scholarly journals NBCe1A dimer assemble visualized by bimolecular fluorescence complementation

2014 ◽  
Vol 306 (6) ◽  
pp. F672-F680 ◽  
Author(s):  
Min-Hwang Chang ◽  
An-Ping Chen ◽  
Michael F. Romero
Keyword(s):  
Fluorescent Protein ◽  
Functional Unit ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Bimolecular Fluorescence Complementation ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Enhanced Yellow Fluorescent Protein ◽  
Type Function ◽  
Fluorescence Complementation

Mutations in the electrogenic Na+/HCO3−cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1–158, EYFPN) or (159–238, EYFPC) was fused to the NH2or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2terminus of NBCe1A (EYFPN-N-NBCe1A w/ EYFPC-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.

scholarly journals A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State

1999 ◽  
Vol 114 (5) ◽  
pp. 685-700 ◽  
Author(s):  
Thomas P. Flagg ◽  
Margaret Tate ◽  
Jean Merot ◽  
Paul A. Welling
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Closed State ◽  
Negative Effect

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

scholarly journals The NHERF1 PDZ1 domain and IRBIT interact and mediate the activation of Na+/H+ exchanger 3 by ANG II

2016 ◽  
Vol 311 (2) ◽  
pp. F343-F351 ◽  
Author(s):  
Peijian He ◽  
Luqing Zhao ◽  
Yi Ran No ◽  
Serhan Karvar ◽  
C. Chris Yun
Keyword(s):  
Proximal Tubule ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Yellow Fluorescent Protein ◽  
Protein S ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Ang Ii ◽  
Renal Brush Border Membrane ◽  
Inositol 1,4,5 Trisphosphate

Na+/H+ exchanger (NHE)3, a major Na+ transporter in the luminal membrane of the proximal tubule, is subject to ANG II regulation in renal Na+/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that NHE regulatory factor (NHERF)1, NHE3, and IRBIT proteins were coexpressed in the same macrocomplexes and that loss of ANG II type 1 receptors decreased their expression in the renal brush-border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interactions with NHE3 and IRBIT, which were abolished when the NHERF1 PDZ1 domain was removed. Overexpression of a yellow fluorescent protein-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate the ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for the ANG II-provoked increase in NHERF1-NHE3 interactions and that phosphorylation of IRBIT at Ser68 was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and the NHERF1 PDZ1 domain in binding and transporting NHE3.

scholarly journals Actin Depolymerization Disrupts Tight Junctions via Caveolae-mediated Endocytosis

2005 ◽  
Vol 16 (9) ◽  
pp. 3919-3936 ◽  
Author(s):  
Le Shen ◽  
Jerrold R. Turner
Keyword(s):  
Barrier Function ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Yellow Fluorescent Protein ◽  
Membrane Traffic ◽  
Time Lapse ◽  
Dominant Negative ◽  
Enhanced Yellow Fluorescent Protein ◽  
Caveolin 1 ◽  
Actin Depolymerization

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of β-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14°C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Δ95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.

scholarly journals Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits

2004 ◽  
Vol 15 (3) ◽  
pp. 990-1002 ◽  
Author(s):  
Nicola Susann Werner ◽  
Reinhard Windoffer ◽  
Pavel Strnad ◽  
Christine Grund ◽  
Rudolf Eberhard Leube ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Fluorescent Protein ◽  
Dynamic Equilibrium ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epidermolysis Bullosa Simplex ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dominant Negative Mutation

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

scholarly journals Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

2021 ◽  
Author(s):  
Ashley S. Denney ◽  
Andrew D. Weems ◽  
Michael A. McMurray
Keyword(s):  
Three Dimensional ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Type Function ◽  
Negative Effect ◽  
Chaperone Interactions

ABSTRACTLife requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach to identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Chaperone inhibition liberates the mutant to enter the nucleus where it has a slight dominant-negative effect. These findings provide new insights into the effects of missense mutations.

scholarly journals Probing the domain structure of FtsZ by random truncation and insertion of GFP

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4033-4043 ◽  
Author(s):  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Wild Type ◽  
Negative Effects ◽  
Terminal Domain ◽  
Z Ring ◽  
Green Fluorescent

Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1–195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3–5 times the level of wild-type FtsZ. The C-terminal domain, aa 195–383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.

Applicability of superfolder YFP bimolecular fluorescence complementation in vitro

2009 ◽  
Vol 390 (1) ◽  
Author(s):  
Corinna Ottmann ◽  
Michael Weyand ◽  
Alexander Wolf ◽  
Jürgen Kuhlmann ◽  
Christian Ottmann
Keyword(s):  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

Abstract Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) is a widely employed method to study protein-protein interactions in cells. As yet, this technique has not been used in vitro. To evaluate a possible application of BiFC in vitro, we constructed a ‘superfolder split YFP’ system where 15 mutations enhance expression of the fusion proteins in Escherichia coli and enable a native purification due to improved solubility. Here, we present the crystal structure of ‘superfolder YFP’, providing the structural basis for the enhanced folding and stability characteristics. Complementation between the two non-fluorescent YFP fragments fused to HRas and Raf1RBD or to 14-3-3 and PMA2-CT52 resulted in the constitution of the functional fluorophore. The in vivo BiFC with these protein interaction pairs was demonstrated in eukaryotic cell lines as well. Here, we present for the first time BiFC in vitro studies with natively purified superfolder YFP fusion proteins and show the potential and drawbacks of this method for analyzing protein-protein interactions.

scholarly journals Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

2007 ◽  
Vol 82 (2) ◽  
pp. 683-691 ◽  
Author(s):  
Scott P. Kenney ◽  
Timothy L. Lochmann ◽  
Cullen L. Schmid ◽  
Leslie J. Parent
Keyword(s):  
Nuclear Export ◽  
Plasma Membranes ◽  
Dominant Negative ◽  
Confocal Imaging ◽  
Bimolecular Fluorescence Complementation ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Particle Assembly ◽  
Gag Protein ◽  
Gag Proteins

ABSTRACT The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these “trapped” Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.

scholarly journals Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small-Molecule Inhibitors of HIV-1 Nef Dimerization

2013 ◽  
Vol 19 (4) ◽  
pp. 556-565 ◽  
Author(s):  
Jerrod A. Poe ◽  
Laura Vollmer ◽  
Andreas Vogt ◽  
Thomas E. Smithgall
Keyword(s):  
Small Molecules ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Critical Role ◽  
Bimolecular Fluorescence Complementation ◽  
Accessory Factor ◽  
And Function ◽  
Hiv 1 ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus “2A” linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

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