Dataset Information

2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes.

ABSTRACT: The modulation of protein-protein interactions (PPIs) is an essential regulatory activity defining diverse cell functions in development and disease. BioID is an unbiased proximity-dependent biotinylation method making use of a biotin-protein ligase fused to a protein of interest and has become an important tool for mapping of PPIs within cellular contexts. We devised an advanced method, 2C-BioID, in which the biotin-protein ligase is kept separate from the protein of interest, until the two are induced to associate by the addition of a dimerizing agent. As proof of principle, we compared the interactomes of lamina-associated polypeptide 2? (LAP2?) with those of lamins A and C, using 2C- and conventional BioID. 2C-BioID greatly enhanced data robustness by facilitating the in silico elimination of non-specific interactors as well as overcoming the problems associated with aberrant protein localization. 2C-BioID therefore significantly strengthens the specificity and reliability of BioID-based interactome analysis, by the more stringent exclusion of false-positives and more efficient intracellular targeting.

SUBMITTER: Chojnowski A

PROVIDER: S-EPMC6263017 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes.

Chojnowski Alexandre A Sobota Radoslaw M RM Ong Peh Fern PF Xie Wei W Wong Xianrong X Dreesen Oliver O Burke Brian B Stewart Colin L CL

iScience 20181115

The modulation of protein-protein interactions (PPIs) is an essential regulatory activity defining diverse cell functions in development and disease. BioID is an unbiased proximity-dependent biotinylation method making use of a biotin-protein ligase fused to a protein of interest and has become an important tool for mapping of PPIs within cellular contexts. We devised an advanced method, 2C-BioID, in which the biotin-protein ligase is kept separate from the protein of interest, until the two are ...[more]

PMID: 30500481

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Project description:Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS). In response to phosphate (Pi)-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural protein expression levels and feedback regulatory schemes of TCSs are evolved to match the phosphorylation output of the system, which is determined by intrinsic activities of TCS proteins.

Dataset Information

2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes.

Publications

2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes.

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