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 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 Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

2002 ◽  
Vol 83 (3) ◽  
pp. 611-621 ◽  
Author(s):  
Gaie Brown ◽  
James Aitken ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Filamentous Form ◽  
Caveolin 1 ◽  
Immuno Electron Microscopy ◽  
Infected Cells ◽  
Syncytial Virus

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

scholarly journals Cell Surface Orifices of Caveolae and Localization of Caveolin to the Necks of Caveolae in Adipocytes

2003 ◽  
Vol 14 (10) ◽  
pp. 3967-3976 ◽  
Author(s):  
Hans Thorn ◽  
Karin G. Stenkula ◽  
Margareta Karlsson ◽  
Unn Örtegren ◽  
Fredrik H. Nystrom ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Human Fibroblasts ◽  
High Resolution Electron Microscopy ◽  
Proximal Region ◽  
Cholesterol Depletion ◽  
Caveolin 1 ◽  
Resolution Electron ◽  
20 Nm ◽  
Membrane Proximal Region

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.

scholarly journals Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Qiong Deng ◽  
Yong Wu ◽  
Zeng Zhang ◽  
Yue Wang ◽  
Minghua Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Androgen Receptor ◽  
Signaling Pathway ◽  
Membrane Trafficking ◽  
Membrane Fraction ◽  
Cysteine Residue ◽  
Membrane Localization ◽  
Physiological Concentration ◽  
Caveolin 1 ◽  
New Evidence

The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR) localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

scholarly journals Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes

2005 ◽  
Vol 387 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rosaria COSTA ◽  
Derek T. WARREN ◽  
Kathryn R. AYSCOUGH
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Active Form ◽  
Actin Dynamics ◽  
Binding Motif ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Vacuolar Protein ◽  
First Time

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, γ-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a γ-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.

scholarly journals Intracellular Retention of Glycosylphosphatidyl Inositol-Linked Proteins in Caveolin-Deficient Cells

2002 ◽  
Vol 22 (11) ◽  
pp. 3905-3926 ◽  
Author(s):  
Federica Sotgia ◽  
Babak Razani ◽  
Gloria Bonuccelli ◽  
William Schubert ◽  
Michela Battista ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Recombinant Expression ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Null Mouse ◽  
Tissue Samples ◽  
Caveolin 1 ◽  
Glycosylphosphatidyl Inositol

ABSTRACT The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a ∼95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

scholarly journals The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells

2019 ◽  
Author(s):  
Cyril Legrand ◽  
Roberto Saleppico ◽  
Jana Sticht ◽  
Fabio Lolicato ◽  
Hans-Michael Müller ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Cell Surface ◽  
Fibroblast Growth Factor ◽  
Tumor Cells ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Membrane Translocation ◽  
Unconventional Secretion ◽  
Α1 Subunit

SummaryFibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the α1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with α1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that α1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

Endothelial Cell Protein C Receptor Cellular Localization and Trafficking

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1015-1015 ◽  
Author(s):  
Ramesh Nayak ◽  
Prosenjit Sen ◽  
Charles Esmon ◽  
Usha Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Plasma Membrane ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Protein C ◽  
Cellular Localization ◽  
Model Systems ◽  
Factor Vii ◽  
Cell Protein ◽  
Mutant Form ◽  
Caveolin 1

