From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes
Li, Zhihua, doctor of cell and molecular biology
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Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.