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

2008 ◽  
Vol 182 (6) ◽  
pp. 1153-1169 ◽  
Author(s):  
Ting Cai ◽  
Haojie Wang ◽  
Yiliang Chen ◽  
Lijun Liu ◽  
William T Gunning ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Long Range ◽  
Membrane Trafficking ◽  
Endocytic Trafficking ◽  
Binding Motif ◽  
Caveolin 1 ◽  
Pumping Function ◽  
Α1 Subunit

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

scholarly journals Characterization of the interaction between β-catenin and sorting nexin 27: contribution of the type I PDZ-binding motif to Wnt signaling

2019 ◽  
Vol 39 (11) ◽  
Author(s):  
Brian J. DuChez ◽  
Christina L. Hueschen ◽  
Seth P. Zimmerman ◽  
Yvonne Baumer ◽  
Stephen Wincovitch ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Collecting Duct ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Type I ◽  
Cortical Collecting Duct ◽  
Fusion Construct ◽  
Sorting Nexin ◽  
Early Endosomes

Abstract Background: Sorting Nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain common among all of the sorting nexin family, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27–PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells (mpkCCD) using a GST-SNX27 fusion construct as bait. We found that the C-terminal type I PDZ binding motif (DTDL) of β-catenin, an adherens junction scaffolding protein and transcriptional co-activator, interacts directly with SNX27. Using biochemical and immunofluorescent techniques, β-catenin was identified in endosomal compartments where co-localization with SNX27 was observed. Furthermore, E-cadherin, but not Axin, GSK3 or Lef-1 was located in SNX27 protein complexes. While overexpression of wild-type β-catenin protein increased TCF-LEF dependent transcriptional activity, an enhanced transcriptional activity was not observed in cells expressing β-Catenin ΔFDTDL or diminished SNX27 expression. These results imply importance of the C-terminal PDZ binding motif for the transcriptional activity of β-catenin and propose that SNX27 might be involved in the assembly of β-catenin complexes in the endosome.

scholarly journals Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

2018 ◽  
Vol 115 (14) ◽  
pp. 3716-3721 ◽  
Author(s):  
Yuliya Salanenka ◽  
Inge Verstraeten ◽  
Christian Löfke ◽  
Kaori Tabata ◽  
Satoshi Naramoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Trafficking ◽  
Molecular Mechanisms ◽  
Sorting Nexin ◽  
Protein Traffic ◽  
Della Proteins ◽  
Retromer Complex ◽  
Ga Signaling ◽  
Multiple Processes

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses.

scholarly journals Sorting Nexin 27 (SNX27): A Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking

2018 ◽  
Author(s):  
Mark I. McDermott ◽  
William R. Thelin ◽  
Yun Chen ◽  
Patrick T. Lyons ◽  
Gabrielle Reilly ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Pdz Domain ◽  
Type I ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sorting Nexin ◽  
Cystic Fibrosis Transmembrane

AbstractThe underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

scholarly journals Sorting nexin 27 (SNX27) associates with zonula occludens-2 (ZO-2) and modulates the epithelial tight junction

2013 ◽  
Vol 455 (1) ◽  
pp. 95-106 ◽  
Author(s):  
Seth P. Zimmerman ◽  
Christina L. Hueschen ◽  
Daniela Malide ◽  
Sharon L. Milgram ◽  
Martin P. Playford
Keyword(s):  
Tight Junction ◽  
Collecting Duct ◽  
Pdz Domain ◽  
Binding Motif ◽  
Sorting Nexin ◽  
Zonula Occludens ◽  
Proteomic Approach ◽  
Early Endosomes ◽  
Junction Protein ◽  
Epithelial Tight Junction

Proteins of the SNX (sorting nexin) superfamily are characterized by the presence of a PX (Phox homology) domain and associate with PtdIns3P (phosphatidylinositol-3-monophosphate)-rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. In the present study, we used a proteomic approach to identify a novel interaction between SNX27 and ZO-2 [zonula occludens-2; also known as TJP2 (tight junction protein 2)], a component of the epithelial tight junction. The SNX27–ZO-2 interaction requires the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2. When tight junctions were perturbed by chelation of extracellular Ca2+, ZO-2 transiently localized to SNX27-positive early endosomes. Depletion of SNX27 in mpkCCD (mouse primary kidney cortical collecting duct) cell monolayers resulted in a decrease in the rate of ZO-2, but not ZO-1, mobility at cell–cell contact regions after photobleaching and an increase in junctional permeability to large solutes. The findings of the present study identify an important new SNX27-binding partner and suggest a role for endocytic pathways in the intracellular trafficking of ZO-2 and possibly other tight junction proteins. Our results also indicate a role for SNX27–ZO-2 interactions in tight junction maintenance and function.

scholarly journals Cell Surface Heparan Sulfate Proteoglycan Syndecan-2 Induces the Maturation of Dendritic Spines in Rat Hippocampal Neurons

1999 ◽  
Vol 144 (3) ◽  
pp. 575-586 ◽  
Author(s):  
Iryna M. Ethell ◽  
Yu Yamaguchi
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Pdz Domain ◽  
Heparan Sulfate Proteoglycan ◽  
Structural Basis ◽  
Binding Motif ◽  
Sulfate Proteoglycan ◽  
Pdz Domain Proteins

Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

scholarly journals CFTR-associated ligand is a negative regulator of Mrp2 expression

2017 ◽  
Vol 312 (1) ◽  
pp. C40-C46 ◽  
Author(s):  
Man Li ◽  
Carol J. Soroka ◽  
Kathy Harry ◽  
James L. Boyer
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Posttranscriptional Regulation ◽  
Pdz Domain ◽  
Rat Hepatocytes ◽  
Surface Expression ◽  
Negative Regulator ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Sodium Hydrogen Exchanger

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.

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