Interaction Proteomics Suggests a New Role for the Tfs1 Protein in Yeast

• Published in: Journal of proteome research Vol. 11; no. 6; pp. 3211 - 3218
• Main Authors: Beaufour, Martine, Godin, Fabienne, Vallée, Béatrice, Cadene, Martine, Bénédetti, Hélène
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.06.2012
• Subjects: Biochemistry, Molecular Biology; Carrier Proteins - isolation & purification; Carrier Proteins - physiology; Immunoprecipitation; Life Sciences; Metabolic Networks and Pathways; Protein Interaction Mapping; Protein Interaction Maps; Proteomics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - isolation & purification; Saccharomyces cerevisiae Proteins - physiology; Signal Transduction; PEBP/RKIP homologues; cell signaling; targeted proteomics; intermediate metabolism; protein function; protein−protein interactions; Saccharomyces cerevisiae; SUPPRESSOR; CARBOXYPEPTIDASE-Y; LANDSCAPE; RAF KINASE INHIBITOR; GTPASE-ACTIVATING PROTEIN; SACCHAROMYCES-CEREVISIAE; protein-protein interactions; FAMILY; PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN; ADENYLATE-CYCLASE; HOMOLOGY
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr201239t

The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. To further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.