Abstract Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Recent studies from others and us showed that EPCR also acts as a cellular binding site for factor VII (FVII) and activated factor VII (FVIIa). Although much is know about the biochemistry and pathophysiological importance of EPCR, little is know how EPCR interaction with its ligands on cell surfaces affects its expression and facilitate internalization of the ligands. The present study was undertaken to characterize cellular localization and trafficking of EPCR and investigate how FVIIa or APC binding to EPCR influences these processes. The studies employed two cell model systems - primary cultured human umbilical endothelial cells (HUVEC) and CHO cells stably transfected with EPCR. Cellular localization of EPCR and its trafficking was analyzed by immunofluorescence confocal microscopy or monitoring the expression of EPCR tagged with green fluorescence protein (GFP). EPCR endocytosis was evaluated by biotinylation of cell surface proteins with NHS-SS- biotin, followed by monitoring the protection of biotinylated EPCR from a membrane-impermeable reducing agent. FVIIa and APC internalization and recycling was evaluated by monitoring the uptake/release of 125I-labeled ligands or following the intracellular routing of fluorescent dye (AF488)-conjugated ligands added to EPCR expressing cells. Data from these studies showed that a majority of EPCR is localized on the cell surface and distributed in a patchy and punctuate manner. Immunostaining of HUVEC and CHO-EPCR cells with EPCR mAb and caveolin-1 antibodies showed a high degree of co-localization of EPCR and caveolin-1. Depletion of cholesterol from the plasma membrane, which disrupts caveolae, by ß methyl cyclodextrin reduced the extent EPCR and caveloin-1 colocalization. These data indicate EPCR on the cell surface predominantly localizes in caveolae. Inside the cell, EPCR is mainly localized in a small perinuclear structure, which is the site of centrosome. Colocalization of EPCR with tubulin (a marker for centrosome) and rab 11 (a marker of recycling compartment, REC) revealed that EPCR is localized actually in the REC and not in the centrosome. A small fraction of EPCR is also localized in endosomes as evident from colocalization of EPCR with EEA1 and Rab5, early endosome markers. Chasing the cell surface biotinylated proteins showed no significant increase in the biotinylated EPCR in the intracellular pool of proteins, which indicate that EPCR is not actively endocytosed constitutively or that the internalized EPCR is immediately recycled back to the cell surface. FVIIa or APC binding to EPCR promoted the EPCR endocytosis. The endocytosed receptors were first observed in proximity of the plasma membrane after 10 min and by 30 to 60 min most of the endocytosed EPCR was accumulated in the REC. The internalized FVIIa or APC appeared to follow the same route of the endocytosed EPCR. Proteolytically inactive FVIIa or APC behaved same as FVIIa or APC in promoting EPCR endocytosis and its trafficking. EPCR-dependent FVIIa or APC internalization is a dynamin-dependent process as the inhibition of the GTPase activity of dynamin by a specific inhibitor (Dynasore) completely abrogated their internalization and accumulation in the REC. Additional studies revealed that disruption of coated-pit pathway by potassium depletion blocked the endocytosis of transferrin, a classic marker for endocytosis via clathrin-dependent coated-pit pathway, but had no effect on EPCR-dependent FVIIa or APC internalization. In contrast, disruption of caveolae by cholesterol depletion blocked the internalization of FVIIa but not transferrin. Rab11 dominant negative mutant form (S25N) prevented the passage of the endocytosed EPCR or the internalized FVIIa or APC into the REC. After peaking at 30 min, the amount of both the ligands and the receptor in the REC gradually decreased, and some of the internalized ligand re-appeared at the cell surface. Overall the data provided herein suggest that FVIIa or APC binding to EPCR, independent of their protease activity, promotes EPCR endocytosis via the dynamin-dependent caveolar pathway and the activation of rab11 by GTP is required for exit of the endocytosed receptor or the ligands from sorting endosomes to the recycling compartment.

scholarly journals MICAL-L1 is required for cargo protein delivery to the cell surface

Biology Open ◽  
2021 ◽  
Vol 10 (6) ◽  
Author(s):  
R. Sikora ◽  
P. Bun ◽  
L. Danglot ◽  
M. Alqabandi ◽  
P. Bassereau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Trafficking ◽  
Protein Delivery ◽  
Close Association ◽  
Super Resolution ◽  
Small Gtpase ◽  
C Terminus ◽  
Membrane Tubulation

ABSTRACT Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Syntaxin 1A inhibits regulated CFTR trafficking inXenopus oocytes

1999 ◽  
Vol 277 (1) ◽  
pp. C174-C180 ◽  
Author(s):  
Kathryn W. Peters ◽  
Juanjuan Qi ◽  
Simon C. Watkins ◽  
Raymond A. Frizzell
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Functional Expression ◽  
Membrane Capacitance ◽  
K Channel ◽  
Syntaxin 1A ◽  
Viral Hemagglutinin ◽  
Hemagglutinin Protein

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302–305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin’s effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

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