title: A Split-Ubiquitin Based Strategy Selecting for Protein Complex-Interfering Mutations.
abstract: Understanding the topologies and functions of protein interaction networks requires the selective removal of single interactions. We introduce a selection strategy that enriches among a random library of alleles for mutations that impair the binding to a given partner protein. The selection makes use of a split-ubiquitin based protein interaction assay. This assay provides yeast cells that carry protein complex disturbing mutations with the advantage of being able to survive on uracil-lacking media. Applied to the exemplary interaction between the PB domains of the yeast proteins Bem1 and Cdc24, we performed two independent selections. The selections were either analyzed by Sanger sequencing of isolated clones or by next generation sequencing (NGS) of pools of clones. Both screens enriched for the same mutation in position 833 of Cdc24. Biochemical analysis confirmed that this mutation disturbs the interaction with Bem1 but not the fold of the protein. The larger dataset obtained by NGS achieved a more complete representation of the bipartite interaction interface of Cdc24.
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gronemeyer
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Chollet
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Werner
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Glomb
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Bäuerle
authors.affiliation: Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany nils.johnsson@uni-ulm.de.
authors.forename: Nils
authors.initials: N
authors.lastname: Johnsson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics*; D000483:Alleles; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D046912:Multiprotein Complexes / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D060066:Protein Interaction Maps / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics*; D025801:Ubiquitin / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C073594:CDC24 protein, S cerevisiae; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C073129:BEM1 protein, S cerevisiae
keywords: mutagenesis; protein structure; protein-protein interaction; split-ubiquitin