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 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 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.

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.

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 Assembly of the Yeast Vacuolar H+-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex

1998 ◽  
Vol 142 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Laurie A. Graham ◽  
Kathryn J. Hill ◽  
Tom H. Stevens
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cross Linking ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Atpase Subunit ◽  
Mutant Cells

Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of ∼5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein–protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

scholarly journals Prioritization of genes associated with the pathogenesis of leukosis in cattle

2019 ◽  
Vol 22 (8) ◽  
pp. 1063-1069 ◽  
Author(s):  
N. S. Yudin ◽  
N. L. Podkolodnyy ◽  
T. A. Agarkova ◽  
E. V. Ignatieva
Keyword(s):  
Protein Interactions ◽  
Genome Wide Association Study ◽  
Association Studies ◽  
Mammalian Species ◽  
Genome Wide Association ◽  
Farm Animals ◽  
Genome Wide Association Studies ◽  
Protein Protein Interactions ◽  
Genome Wide ◽  
A Genome

Selection by means of genetic markers is a promising approach to the eradication of infectious diseases in farm animals, especially in the absence of effective methods of treatment and prevention. Bovine leukemia virus (BLV) is spread throughout the world and represents one of the biggest problems for the livestock production and food security in Russia. However, recent genome-wide association studies have shown that sensitivity/resistance to BLV is polygenic. The aim of this study was to create a catalog of cattle genes and genes of other mammalian species involved in the pathogenesis of BLV-induced infection and to perform gene prioritization using bioinformatics methods. Based on manually collected information from a range of open sources, a total of 446 genes were included in the catalog of cattle genes and genes of other mammals involved in the pathogenesis of BLV-induced infection. The following criteria were used to prioritize 446 genes from the catalog: (1) the gene is associated with leukemia according to a genome-wide association study; (2) the gene is associated with leukemia according to a case-control study; (3) the role of the gene in leukemia development has been studied using knockout mice; (4) protein-protein interactions exist between the gene-encoded protein and either viral particles or individual viral proteins; (5) the gene is annotated with Gene Ontology terms that are overrepresented for a given list of genes; (6) the gene participates in biological pathways from the KEGG or REACTOME databases, which are over-represented for a given list of genes; (7) the protein encoded by the gene has a high number of protein-protein interactions with proteins encoded by other genes from the catalog. Based on each criterion, a rank was assigned to each gene. Then the ranks were summarized and an overall rank was determined. Prioritization of 446 candidate genes allowed us to identify 5 genes of interest (TNF,LTB,BOLA-DQA1,BOLA-DRB3,ATF2), which can affect the sensitivity/resistance of cattle to leukemia.

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