High-Throughput Yeast Two-Hybrid Screening

Roberts, George G.; Parrish, Jodi R.; Mangiola, Bernardo A.; Finley, Russell L.

doi:10.1007/978-1-61779-455-1_3

Cited by 13 publications

(8 citation statements)

References 26 publications

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“…Binary protein interactions were tested by direct mating of a set of baits with a set of preys expressed in opposite yeast mating types6975. Diploids containing both bait and prey constructs were inoculated on synthetic dextrose plates without Leu, Trp and His containing 3-aminotriazole69 and colony formation was scored following 3, 5 and 8 days.…”

Section: Methodsmentioning

confidence: 99%

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

et al. 2017

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Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein–protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms.

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Section: Methodsmentioning

confidence: 99%

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

et al. 2017

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“…The expression clones for the Y2H assay were transformed into yeast mating strains AH109 and Y187 as described by Roberts et al (2012). Binary protein interactions were tested by direct mating of a set of baits with a set of preys expressed in opposite yeast mating types (Stellberger et al, 2010).…”

Section: Y2h Assaymentioning

confidence: 99%

The Extra-Pathway Interactome of the TCA Cycle: Expected and Unexpected Metabolic Interactions

et al. 2018

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The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis () mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.

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“…This could elucidate mitochondrial and astrocyte processes specifically dysregulated in ALS. Furthermore, constructing a unique PPI network for known mitochondrial proteins, utilizing high throughput yeast 2 hybrid methods, could allow for dysregulated processes in ALS mitochondria to be predicted [140].…”

Section: Molecular Data and Analyses Applied To Other Als-related mentioning

confidence: 99%

Personalized Medicine and Molecular Interaction Networks in Amyotrophic Lateral Sclerosis (ALS): Current Knowledge

Morgan

Duguez

Duddy

2018

JPM

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Multiple genes and mechanisms of pathophysiology have been implicated in amyotrophic lateral sclerosis (ALS), suggesting it is a complex systemic disease. With this in mind, applying personalized medicine (PM) approaches to tailor treatment pipelines for ALS patients may be necessary. The modelling and analysis of molecular interaction networks could represent valuable resources in defining ALS-associated pathways and discovering novel therapeutic targets. Here we review existing omics datasets and analytical approaches, in order to consider how molecular interaction networks could improve our understanding of the molecular pathophysiology of this fatal neuromuscular disorder.

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High-Throughput Yeast Two-Hybrid Screening

Cited by 13 publications

References 26 publications

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

The Extra-Pathway Interactome of the TCA Cycle: Expected and Unexpected Metabolic Interactions

Personalized Medicine and Molecular Interaction Networks in Amyotrophic Lateral Sclerosis (ALS): Current Knowledge

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