Elucidation
of the interaction network between yeast DNA processing proteins using endogenously
tagged ORFs and the LiquiChip technology.
Mike Fetchko, Ingrid Studer, Jacqueline Hort, Igor Stagljar
I. of V. Bioch. & Mol. Biol., University of Zurich, Winterhrurerstr. 190,
Zürich, 8057, Switzerland (stagljar@vetbio.unizh.ch)
Protein complexes mediate many cellular processes such as division, growth and signaling. Dissecting protein interactions among protein complex components generates insight into the composition, regulation and function of the complexes. In the yeast Saccharomyces cerevisiae, we are in the process of examining interactions of proteins involved in DNA replication, recombination and repair in a high throughput manner. The project uses the LiquiChip system from Qiagen to test for binary interactions between endogenously tagged 6-His 'bait' proteins and endogenously tagged VSV 'prey' proteins. To generate yeast containing the bait and prey proteins, individual haploid bait strains are robotically mated to an array of haploid prey strains. Extracts from the diploids are incubated with fluorescently labeled beads which bind the bait and a different fluorescently labeled antibody which recognizes the prey. The labeled extracts are analyzed by the LiquiChip machine which uses lasers to examine single beads loaded with bait for the presence or absence of the fluorescently labeled prey protein, thus determining the interaction status between bait and prey. To test the method, we have generated control strains of proteins with well described interactions and are performing LiquiChip analysis. Furthermore, these strains could be mated in the same manner as above to test for in vivo colocalization of the endogenously tagged proteins. The current state of the project will be presented.