title: Analysis of phosphoinositide-binding properties and subcellular localization of GFP-fusion proteins.
abstract: Specific protein-phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP-fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP-fusion proteins could be used for in vitro protein-PI binding assays. We applied this approach to examine the PI-binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did specifically bind to PI(3,4,5)P3 in an in vitro lipid-coated bead assay. Overexpression of EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P3-binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P3 to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP-ApSec7 via the interaction between its PH domain and PI(3,4,5)P3 might play a key role in neurite outgrowth in Aplysia.
authors: Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sang-ju, Korea.
authors: Yong-Woo
authors: YW
authors: Jun
authors: Sangyeol
authors: S
authors: Kim
authors: Kun-Hyung
authors: KH
authors: Kim
authors: Jin-A
authors: JA
authors: Lee
authors: Chae-Seok
authors: CS
authors: Lim
authors: Iksoo
authors: I
authors: Chang
authors: Byung-Chang
authors: BC
authors: Suh
authors: Bong-Kiun
authors: BK
authors: Kaang
authors: Deok-Jin
authors: DJ
authors: Jang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins