Homo- and hetero-dimeric architecture of the human liver Na+-dependent taurocholate co-transporting protein

2012 ◽  
Vol 441 (3) ◽  
pp. 1007-1016 ◽  
Author(s):  
Ingrid T. G. W. Bijsmans ◽  
Rianne A. M. Bouwmeester ◽  
Joachim Geyer ◽  
Klaas Nico Faber ◽  
Stan F. J. van de Graaf
Keyword(s):  
Plasma Membrane ◽  
Bile Salt ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Physical Interaction ◽  
Oligomeric State ◽  
Salt Uptake ◽  
Transporter Family ◽  
C Terminus ◽  
U2os Cells

The NTCP (Na+–taurocholate co-transporting protein)/SLC10A [solute carrier family 10 (Na+/bile acid co-transporter family)] 1 is tightly controlled to ensure hepatic bile salt uptake while preventing toxic bile salt accumulation. Many transport proteins require oligomerization for their activity and regulation. This is not yet established for bile salt transporters. The present study was conducted to elucidate the oligomeric state of NTCP. Chemical cross-linking revealed the presence of NTCP dimers in rat liver membranes and U2OS cells stably expressing NTCP. Co-immunoprecipitation of tagged NTCP proteins revealed a physical interaction between subunits. The C-terminus of NTCP was not required for subunit interaction, but was essential for exit from the ER (endoplasmic reticulum). NTCP without its C-terminus (NTCP Y307X) retained full-length wtNTCP (wild-type NTCP) in the ER in a dominant fashion, suggesting that dimerization occurs early in the secretory pathway. FRET (fluorescence resonance energy transfer) using fluorescently labelled subunits further demonstrated that dimerization persists at the plasma membrane. NTCP belongs to the SLC10A protein family which consists of seven members. NTCP co-localized in U2OS cells with SLC10A4 and SLC10A6, but not with SLC10A3, SLC10A5 or SLC10A7. SLC10A4 and SLC10A6 co-immunoprecipitated with NTCP, demonstrating that heteromeric complexes can be formed between SLC10A family members in vitro. Expression of SLC10A4 and NTCP Y307X resulted in a reduction of NTCP abundance at the plasma membrane and NTCP-mediated taurocholate uptake, whereas expression of SLC10A6 or NTCP E257N, an inactive mutant, did not affect NTCP function. In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by heterodimerization when this impairs NTCP plasma membrane trafficking.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

2018 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Pradhipa Karuna M ◽  
Leonie Witte ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Long Range ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Multivesicular Bodies ◽  
Phosphorylation Sites ◽  
Wnt Proteins ◽  
C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

scholarly journals Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

2018 ◽  
Vol 115 (31) ◽  
pp. E7331-E7340 ◽  
Author(s):  
Ben Johnson ◽  
Ashley N. Leek ◽  
Laura Solé ◽  
Emily E. Maverick ◽  
Tim P. Levine ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Neuronal Cultures ◽  
Physical Relationship ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Hek Cells ◽  
C Terminus ◽  
Membrane Contact

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

scholarly journals Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

2002 ◽  
Vol 76 (8) ◽  
pp. 3720-3730 ◽  
Author(s):  
Séverine Carrère-Kremer ◽  
Claire Montpellier-Pala ◽  
Laurence Cocquerel ◽  
Czeslaw Wychowski ◽  
François Penin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Subcellular Localization ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
C Terminus ◽  
Membrane Spanning ◽  
Polytopic Membrane Protein

ABSTRACT Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

Molecular mechanisms of hepatic bile salt transport from sinusoidal blood into bile

1995 ◽  
Vol 269 (6) ◽  
pp. G801-G812 ◽  
Author(s):  
P. J. Meier
Keyword(s):  
Plasma Membrane ◽  
Bile Salt ◽  
Bile Salts ◽  
Organic Anion ◽  
Salt Transport ◽  
Salt Flux ◽  
Salt Uptake ◽  
Canalicular Bile ◽  
Salt Secretion ◽  
Bile Salt Transport

