scholarly journals The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 986
Author(s):  
Monique D. Appelman ◽  
Marion J.D. Robin ◽  
Esther W.M. Vogels ◽  
Christie Wolzak ◽  
Winnie G. Vos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bile Salt ◽  
Protein Interactions ◽  
Basolateral Membrane ◽  
Sodium Taurocholate ◽  
Salt Transport ◽  
Protein Protein Interactions ◽  
Cell Surface Biotinylation ◽  
Detergent Resistant Membranes ◽  
Entry Receptor

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein–protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.

scholarly journals N-Terminal Protein Acylation Confers Localization to Cholesterol, Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae

2001 ◽  
Vol 12 (11) ◽  
pp. 3601-3617 ◽  
Author(s):  
James B. McCabe ◽  
Luc G. Berthiaume
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Protein Interactions ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Fatty Acylation ◽  
Detergent Resistant Membranes

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM1, but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM1at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62c-yesand caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.

scholarly journals The NHR1 Domain of Neuralized Binds Delta and Mediates Delta Trafficking and Notch Signaling

2007 ◽  
Vol 18 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Cosimo Commisso ◽  
Gabrielle L. Boulianne
Keyword(s):  
Plasma Membrane ◽  
Notch Signaling ◽  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Neural Development ◽  
Ring Domain ◽  
Protein Protein Interactions ◽  
Necessary And Sufficient ◽  
Conserved Residue ◽  
Notch Ligands

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized1, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein–protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.

Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

2003 ◽  
Vol 284 (6) ◽  
pp. C1633-C1644 ◽  
Author(s):  
Mohammed A. Khadeer ◽  
Zhihui Tang ◽  
Harriet S. Tenenhouse ◽  
Maribeth V. Eiden ◽  
Heini Murer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bone Resorption ◽  
Protein Interactions ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Basolateral Membrane ◽  
Atp Synthesis ◽  
Cellular Distribution ◽  
Green Fluorescent ◽  
Carboxy Terminal

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.

scholarly journals Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

2016 ◽  
Vol 90 (9) ◽  
pp. 4544-4555 ◽  
Author(s):  
Marilia Barros ◽  
Frank Heinrich ◽  
Siddhartha A. K. Datta ◽  
Alan Rein ◽  
Ioannis Karageorgos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Matrix Protein ◽  
Full Length ◽  
Protein Protein Interactions ◽  
Binding Studies ◽  
Protein Protein Interaction ◽  
Gag Polyprotein ◽  
Protein Lipidation ◽  
Hiv 1

ABSTRACTBy assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities.IMPORTANCELike other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.

Identification of taurocholate binding sites in ileal plasma membrane

1990 ◽  
Vol 259 (3) ◽  
pp. G394-G401
Author(s):  
F. R. Simon ◽  
J. Sutherland ◽  
E. Sutherland
Keyword(s):  
Plasma Membrane ◽  
Bile Salt ◽  
Bile Acids ◽  
Brush Border ◽  
Binding Sites ◽  
Alkylating Agents ◽  
Basolateral Membrane ◽  
Specific Marker ◽  
Small Intestinal Mucosa ◽  
Marker Enzyme

Intestinal absorption of bile salts occurs by passive processes throughout the length of the small intestine, whereas active carrier-mediated uptake is localized to the ileum. Although previous studies have extensively characterized brush-border transport of bile acids, their extrusion across the basolateral membrane is less well understood. Because previous reports had failed to show specific bile acid binding sites except with the use of photolabeled bile salt derivatives, we sought to identify and characterize the binding parameters of the physiological bile salt taurocholate in ileal and jejunal plasma membrane subfractions. Brush-border membrane (BBM) and basolateral membrane (BLM) fractions were rapidly and simultaneously isolated from the small intestinal mucosa. BBM fractions were isolated with enrichments of 50- to 54-fold for leucine aminopeptidase, whereas the basolateral membrane enrichment of Na(+)-K(+)-ATPase, its specific marker enzyme, was 22- to 25-fold. Contamination from intracellular organelles was minimal. Binding of [14C]taurocholate was demonstrated in both jejunal as well as ileal plasma membrane fractions. However, only ileal binding demonstrated saturation, reversibility, and susceptibility to proteolytic enzymes. [14C]taurocholate binding to BBM fractions also showed competition with bile acids but was not altered by pH or alkylating agents. In contrast, binding of taurocholate to the basolateral membrane showed optimal pH between 6.5 and 7.5 and was inhibited by thiol and alkylating agents. Kinetic analysis of specific ileal BBM and BLM binding showed the parameters for BBM as 288 +/- 70 microM and 2.4 +/- 0.6 nmol/mg protein and for BLM as 6.6 +/- 0.7 microM and 0.56 +/- 0.01 nmol/mg protein for dissociation constant and maximum binding capacity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-protein interactions and membrane localization of the human organic solute transporter

