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

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Lipids

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.

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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

References

  1. Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443:651–657

    Article  PubMed  Google Scholar 

  2. Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9:99–111

    Article  CAS  PubMed  Google Scholar 

  3. Jang DJ, Park SW, Kaang BK (2009) The role of lipid binding for the targeting of synaptic proteins into synaptic vesicles. BMB Rep 42:1–5

    Article  CAS  PubMed  Google Scholar 

  4. Godi A, Di Campli A, Konstantakopoulos A, Di Tullio G, Alessi DR, Kular GS, Daniele T, Marra P, Lucocq JM, De Matteis MA (2004) FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nat Cell Biol 6:393–404

    Article  CAS  PubMed  Google Scholar 

  5. Levine TP, Munro S (2002) Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr Biol 12:695–704

    Article  CAS  PubMed  Google Scholar 

  6. Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M, Nishijima M (2003) Molecular machinery for non-vesicular trafficking of ceramide. Nature 426:803–809

    Article  CAS  PubMed  Google Scholar 

  7. Lemmon MA, Ferguson KM, O’Brien R, Sigler PB, Schlessinger J (1995) Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc Natl Acad Sci USA 92:10472–10476

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Garcia P, Gupta R, Shah S, Morris AJ, Rudge SA, Scarlata S, Petrova V, McLaughlin S, Rebecchi MJ (1995) The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes. Biochemistry 34:16228–16234

    Article  CAS  PubMed  Google Scholar 

  9. Franke TF, Kaplan DR, Cantley LC, Toker A (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665–668

    Article  CAS  PubMed  Google Scholar 

  10. Jang DJ, Park SW, Lee JA, Lee C, Chae YS, Park H, Kim MJ, Choi SL, Lee N, Kim H, Kaang BK (2010) N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes. Learn Mem 17:469–479

    Article  CAS  PubMed  Google Scholar 

  11. Wang X, Li W, Zhao D, Liu B, Shi Y, Chen B, Yang H, Guo P, Geng X, Shang Z, Peden E, Kage-Nakadai E, Mitani S, Xue D (2010) Caenorhabditis elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor. Nat Cell Biol 12:655–664

    Article  PubMed Central  PubMed  Google Scholar 

  12. Fairn GD, Ogata K, Botelho RJ, Stahl PD, Anderson RA, De Camilli P, Meyer T, Wodak S, Grinstein S (2009) An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis. J Cell Biol 187:701–714

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Lee SH, Shim J, Choi SL, Lee N, Lee CH, Bailey CH, Kandel ER, Jang DJ, Kaang BK (2012) Learning-related synaptic growth mediated by internalization of Aplysia cell adhesion molecule is controlled by membrane phosphatidylinositol 4,5-bisphosphate synthetic pathway. J Neurosci 32:16296–16305

    Article  CAS  PubMed  Google Scholar 

  14. Frank S, Upender S, Hansen SH, Casanova JE (1998) ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6. J Biol Chem 273:23–27

    Article  CAS  PubMed  Google Scholar 

  15. Venkateswarlu K, Oatey PB, Tavare JM, Cullen PJ (1998) Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr Biol 8:463–466

    Article  CAS  PubMed  Google Scholar 

  16. Santy LC, Casanova JE (2001) Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J Cell Biol 154:599–610

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Lee YS (2014) Genes and signaling pathways involved in memory enhancement in mutant mice. Mol Brain 7:43

    Article  PubMed Central  PubMed  Google Scholar 

  18. Choi JH, Park P, Baek GC, Sim SE, Kang S, Lee Y, Ahn SH, Lim CS, Lee YS, Collingridge GL, Kaang BK (2014) Effects of PI3K inverted question mark overexpression in the hippocampus on synaptic plasticity and spatial learning. Mol Brain 7:78

    Article  PubMed Central  PubMed  Google Scholar 

  19. Huh M, Han JH, Lim CS, Lee SH, Kim S, Kim E, Kaang BK (2003) Regulation of neuritogenesis and synaptic transmission by msec7-1, a guanine nucleotide exchange factor, in cultured Aplysia neurons. J Neurochem 85:282–285

    Article  CAS  PubMed  Google Scholar 

  20. Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP, Meyer T (2000) Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221–228

    Article  CAS  PubMed  Google Scholar 

  21. Balla A, Kim YJ, Varnai P, Szentpetery Z, Knight Z, Shokat KM, Balla T (2008) Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha. Mol Biol Cell 19:711–721

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Lee JA, Kim HK, Kim KH, Han JH, Lee YS, Lim CS, Chang DJ, Kubo T, Kaang BK (2001) Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses. Learn Mem 8:220–226

    Article  PubMed Central  PubMed  Google Scholar 

  23. Kaang BK (1996) Parameters influencing ectopic gene expression in Aplysia neurons. Neurosci Lett 221:29–32

    Article  CAS  PubMed  Google Scholar 

  24. Roy A, Levine TP (2004) Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p. J Biol Chem 279:44683–44689

    Article  CAS  PubMed  Google Scholar 

  25. Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, Grinstein S (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319:210–213

    Article  CAS  PubMed  Google Scholar 

  26. Kim KH, Jun YW, Park Y, Lee JA, Suh BC, Lim CS, Lee YS, Kaang BK, Jang DJ (2014) Intracellular membrane association of the Aplysia cAMP phosphodiesterase long and short forms via different targeting mechanisms. J Biol Chem 289:25797–25811

    Article  CAS  PubMed  Google Scholar 

  27. Suh BC, Inoue T, Meyer T, Hille B (2006) Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314:1454–1457

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Ferguson KM, Lemmon MA, Schlessinger J, Sigler PB (1995) Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83:1037–1046

    Article  CAS  PubMed  Google Scholar 

  29. Klarlund JK, Tsiaras W, Holik JJ, Chawla A, Czech MP (2000) Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs. J Biol Chem 275:32816–32821

    Article  CAS  PubMed  Google Scholar 

  30. Ogasawara M, Kim SC, Adamik R, Togawa A, Ferrans VJ, Takeda K, Kirby M, Moss J, Vaughan M (2000) Similarities in function and gene structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors. J Biol Chem 275:3221–3230

    Article  CAS  PubMed  Google Scholar 

  31. Suh BC, Hille B (2008) PIP2 is a necessary cofactor for ion channel function: how and why? Annu Rev Biophys 37:175–195

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

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).

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There is no conflict of interest.

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Authors and Affiliations

  1. Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sang-ju, Korea

    Yong-Woo Jun, Kun-Hyung Kim & Deok-Jin Jang

  2. Center for Proteome Biophysics, Daegu, Korea

    Sangyeol Kim & Iksoo Chang

  3. Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Korea

    Iksoo Chang & Byung-Chang Suh

  4. Department of Physics, Pusan National University, Pusan, Korea

    Sangyeol Kim

  5. Department of Biotechnology, College of Life Science and Nano Technology, Hannam University, Daejeon, Korea

    Jin-A Lee

  6. Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea

    Chae-Seok Lim & Bong-Kiun Kaang

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  1. Yong-Woo Jun
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  2. Sangyeol Kim
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Correspondence to Deok-Jin Jang.

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Jun, YW., Kim, S., Kim, KH. et al. Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins. Lipids 50, 427–436 (2015). https://doi.org/10.1007/s11745-015-3994-z

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  • DOI: https://doi.org/10.1007/s11745-015-3994-z

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