The social architecture of an in-depth cellular protein interactome

Nearly all cellular functions are mediated by protein-protein interactions and mapping the interactome provides fundamental insights into the regulation and structure of biological systems. In principle, affinity purification coupled to mass spectrometry (AP-MS) is an ideal and scalable tool, however, it has been difficult to identify low copy number complexes, membrane complexes and those disturbed by protein-tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive, high-throughput, and highly reproducible AP-MS technology combined with a quantitative two-dimensional analysis strategy for comprehensive interactome mapping of Saccharomyces cerevisiae. We reduced required cell culture volumes thousand-fold and employed 96-well formats throughout, allowing replicate analysis of the endogenous green fluorescent protein (GFP) tagged library covering the entire expressed yeast proteome. The 4159 pull-downs generated a highly structured network of 3,909 proteins connected by 29,710 interactions. Compared to previous large-scale studies, we double the number of proteins (nodes in the network) and triple the number of reliable interactions (edges), including very low abundant epigenetic complexes, organellar membrane complexes and non-taggable complexes interfered by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 15 interactors, the majority of them unreported so far. Similar to social networks between humans, the average shortest distance is 4.2 interactions. A web portal (www.yeast-interactome.org) enables exploration of our dataset by the network and biological communities and variations of our AP-MS technology can be employed in any organism or dynamic conditions.

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Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

Comparative and Functional Genomics ◽

10.1002/cfg.449 ◽

2005 ◽

Vol 6 (1-2) ◽

pp. 2-16 ◽

Cited By ~ 55

Author(s):

Russell Howson ◽

Won-Ki Huh ◽

Sina Ghaemmaghami ◽

James V. Falvo ◽

Kiowa Bower ◽

...

Keyword(s):

Protein Interactions ◽

Fluorescent Protein ◽

Budding Yeast ◽

Affinity Purification ◽

Reading Frame ◽

Yeast Strains ◽

Yeast Community ◽

C Terminus ◽

Protein Properties ◽

Green Fluorescent

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

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Engineering an efficient and bright split Corynactis californica green fluorescent protein

Scientific Reports ◽

10.1038/s41598-021-98149-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hau B. Nguyen ◽

Thomas C. Terwilliger ◽

Geoffrey S. Waldo

Keyword(s):

Green Fluorescent Protein ◽

Protein Interactions ◽

Large Scale ◽

Fluorescent Protein ◽

Protein Translocation ◽

Fluorescent Proteins ◽

Protein Crystallization ◽

Protein Solubility ◽

Sea Anemones ◽

Green Fluorescent

AbstractSplit green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

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Subpopulation-Specific Metabolic Pathway Usage in Mixed Cultures as Revealed by Reporter Protein-Based13C Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.02696-10 ◽

2011 ◽

Vol 77 (5) ◽

pp. 1816-1821 ◽

Cited By ~ 27

Author(s):

Martin Rühl ◽

Wolf-Dietrich Hardt ◽

Uwe Sauer

Keyword(s):

Amino Acids ◽

Large Scale ◽

Fluorescent Protein ◽

Affinity Purification ◽

Cellular Protein ◽

Mixed Cultures ◽

Microbial Consortia ◽

Flux Analysis ◽

Reporter Protein ◽

Element Cycling

ABSTRACTMost large-scale biological processes, like global element cycling or decomposition of organic matter, are mediated by microbial consortia. Commonly, the different species in such consortia exhibit mutual metabolic dependencies that include the exchange of nutrients. Despite the global importance, surprisingly little is known about the metabolic interplay between species in particular subpopulations. To gain insight into the intracellular fluxes of subpopulations and their interplay within such mixed cultures, we developed here a13C flux analysis approach based on affinity purification of the recombinant fusion glutathioneS-transferase (GST) and green fluorescent protein (GFP) as a reporter protein. Instead of detecting the13C labeling patterns in the typically used amino acids from the total cellular protein, our method detects these13C patterns in amino acids from the reporter protein that has been expressed in only one species of the consortium. As a proof of principle, we validated our approach by mixed-culture experiments of anEscherichia coliwild type with two metabolic mutants. The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%.

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A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211018198 ◽

2021 ◽

pp. 247255522110181

Author(s):

Andreas Vogt ◽

Samantha L. Eicher ◽

Tracey D. Myers ◽

Stacy L. Hrizo ◽

Laura L. Vollmer ◽

...

Keyword(s):

High Throughput Screening ◽

Large Scale ◽

Protein Quality ◽

Fluorescent Protein ◽

Triose Phosphate Isomerase ◽

Pharmacological Intervention ◽

Screening Assay ◽

Triose Phosphate ◽

The Common ◽

Green Fluorescent

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

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The Fe-Core/Carbon-Shell Ultrafine Nanopowders as Platform for Biomolecules Grafting

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1040.194 ◽

2014 ◽

Vol 1040 ◽

pp. 194-198

Author(s):

N.S. Surgutskaya ◽

P.S. Postnikov ◽

Alexandra G. Pershina ◽

A.I. Galanov ◽

Marina E. Trusova ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Large Scale ◽

Fluorescent Protein ◽

Carbon Shell ◽

Shell Nanoparticles ◽

Powder Surface ◽

Metal Precursors ◽

Covalent Grafting ◽

Large Scale Synthesis ◽

Green Fluorescent

The Fe-core/carbon-shell nanopowders are excellent platform for covalent grafting of biomolecules. The large-scale synthesis of Fe-core/carbon-shell nanoparticles via electropulse erosion of metal precursors in hydrocarbons was developed. The green fluorescent protein was covalently attached to the powder surface via diazonium functionalization and further carbodiimide activation.

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Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

10.1101/367110 ◽

2018 ◽

Author(s):

Clémence Roggo ◽

Jan Roelof van der Meer

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Bacterial Chemotaxis ◽

Ph Sensitive ◽

Flagellar Motors ◽

Receptor Interactions ◽

Split Egfp ◽

Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

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Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

AJP Cell Physiology ◽

10.1152/ajpcell.00580.2002 ◽

2003 ◽

Vol 284 (6) ◽

pp. C1633-C1644 ◽

Cited By ~ 37

Author(s):

Mohammed A. Khadeer ◽

Zhihui Tang ◽

Harriet S. Tenenhouse ◽

Maribeth V. Eiden ◽

Heini Murer ◽

...

Keyword(s):

Plasma Membrane ◽

Bone Resorption ◽

Protein Interactions ◽

Cellular Localization ◽

Fluorescent Protein ◽

Basolateral Membrane ◽

Atp Synthesis ◽

Cellular Distribution ◽

Green Fluorescent ◽

Carboxy Terminal

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.

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