2007 ◽  
Vol 292 (6) ◽  
pp. G1586-G1593 ◽  
Author(s):  
An-Qiang Sun ◽  
Natarajan Balasubramaniyan ◽  
Ke Xu ◽  
Chuan Ju Liu ◽  
Vijaya M. Ponamgi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Protein Interactions ◽  
Membrane Localization ◽  
Protein Protein Interactions ◽  
Amino Terminal ◽  
Organic Solute Transporter ◽  
Organic Solute ◽  
Carboxyl Terminal ◽  
Solute Transporter

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-α and -β, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-α and -β proteins were examined. The results demonstrated that coexpression of hOST-α and -β in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-α and -β proteins in cells cotransfected with hOST-α and -β cDNAs. Protein-protein interactions between hOST-α and -β were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-α abolished its interaction with hOST-β and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-α still interacted with hOST-β, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-β protein in Madin-Darby canine kidney cells. In summary, hOST-α and -β proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-α is not essential for this interaction with hOST-β. The extracellular amino-terminal fragment of hOST-α may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane.

scholarly journals A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

2004 ◽  
Vol 78 (3) ◽  
pp. 1230-1242 ◽  
Author(s):  
Aaron Derdowski ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

scholarly journals S-Acylation of plant immune receptors mediates immune signaling in plasma membrane nanodomains

2019 ◽  
Author(s):  
Dongqin Chen ◽  
Nagib Ahsan ◽  
Jay J. Thelen ◽  
Gary Stacey
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Critical Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Immune Signaling ◽  
Immune Receptors ◽  
Similar Phenotype ◽  
External Stimuli

SUMMARYS-Acylation is a reversible protein post-translational modification mediated by Protein S-acyltransferases (PATs). Here, we demonstrate that three plant immune receptors P2K1 (DORN1), FLS2 and CERK1, representing three distinct families of receptor like-kinases, are S-acylated by Arabidopsis PAT5 and PAT9 on the plasma membrane (PM). Mutations in Atpat5 and Atpat9 resulted in an elevated plant immune response, increased phosphorylation and decreased degradation of FLS2, P2K1 and CERK1 during immune signaling. Mutation of key cysteine residues S-acylated in these receptors resulted in a similar phenotype as exhibited by Atpat5/Atpat9 mutant plants. The data indicate that S-acylation also controls localization of the receptors in distinct PM nanodomains and, thereby, controls the formation of specific protein-protein interactions. Our study reveals that S-acylation plays a critical role in mediating spatiotemporal dynamics of plant receptors within PM nanodomains, suggesting a key role for this modification in regulating the ability of plants in respond to external stimuli.

scholarly journals Differential regulation of the Menkes and Wilson disease copper transporters by hormones: an integrated model of metal transport in the placenta

2007 ◽  
Vol 402 (2) ◽  
Author(s):  
Des R. Richardson ◽  
Yohan Suryo rahmanto
Keyword(s):  
Protein Interactions ◽  
Wilson Disease ◽  
Hormonal Regulation ◽  
Basolateral Membrane ◽  
Critical Role ◽  
Menkes Disease ◽  
Metal Transport ◽  
Integrated Model ◽  
Protein Protein Interactions ◽  
Biochemical Journal

Copper (Cu) plays a critical role in the developing foetus, but virtually nothing is known concerning the regulation of its uptake and metabolism in the placenta. In this issue of the Biochemical Journal, Hardman and colleagues, using a model of placental trophoblasts in culture, identify differential hormonal regulation of two copper-transporting ATPases; namely, those responsible for Menkes disease (ATP7A; MNK) and Wilson disease (ATP7B; WND). Insulin and oestrogen, which are essential during gestation, up-regulate MNK and this leads to trafficking of the MNK protein from the Golgi to the basolateral membrane, resulting in increased Cu efflux. At the same time, insulin decreased WND levels, and this leads to intracellular sequestration of the protein to a perinuclear region that reduces apical Cu release. As such, this results in a concerted flux of Cu from the basolateral surface of the trophoblast that would potentially be used by the developing foetus. An integrated model of vectorized Cu transport is proposed, which involves co-ordinated expression of transporters, organelle interactions and probable protein–protein interactions. The findings have wider implications for considering general models of intracellular metal transport.

Sign in / Sign up

Export Citation Format

Share Document