title: Examining protein protein interactions using endogenously tagged yeast arrays: the cross-and-capture system.
abstract: Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Suter
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Fetchko
authors.forename: Ralph
authors.initials: R
authors.lastname: Imhof
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Graham
authors.forename: Ingrid
authors.initials: I
authors.lastname: Stoffel-Studer
authors.forename: Caroline
authors.initials: C
authors.lastname: Zbinden
authors.forename: Maanasa
authors.initials: M
authors.lastname: Raghavan
authors.forename: Lianet
authors.initials: L
authors.lastname: Lopez
authors.forename: Lucija
authors.initials: L
authors.lastname: Beneti
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Hort
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fillingham
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016324:Sequence Tagged Sites; D046888:Tissue Array Analysis / Q000379:methods; D020798:Two-Hybrid System Techniques
chemical_list: D029701:Saccharomyces cerevisiae Proteins