Interrogating yeast surface-displayed human proteome to identify small molecule-binding proteins.
Identifying proteins that interact with small molecules is often a challenging step in understanding cellular signaling pathways or molecular mechanisms of drug action. In this report, we describe the construction of libraries displaying human protein fragments on the surface of yeast cells and demonstrate the utility of these libraries for the study of small molecule/protein interactions. The libraries were used to select protein fragments with affinity for the phosphatidylinositides phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). We recovered cDNA inserts encoding pleckstrin homology domains, a phosphotyrosine-binding domain, and a fragment of apolipoprotein H. The pleckstrin homology and phosphotyrosine-binding domains are known phosphatidylinositide-binding domains, demonstrating the effectiveness of our approach. Binding of apolipoprotein H to PtdIns(4,5)P2 and PtdIns(3,4,5)P3 has not been reported previously and thus represents novel interactions. We expect that this method will be generally applicable to the study of small molecule/protein interactions and may facilitate the study of cellular signaling pathways and mechanisms of drug action or toxicity.
Department of Anesthesia, UCSF Comprehensive Cancer Center, University of California, San Francisco, California 94110, USA.
Scott
S
Bidlingmaier
Bin
B
Liu
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D002352:Carrier Proteins; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D020543:Proteome; C060974:phosphatidylinositol 3,4,5-triphosphate