scholarly journals Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain

2017 ◽  
Vol 150 (2) ◽  
pp. 293-306 ◽  
Author(s):  
Alexander Polster ◽  
Stefano Perni ◽  
Dilyana Filipova ◽  
Ong Moua ◽  
Joshua D. Ohrtman ◽  
...  
Keyword(s):  
Large Fraction ◽  
Freeze Fracture ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cytoplasmic Domain ◽  
Retrograde Signaling ◽  
Bidirectional Signaling ◽  
Retrograde Signal ◽  
Fracture Electron

The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an “orthograde” signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a “retrograde” signal from RyR1 increases the amplitude of the Ca2+ current produced by CaV1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with CaV1.1 by producing an N-terminal construct (RyR11:4300) that lacks the C-terminal membrane domain. In CaV1.1-null (dysgenic) myotubes, RyR11:4300 is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR–PM junctions containing endogenous CaV1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR11:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR11:4300 stably associates with PM–SR junctions. Strikingly, expression of RyR11:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through CaV1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that ∼25% of RyR11:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR11:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RyR11:4300. In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR–PM junctions in myotubes only if CaV1.1 is also present and is fully functional in retrograde signaling to CaV1.1.

scholarly journals Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

2009 ◽  
Vol 83 (14) ◽  
pp. 7322-7336 ◽  
Author(s):  
Ian B. Hogue ◽  
Adam Hoppe ◽  
Akira Ono
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.

scholarly journals Structural Mapping of Divergent Regions in the Type 1 Ryanodine Receptor Using Fluorescence Resonance Energy Transfer

Structure ◽  
2014 ◽  
Vol 22 (9) ◽  
pp. 1322-1332 ◽  
Author(s):  
Mohana Mahalingam ◽  
Tanya Girgenrath ◽  
Bengt Svensson ◽  
David D. Thomas ◽  
Razvan L. Cornea ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Ryanodine Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Mapping ◽  
Fluorescence Resonance

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

2013 ◽  
Vol 452 (2) ◽  
pp. 303-312 ◽  
Author(s):  
Suparna Patowary ◽  
Elisa Alvarez-Curto ◽  
Tian-Rui Xu ◽  
Jessica D. Holz ◽  
Julie A. Oliver ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Acetylcholine Receptors ◽  
Quaternary Structure ◽  
Large Fraction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dimeric Complexes ◽  
Novel Method ◽  
Quaternary Structures ◽  
G Protein Coupled

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M3Rs (muscarinic M3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M3R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

scholarly journals Minimal requirements for ubiquitination-mediated regulation of thyroid hormone activation

2014 ◽  
Vol 53 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Péter Egri ◽  
Balázs Gereben
Keyword(s):  
Thyroid Hormone ◽  
Resonance Energy Transfer ◽  
E3 Ligase ◽  
Resonance Energy ◽  
Globular Domain ◽  
Native Form ◽  
Molecular Features ◽  
Molecular Machinery

Activation of thyroxine by outer ring deiodination is the crucial first step of thyroid hormone action. Substrate-induced ubiquitination of type 2 deiodinase (D2) is the most rapid and sensitive mechanism known to regulate thyroid hormone activation. While the molecular machinery responsible for D2 ubiquitination has been extensively studied, the combination of molecular features sufficient and required to allow D2 ubiquitination have not previously been determined. To address this question, we constructed chimeric deiodinases by introducing different combinations of D2-specific elements into type 1 deiodinase (D1), another member of the deiodinase enzyme family, which, however, does not undergo ubiquitination in its native form. Studies on the chimeric proteins expressed transiently in HEK-293T cells revealed that combined insertion of the D2-specific instability loop and the K237/K244 D2 ubiquitin carrier lysines into the corresponding positions of D1 could not ubiquitinate D1 unless the chimera was directed to the endoplasmic reticulum (ER). Fluorescence resonance energy transfer measurements demonstrated that the C-terminal globular domain of the ER-directed chimera was able to interact with the E3 ligase subunit WSB1. However, this interaction did not occur between the chimera and the TEB4 (MARCH6) E3 ligase, although a native D2 could readily interact with the N-terminus of TEB4. In conclusion, insertion of the instability loop and ubiquitin carrier lysines in combination with direction to the ER are sufficient and required to govern WSB1-mediated ubiquitination of an activating deiodinase enzyme.

scholarly journals Hepatitis C Genotype Determination by Melting Curve Analysis with a Single Set of Fluorescence Resonance Energy Transfer Probes

2002 ◽  
Vol 48 (12) ◽  
pp. 2147-2154 ◽  
Author(s):  
Grant C Bullock ◽  
David E Bruns ◽  
Doris M Haverstick
Keyword(s):  
Energy Transfer ◽  
Hepatitis C ◽  
Real Time ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Comparison Method ◽  
Curve Analysis ◽  
Melting Curve Analysis

Abstract Background: The genotype of hepatitis C virus (HCV) is a predictor of antiviral therapeutic response. We describe an approach for HCV genotype determination by real-time PCR and melting curve analysis. Methods: After automated nucleic acid extraction, we used reverse transcription-PCR in a block cycler to amplify nucleotides 6–329 of the 5′-untranslated region of HCV. The product was further amplified by single-tube real-time seminested PCR in a LightCyclerTM instrument (Roche). The final product was analyzed by melting curves with the use of fluorescence resonance energy transfer (FRET) probes. The FRET sensor probe was directed at nucleotides 151–170 of type 1 HCV and was designed to distinguish types 1a/b, 2a/c, 2b, 3a, and 4, with melting temperatures (Tms) predicted to differ by 1 °C. Genotypes were compared in a blinded fashion with those of the INNO-LiPATM test (Bayer Diagnostics) on 111 serum samples. Results: In preliminary experiments, the Mg2+ concentration was found to be critical in allowing clear separation of melting points, with the best separation at a Mg2+ concentration of 2 mmol/L. The results for 111 samples clustered at expected Tms for genotypes 1a/b (n = 78), 2a/c (n = 2), 2b (n = 11), 3a (n = 14), and 4 (n = 2). Of the 111 samples, results for 110 were concordant with the comparison method at the level of type 1, 2, 3, or 4. Subtyping results were discordant for two samples, both of type 2. For 108 samples concordant with INNO-LiPA at the genotype and subtype levels, the mean Tms were 64.1, 59.5, 54.2, 52.6, and 50.1 °C for types 1a/b, 2a/c, 4, 2b, and 3a, respectively, with SDs of 0.2, 0.3, 0.3, 0.2, and 0.3 °C. All 78 samples identified as type 1 were concordant with results of the comparison method. Conclusions: Melting analysis with a single pair of FRET probes can rapidly provide information about HCV genotypes and identifies type 1 samples with high specificity.

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.

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