scholarly journals Agonist-evoked inositol trisphosphate receptor (IP3R) clustering is not dependent on changes in the structure of the endoplasmic reticulum

2006 ◽  
Vol 394 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Mark Chalmers ◽  
Michael J. Schell ◽  
Peter Thorn
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Cell Activation ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Yellow Fluorescent Protein ◽  
Resting Cells ◽  
Trisphosphate Receptor ◽  
Er Membrane

The size and number of IP3R (inositol 1,4,5-trisphosphate receptor) clusters located on the surface of the ER (endoplasmic reticulum) is hypothesized to regulate the propagation of Ca2+ waves in cells, but the mechanisms by which the receptors cluster are not understood. Using immunocytochemistry, live-cell imaging and heterologous expression of ER membrane proteins we have investigated IP3R clustering in the basophilic cell line RBL-2H3 following the activation of native cell-surface antigen receptors. IP3R clusters are present in resting cells, and upon receptor stimulation, form larger aggregates. Cluster formation and maintenance required the presence of extracellular Ca2+ in both resting and stimulated cells. Using transfection with a marker of the ER, we found that the ER itself also showed structural changes, leading to an increased number of ‘hotspots’, following antigen stimulation. Surprisingly, however, when we compared the ER hotspots and IP3R clusters, we found them to be distinct. Imaging of YFP (yellow fluorescent protein)–IP3R transfected in to living cells confirmed that IP3R clustering increased upon stimulation. Photobleaching experiments showed that the IP3R occupied a single contiguous ER compartment both before and after stimulation, suggesting a dynamic exchange of IP3R molecules between the clusters and the surrounding ER membrane. It also showed a decrease in the mobile fraction after cell activation, consistent with receptor anchoring within clusters. We conclude that IP3R clustering in RBL-2H3 cells is not simply a reflection of bulk-changes in ER structure, but rather is due to the receptor undergoing homotypic or heterotypic protein–protein interactions in response to agonist stimulation.

Visualization of protein interactions inside the secretory pathway

2007 ◽  
Vol 35 (5) ◽  
pp. 970-973 ◽  
Author(s):  
B. Nyfeler ◽  
H.-P. Hauri
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Coagulation Factor ◽  
Cdna Libraries ◽  
Major Protein ◽  
Protein Protein Interactions ◽  
A Genome

The ER (endoplasmic reticulum) is a major protein folding and modification organelle. In its lumen, the ER processes a third of all newly synthesized proteins. To accomplish this task, numerous resident proteins capture the nascent and newly synthesized proteins. The underlying luminal protein–protein interactions, however, are inherently difficult to analyse, mainly due to their transient nature and the rather specialized environment of the ER. To overcome these limitations, we developed a PCA (protein fragment complementation assay) based on the citrine variant of YFP (yellow fluorescent protein). YFP PCA was successfully applied to visualize the protein interactions of the cargo transport receptor ERGIC-53 (endoplasmic reticulum–Golgi intermediate compartment protein of 53 kDa) with its luminal interaction partner MCFD2 (multiple coagulation factor deficiency protein 2) and its cargo proteins cathepsin Z and cathepsin C in a specific manner. With the prospect of screening cDNA libraries for novel protein–protein interactions, YFP PCA is a promising emerging technique for mapping protein interactions inside the secretory pathway in a genome-wide setting.

scholarly journals Insulin Promotes the Association of Heat Shock Protein 90 with the Inositol 1,4,5-Trisphosphate Receptor to Dampen Its Ca2+ Release Activity

