title: Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods.
abstract: We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.
authors: Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
authors: Ricarda A
authors: RA
authors: Busse
authors: Andreea
authors: A
authors: Scacioc
authors: Javier M
authors: JM
authors: Hernandez
authors: Roswitha
authors: R
authors: Krick
authors: Milena
authors: M
authors: Stephan
authors: Andreas
authors: A
authors: Janshoff
authors: Michael
authors: M
authors: Thumm
authors: Karin
authors: K
authors: Kühnel
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002151:Calorimetry; D002352:Carrier Proteins / Q000378:metabolism*; D031521:Fractionation, Field Flow; D008027:Light; D008081:Liposomes / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012542:Scattering, Radiation
chemical_list: D002352:Carrier Proteins; C532949:HSV2 protein, S cerevisiae; D008081:Liposomes; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; C055525:phosphatidylinositol 3-phosphate
keywords: PROPPIN; isothermal titration calorimetry; liposome flotation assays; multi-angle laser light scattering; small unilamellar vesicle