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Lipids

, Volume 50, Issue 4, pp 427–436 | Cite as

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

  • Yong-Woo Jun
  • Sangyeol Kim
  • Kun-Hyung Kim
  • Jin-A Lee
  • Chae-Seok Lim
  • Iksoo Chang
  • Byung-Chang Suh
  • Bong-Kiun Kaang
  • Deok-Jin JangEmail author
Methods

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.

Keywords

GFP-fusion protein Phosphoinositide PI(3,4,5)P3 Aplysia Sec7 In vitro protein-phosphoinositide binding Neurite outgrowth 

Abbreviations

ApSec7

Aplysia Sec7 protein

PH

Pleckstrin homology

PI

Phosphoinositide

ER

Endoplasmic reticulum

OSBP

Oxysterol-binding protein

FAPP1/2

PI4P adaptor protein ½

CERT

Ceramide transfer protein

TGN

Trans-Golgi network

PLC

Phospholipase

GEF

GDP/GTP exchange factor

ARF

ADP ribosylation factor

ARNO

ARF binding site opener

FRB

Rapamycin-binding domain of mTOR

FKBP

FK506 binding protein 12

PJ

Pseudojanin

INPP5E

Polyphosphate 5-phosphatase E

PAO

Phenylarsine oxide

PI4K

PI4 kinase

MD

Molecular dynamics

Notes

Acknowledgments

This work was supported by Basic Science Research Program through NRF (2014-R1A1A2012804) funded by the Ministry of Education (D.-J. J), Science and Technology, and by the National Honor Scientist Program of Korea (D.-J. J & B.-K. K). S.K and I.C are supported by the MIREBraiN program of DGIST and the National Creative Research Initiatives (Center for Proteome Biophysics) of National Research Foundation, Korea (No. 2008-0061984).

Conflict of interest

There is no conflict of interest.

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Copyright information

© AOCS 2015

Authors and Affiliations

  • Yong-Woo Jun
    • 1
  • Sangyeol Kim
    • 2
    • 4
  • Kun-Hyung Kim
    • 1
  • Jin-A Lee
    • 5
  • Chae-Seok Lim
    • 6
  • Iksoo Chang
    • 2
    • 3
  • Byung-Chang Suh
    • 3
  • Bong-Kiun Kaang
    • 6
  • Deok-Jin Jang
    • 1
    Email author
  1. 1.Department of Ecological Science, College of Ecology and EnvironmentKyungpook National UniversitySang-juKorea
  2. 2.Center for Proteome BiophysicsDaeguKorea
  3. 3.Department of Brain ScienceDaegu Gyeongbuk Institute of Science and TechnologyDaeguKorea
  4. 4.Department of PhysicsPusan National UniversityPusanKorea
  5. 5.Department of Biotechnology, College of Life Science and Nano TechnologyHannam UniversityDaejeonKorea
  6. 6.Department of Biological Sciences, College of Natural SciencesSeoul National UniversitySeoulKorea

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