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Methods

Examining protein–protein interactions using endogenously tagged yeast arrays: The Cross-and-Capture system

¹ Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), Department of Biochemistry and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada; ² Institute of Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland; ³ Institute of Biochemistry, Biology Department, ETH Zurich CH-8093, Switzerland; ⁴ Laboratory of Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia; ⁵ VetSuisse Faculty, University of Zurich, Zurich CH-8057, Switzerland; ⁶ Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5A 3E1, Canada

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.

⁸ Corresponding author.

E-mail igor.stagljar{at}utoronto.ca; fax (416) 978-8287.

[Supplemental material is available online at www.genome.org.]

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.6667007

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