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Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins

Title
Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins
Authors
Jun, YW[Jun, Yong-Woo]Kim, S[Kim, Sangyeol]Kim, KH[Kim, Kun-Hyung]Lee, JA[Lee, Jin-A]Lim, CS[Lim, Chae-Seok]Chang, I[Chang, Iksoo]Suh, BC[Suh, Byung-Chang]Kaang, BK[Kaang, Bong-Kiun]Jang, DJ[Jang, Deok-Jin]
DGIST Authors
Chang, I[Chang, Iksoo]Suh, BC[Suh, Byung-Chang]
Issue Date
2015-04
Citation
Lipids, 50(4), 427-436
Type
Article
Article Type
Article
Keywords
Amino Acid SequenceAnimalAnimalsAplysiaAplysia Sec7Chemical StructureCytologyEnhanced Green Fluorescent ProteinGeneticsGFP-Fusion ProteinGreen Fluorescent ProteinGreen Fluorescent ProteinsHEK293 Cell LineHEK293 CellsHumanHumansHybrid ProteinIn Vitro Protein-Phosphoinositide BindingMetabolismModels, MolecularMolecular GeneticsMolecular Sequence DataNeurite OutgrowthPhosphatidylinositolPhosphatidylinositolsPhosphoinositidePI(3 4 5)P3Protein BindingRecombinant Fusion Proteins
ISSN
0024-4201
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. © 2015 AOCS.
URI
http://hdl.handle.net/20.500.11750/1574
DOI
10.1007/s11745-015-3994-z
Publisher
SPRINGER HEIDELBERG
Related Researcher
  • Author Chang, Ik Soo Theoretical and Computational Biophysics Laboratory
  • Research Interests
Files:
There are no files associated with this item.
Collection:
Brain and Cognitive SciencesCurrent Lab1. Journal Articles


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