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2190-2196 ◽  
Author(s):  
Nathalie Nguyen ◽  
Nancy Francoeur ◽  
Valérie Chartrand ◽  
Klaus Klarskov ◽  
Gaétan Guillemette ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Mammalian Target Of Rapamycin ◽  
Src Kinase ◽  
Target Of Rapamycin ◽  
Resting Cells ◽  
Release Channel ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (IP3R) is a Ca2+ release channel that plays a pivotal role in regulating intracellular Ca2+ levels in resting cells. Three isoforms of IP3Rs have been identified, and they all possess a large regulatory domain that covers about 60% of the protein. This regulation is accomplished by interaction with small molecules, posttranslational modifications, and mostly protein-protein interactions. In our search for new binding partners of the IP3R, we found that 90-kDa heat-shock protein (Hsp90) binds to the IP3R. This interaction increased on stimulation of HEK293T6.11 cells with insulin but not with Gq protein-coupled receptor (GqPCR) agonists. Moreover, the Hsp90 inhibitor geldanamycin (GA) disrupted the interaction between Hsp90 and the IP3R. Pretreatment of HEK293T6.11 cells with GA greatly increased the intracellular Ca2+ release induced by a GqPCR agonist. Insulin alone did not induce any intracellular Ca2+ release. However, insulin diminished the intracellular Ca2+ release induced by a GqPCR agonist. Interestingly, GA abolished the inhibitory effect of insulin on GqPCR-induced intracellular Ca2+ release. Furthermore, in our search for a mechanistic explanation to this phenomenon, we found that inhibition of kinases activated downstream of the insulin receptor greatly increased the interaction between Hsp90 and the IP3R. Of greater interest, we found that the simultaneous inhibition of mammalian target of rapamycin and the Src kinase almost completely disrupted the interaction between Hsp90 and the IP3R. These results demonstrate that insulin promotes the interaction of Hsp90 with the IP3R to dampen its Ca2+ release activity by a complex mechanism involving mammalian target of rapamycin and the Src kinase.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

scholarly journals Intracellular Targeting of Gag Proteins of the Drosophila Telomeric Retrotransposons

2003 ◽  
Vol 77 (11) ◽  
pp. 6376-6384 ◽  
Author(s):  
S. Rashkova ◽  
A. Athanasiadis ◽  
M.-L. Pardue
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Intracellular Localization ◽  
Ltr Retrotransposons ◽  
Protein Protein Interactions ◽  
Gag Proteins ◽  
Intracellular Targeting ◽  
Identify Amino Acid ◽  
Green Fluorescent

ABSTRACT Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.

scholarly journals Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

1996 ◽  
Vol 7 (5) ◽  
pp. 769-789 ◽  
Author(s):  
A J Koning ◽  
C J Roberts ◽  
R L Wright
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Endogenous Expression ◽  
Coa Reductase ◽  
Er Membrane

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

scholarly journals Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Emma J Fenech ◽  
Federica Lari ◽  
Philip D Charles ◽  
Roman Fischer ◽  
Marie Laétitia-Thézénas ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Protein Quality ◽  
Transmembrane Domain ◽  
Ubiquitin Ligases ◽  
Bioinformatic Analysis ◽  
Innate Immune ◽  
Calcium Flux ◽  
Sting Pathway ◽  
Er Membrane

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

scholarly journals Identification of ERGIC-53 as an intracellular transport receptor of α1-antitrypsin

2008 ◽  
Vol 180 (4) ◽  
pp. 705-712 ◽  
Author(s):  
Beat Nyfeler ◽  
Veronika Reiterer ◽  
Markus W. Wendeler ◽  
Eduard Stefan ◽  
Bin Zhang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Er Export ◽  
Transport Receptor

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

Ezrin oligomers are the membrane-bound dormant form in gastric parietal cells

2005 ◽  
Vol 288 (6) ◽  
pp. C1242-C1254 ◽  
Author(s):  
Lixin Zhu ◽  
Yuechueng Liu ◽  
John G. Forte
Keyword(s):  
Cell Activation ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Primary Cultures ◽  
Parietal Cells ◽  
Erm Proteins ◽  
Membrane Bound ◽  
Gastric Parietal Cells

Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH2- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH2 terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH2- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells.

scholarly journals The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum

2003 ◽  
Vol 370 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Neil M. BROADWAY ◽  
Richard J. PEASE ◽  
Graeme BIRDSEY ◽  
Majid SHAYEGHI ◽  
Nigel A. TURNER ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Contact Sites ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase 1 ◽  
Er Membrane

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69—74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403—5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1—328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.

scholarly journals In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation

2006 ◽  
Vol 87 (6) ◽  
pp. 1745-1750 ◽  
Author(s):  
Frederic Aparicio ◽  
Jesús A. Sánchez-Navarro ◽  
Vicente Pallás
Keyword(s):  
Coat Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Critical Role ◽  
Ringspot Virus ◽  
Bimolecular Fluorescence Complementation ◽  
Dimerization Domain ◽  
Prunus Necrotic Ringspot Virus

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

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