DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.
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.
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Natasha
N
Pashkova
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Tabitha A
TA
Peterson
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Venkatramanan
V
Krishnamani
Department of Biostatistics, University of Iowa, Iowa City, IA 52242, USA.
Patrick
P
Breheny
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Mark
M
Stamnes
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA. Electronic address: robert-piper@uiowa.edu.
Robert C
RC
Piper
D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods* / Q000706:statistics & numerical data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012984:Software; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D020696:rab5 GTP-Binding Proteins
protein interactions; protein networks; ubiquitin
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