Analysis of Phosphoinositide-binding Properties and Subcellular Localization of GFP-fusion Proteins

Overview

Journal Lipids

Specialty Biochemistry

Date 2015 Feb 18

PMID 25688026

Citations 2

Authors

Yong-Woo Jun

Sangyeol Kim

Kun-Hyung Kim

Jin-A Lee

Chae-Seok Lim

Iksoo Chang

Byung-Chang Suh

Bong-Kiun Kaang

Deok-Jin Jang

Affiliations

Soon will be listed here.

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.

Citing Articles

Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane.

Jun Y, Lee J, Jang D Anim Cells Syst (Seoul). 2019; 23(3):164-169.

PMID: 31231579 PMC: 6567041. DOI: 10.1080/19768354.2019.1599424.

Distinct regulations of ARF1 by two Sec7 isoforms.

Jun Y, Lee Y, Lee J, Kaang B, Jang D Anim Cells Syst (Seoul). 2018; 21(1):10-16.

PMID: 30460046 PMC: 6138325. DOI: 10.1080/19768354.2016.1276025.

References

Ferguson K, Lemmon M, Schlessinger J, SIGLER P . Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell. 1995; 83(6):1037-46. DOI: 10.1016/0092-8674(95)90219-8. View

Suh B, Hille B . PIP2 is a necessary cofactor for ion channel function: how and why?. Annu Rev Biophys. 2008; 37:175-95. PMC: 2692585. DOI: 10.1146/annurev.biophys.37.032807.125859. View

Venkateswarlu K, Oatey P, Tavare J, Cullen P . Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr Biol. 1998; 8(8):463-6. DOI: 10.1016/s0960-9822(98)70181-2. View

Balla A, Kim Y, Varnai P, Szentpetery Z, Knight Z, Shokat K . Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha. Mol Biol Cell. 2007; 19(2):711-21. PMC: 2230591. DOI: 10.1091/mbc.e07-07-0713. View

Lemmon M . Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol. 2008; 9(2):99-111. DOI: 10.1038/nrm2328. View

Lee S, Shim J, Choi S, Lee N, Lee C, Bailey C . Learning-related synaptic growth mediated by internalization of Aplysia cell adhesion molecule is controlled by membrane phosphatidylinositol 4,5-bisphosphate synthetic pathway. J Neurosci. 2012; 32(46):16296-305. PMC: 6794033. DOI: 10.1523/JNEUROSCI.1872-12.2012. View

Jang D, Park S, Lee J, Lee C, Chae Y, Park H . N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes. Learn Mem. 2010; 17(9):469-79. DOI: 10.1101/lm.1899410. View

Wang X, Li W, Zhao D, Liu B, Shi Y, Chen B . Caenorhabditis elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor. Nat Cell Biol. 2010; 12(7):655-64. PMC: 2896453. DOI: 10.1038/ncb2068. View

Frank S, Upender S, Hansen S, Casanova J . ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6. J Biol Chem. 1998; 273(1):23-7. DOI: 10.1074/jbc.273.1.23. View

10.

Franke T, Kaplan D, Cantley L, Toker A . Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997; 275(5300):665-8. DOI: 10.1126/science.275.5300.665. View

11.

Kim K, Jun Y, Park Y, Lee J, Suh B, Lim C . Intracellular membrane association of the Aplysia cAMP phosphodiesterase long and short forms via different targeting mechanisms. J Biol Chem. 2014; 289(37):25797-811. PMC: 4162181. DOI: 10.1074/jbc.M114.572222. View

12.

Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M . Molecular machinery for non-vesicular trafficking of ceramide. Nature. 2003; 426(6968):803-9. DOI: 10.1038/nature02188. View

13.

Choi J, Park P, Baek G, Sim S, Kang S, Lee Y . Effects of PI3Kγ overexpression in the hippocampus on synaptic plasticity and spatial learning. Mol Brain. 2014; 7:78. PMC: 4226891. DOI: 10.1186/s13041-014-0078-6. View

14.

Raucher D, Stauffer T, Chen W, Shen K, Guo S, York J . Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell. 2000; 100(2):221-8. DOI: 10.1016/s0092-8674(00)81560-3. View

15.

Huh M, Han J, Lim C, Lee S, Kim S, Kim E . Regulation of neuritogenesis and synaptic transmission by msec7-1, a guanine nucleotide exchange factor, in cultured Aplysia neurons. J Neurochem. 2003; 85(1):282-5. DOI: 10.1046/j.1471-4159.2003.01718.x. View

16.

Levine T, Munro S . Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr Biol. 2002; 12(9):695-704. DOI: 10.1016/s0960-9822(02)00779-0. View

17.

Santy L, Casanova J . Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J Cell Biol. 2001; 154(3):599-610. PMC: 2196419. DOI: 10.1083/jcb.200104019. View

18.

Lemmon M, Ferguson K, OBrien R, SIGLER P, Schlessinger J . Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc Natl Acad Sci U S A. 1995; 92(23):10472-6. PMC: 40633. DOI: 10.1073/pnas.92.23.10472. View

19.

Suh B, Inoue T, Meyer T, Hille B . Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science. 2006; 314(5804):1454-7. PMC: 3579521. DOI: 10.1126/science.1131163. View

20.

Garcia P, Gupta R, Shah S, Morris A, Rudge S, Scarlata S . The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes. Biochemistry. 1995; 34(49):16228-34. DOI: 10.1021/bi00049a039. View