DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

Natasha Pashkova; Tabitha A Peterson; Venkatramanan Krishnamani; Patrick Breheny; Mark Stamnes; Robert C Piper

doi:10.1016/j.celrep.2016.08.095

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DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

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DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

Natasha Pashkova, Tabitha A Peterson, Venkatramanan Krishnamani, Patrick Breheny, Mark Stamnes and Robert C Piper

Cell reports (Cambridge), Vol.17(1), pp.303-315

09/27/2016

DOI: 10.1016/j.celrep.2016.08.095

PMCID: PMC5594928

PMID: 27681439

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Abstract

We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiquitin-binding proteins, including interactors that we verify biochemically. To demonstrate the specificity of DEEPN, we show that DEEPN allows simultaneous comparison of candidate interactors across multiple bait proteins, allowing differential interactions to be identified. This feature was used to identify interactors that distinguish between GTP- and GDP-bound conformations of Rab5. [Display omitted] •Parameters of the batch Y2H approach are defined•Data processing and statistical analysis software packages are outlined•A DEEPN screen identifies multiple proteins that interact with ubiquitin•A DEEPN screen differentiates proteins that bind Rab5-GTP versus Rab5-GDP Using deep sequencing to follow a highly complex library upon selection for yeast 2-hybrid interactions provides a way to compare the interactome of one protein to another in parallel. Pashkova et al. provide a comprehensive workflow complete with a bioinformatics package that makes this approach accessible to a variety of investigators.

protein interactions

ubiquitin

protein networks

Details

Title: Subtitle: DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions
Creators: Natasha Pashkova - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
Tabitha A Peterson - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
Venkatramanan Krishnamani - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
Patrick Breheny - Department of Biostatistics, University of Iowa, Iowa City, IA 52242, USA
Mark Stamnes - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
Robert C Piper - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Cell reports (Cambridge), Vol.17(1), pp.303-315
DOI: 10.1016/j.celrep.2016.08.095
PMID: 27681439
PMCID: PMC5594928
NLM abbreviation: Cell Rep
ISSN: 2211-1247
eISSN: 2211-1247
Publisher: Elsevier Inc
Language: English
Date published: 09/27/2016
Academic Unit: Molecular Physiology and Biophysics; Biostatistics; Medicine Administration; Internal Medicine
Record Identifier: 9983997442402771

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