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

Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins
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]
Issued Date
Article Type
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
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.
Related Researcher
  • 장익수 Chang, Iksoo 뇌과학과
  • Research Interests Theoretical and Computational Biophysics; Supercomputing Simulation of Biomolecules; 이론?계산 생물물리학; 통계물리학; 단백질체의 슈퍼컴퓨터 모델링 및 시물레이션
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Department of Brain Sciences Theoretical and Computational Biophysics Laboratory 1. Journal Articles
Department of Brain Sciences Laboratory of Brain Signal and Synapse Research 1. Journal Articles


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