Phosphatidylinositol 4,5-bisphosphate directly interacts with the β and γ subunits of the sodium channel ENaC

The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na+ channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, remains obscure. To date, possible interaction of PIP2 with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady-state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the N termini of β- (Kd ∼5.2 μm) and γ-ENaC (Kd ∼13 μm). A weaker interaction site within the carboxyl terminus of γ-ENaC (Kd ∼800 μm) was also observed. These results support that PIP2 regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.

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A model for the extracellular release of PAF: The influence of plasma membrane phospholipid asymmetry

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(91)90330-b ◽

1991 ◽

Vol 1062 (1) ◽

pp. 24-34 ◽

Cited By ~ 22

Author(s):

Donna L. Bratton ◽

Jenai M. Kailey ◽

Keith L. Clay ◽

Peter M. Henson

Keyword(s):

Plasma Membrane ◽

Membrane Phospholipid ◽

Phospholipid Asymmetry ◽

Extracellular Release ◽

Plasma Membrane Phospholipid

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Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)99052-6 ◽

1991 ◽

Vol 266 (18) ◽

pp. 11975-11979

Author(s):

F.C. Church ◽

J.E. Phillips ◽

J.L. Woods

Keyword(s):

Synthetic Peptides ◽

Carboxyl Terminus ◽

Peptide Characterization

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Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

Journal of Virology ◽

10.1128/jvi.02000-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 20

Author(s):

Jolene Ramsey ◽

Emily C. Renzi ◽

Randy J. Arnold ◽

Jonathan C. Trinidad ◽

Suchetana Mukhopadhyay

Keyword(s):

Plasma Membrane ◽

Sindbis Virus ◽

Molecular Mechanisms ◽

Wild Type Virus ◽

Membrane Localization ◽

Clear Understanding ◽

Wild Type ◽

Plasma Membrane Localization ◽

C Terminus ◽

N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

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Functional Communication between IP3R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

10.1101/2021.09.24.461545 ◽

2021 ◽

Author(s):

Moaz Ahmad ◽

Hwei Ling Ong ◽

Hassan Saadi ◽

Ga-Yeon Son ◽

Zahra Shokatian ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Functional Communication ◽

Functional Link ◽

N Terminus

STIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)-plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays pre-clustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that is mediated by ambient activity of IP3R and sensed by the STIM2 N-terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at sub-threshold stimuli with activation of Ca2+ entry.

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Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting

Journal of Virology ◽

10.1128/jvi.73.4.2604-2612.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 2604-2612 ◽

Cited By ~ 117

Author(s):

Jean-Christophe Paillart ◽

Heinrich G. Göttlinger

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Assembly ◽

Membrane Binding ◽

Single Amino Acid ◽

Immunodeficiency Virus ◽

N Terminus

ABSTRACT Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55 gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.

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What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

Microscopy and Microanalysis ◽

10.1017/s1431927600031226 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1030-1031

Author(s):

J.M. Robinson

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Biochemical Characterization ◽

Cell Types ◽

Membrane Microdomains ◽

Truncated Form ◽

Coated Pits ◽

N Terminus ◽

Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

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Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

Molecular Biology of the Cell ◽

10.1091/mbc.e10-03-0256 ◽

2011 ◽

Vol 22 (2) ◽

pp. 189-201 ◽

Cited By ~ 40

Author(s):

Roman Gorelik ◽

Changsong Yang ◽

Vasumathi Kameswaran ◽

Roberto Dominguez ◽

Tatyana Svitkina

Keyword(s):

Plasma Membrane ◽

Electrostatic Interactions ◽

Coiled Coil ◽

Small Gtpases ◽

Coincidence Detection ◽

Membrane Targeting ◽

Amino Terminal ◽

N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

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Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Blood ◽

10.1182/blood.v73.5.1226.1226 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1226-1234 ◽

Cited By ~ 39

Author(s):

C Legrand ◽

V Dubernard ◽

AT Nurden

Keyword(s):

Monoclonal Antibody ◽

Plasma Membrane ◽

Synthetic Peptides ◽

Divalent Cations ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Plasma Fibrinogen ◽

Fab Fragments ◽

Polymer Formation ◽

Activated Platelets

Abstract Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400–411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.

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