An increasingly complex picture has emerged in recent years regarding the bile salt transport polarity of hepatocytes. At the sinusoidal (or basolateral) plasma membrane two bile salt-transporting polypeptides have been cloned. The Na(+)-taurocholate-cotransporting polypeptide (Ntcp) can account for most, if not all, physiological properties of the Na(+)-dependent bile salt uptake function in mammalian hepatocytes. The cloned organic anion-transporting protein (Oatp1) can mediate Na(+)-independent transport of bile salts, sulfobromophthalein, estrogen conjugates, and a variety of other amphipathic cholephilic compounds. Hence, Oatp1 appears to correspond to the previously suggested basolateral multispecific bile sale transporter. Intracellular bile salt transport can be mediated by different pathways. Under basal bile salt flux conditions, conjugated trihydroxy bile salts bind to cytoplasmic binding proteins and reach the canalicular plasma membrane predominantly via cytoplasmic diffusion. More hydrophobic mono- and dihydroxy and high concentrations of trihydroxy bile salts associate with intracellular membrane-bound compartments, including transcytotic vesicles, endoplasmic reticulum (ER), and Golgi complex. A facilitated bile salt diffusion pathway has been demonstrated in the ER. The exact role of these and other (e.g., lysosomes, "tubulovesicular structures") organelles in overall vectorial transport of bile salts across hepatocytes is not yet known. Canalicular bile salt secretion is mediated by two ATP-dependent transport systems, one for monovalent bile salts and the second for divalent sulfated or glucuronidated bile salt conjugates. The latter is identical with the canalicular multispecific organic anion transporter, which also transports other divalent organic anions, such as glutathione S-conjugates. Potential dependent canalicular bile salt secretion has also been suggested to occur, but its exact mechanism and physiological significance remain unclear, since a potential driven bile salt uptake system has also been identified in the ER. Hypothetically, and similar to changes in cell volume, the intracellular potential could also play a role in the regulation of the number of bile salt carriers at the canalicular membrane and thereby indirectly influence the maximal canalicular bile salt transport capacity of hepatocytes.

scholarly journals The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Cyril Legrand ◽  
Roberto Saleppico ◽  
Jana Sticht ◽  
Fabio Lolicato ◽  
Hans-Michael Müller ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Underlying Mechanism ◽  
Physical Interaction ◽  
Structural Elements ◽  
Intact Cells ◽  
Unconventional Secretion ◽  
Mutant Forms ◽  
Α1 Subunit ◽  
Cell Survival Factor

AbstractFGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.

scholarly journals Interaction Between the SNARE SYP121 and the Plasma Membrane Aquaporin PIP2;7 Involves Different Protein Domains

2021 ◽  
Vol 11 ◽  
Author(s):  
Timothée Laloux ◽  
Irwin Matyjaszczyk ◽  
Simon Beaudelot ◽  
Charles Hachez ◽  
François Chaumont
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Interaction Mechanism ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Amino Acid Residues ◽  
Interaction Domain ◽  
C Terminus ◽  
Full Interaction ◽  
Plasma Membrane Intrinsic Proteins

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

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.

Trafficking of tail-anchored proteins: transport from the endoplasmic reticulum to the plasma membrane and sorting between surface domains in polarised epithelial cells

2002 ◽  
Vol 115 (8) ◽  
pp. 1689-1702 ◽  
Author(s):  
Alessandra Bulbarelli ◽  
Teresa Sprocati ◽  
Massimo Barberi ◽  
Emanuela Pedrazzini ◽  
Nica Borgese
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Apical Surface ◽  
Tail Region ◽  
Surface Distribution ◽  
C Terminus ◽  
Ta Proteins

Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal transmembrane domain (TMD), provide useful models for studying the role of the TMD in sorting within the exo-endocytic system. Previous work has shown that a short TMD is required to keep ER-resident TA proteins from escaping to downstream compartments of the secretory pathway. To investigate the role of the TMD in TA protein sorting, we used model constructs, which consisted of GFP linked at its C-terminus to the tail region of cytochrome b(5) with TMDs of differing length or hydrophobicity. Expression of these constructs in CV-1 cells demonstrated that the feature determining exit from the ER is hydrophobicity and that if exit occurs, at least a part of the protein reaches the cell surface. To investigate which pathway to the surface is followed by plasma-membrane-directed TA constructs, we expressed the TA constructs in polarised Madin Darby Canine Kidney (MDCK) cells. The constructs with 22 and 25 residue TMDs were localised basolaterally, but addition at the C-terminus of a 20-residue peptide containing an N-glycosylation site resulted in glycosylation-dependent relocation of∼50% of the protein to the apical surface. This result suggests that TA proteins may reach the basolateral surface without a signal or that our constructs contain a weak basolateral determinant that is recessive to the apical information carried by the glycan. To assess the effect of the TMDs of endogenous TA proteins, GFP was linked to the tails of syntaxin 3 and 4, which localise to the apical and basolateral surface, respectively, of MDCK cells. The two GFP fusion proteins showed a different surface distribution, which is consistent with a role for the two syntaxin TMDs in polarised sorting.

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