생명과학회지 (Journal of Life Science)

  • 제28권2호
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  • Pages.162-169
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  • 2018
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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Phenylarsine oxide와 adenosine에 민감한 sulfatide 결합 펩타이드의 trans-golgi network 타기팅

Phenylarsine Oxide and Adenosine-sensitive Trans-golgi Complex Targeting of GFP Fused to Modified Sulfatide-binding Peptide

  • 전용우 (경북대학교 생태환경대학 생태과학과) ;
  • 이진아 (한남대학교 생명나노과학대학 생명시스템과학과) ;
  • 장덕진 (경북대학교 생태환경대학 생태과학과)
  • Jun, Yong-Woo (Department of Ecological Science, College of Ecology and Environment, Kyungpook National University) ;
  • Lee, Jin-A (Department of Biotechnology and Biological Science, College of Life Science and Nanotechnology, Hannam University) ;
  • Jang, Deok-Jin (Department of Ecological Science, College of Ecology and Environment, Kyungpook National University)
  • 투고 : 2017.09.28
  • 심사 : 2017.12.07
  • 발행 : 2018.02.28
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초록

세포기질에 존재하는 많은 종류의 단백질들은 N-말단에 존재하는 짧은 펩타이드들에 의해서 trans-golgi network(TGN)의 세포질쪽 막에 타기팅될때 중요한 역할을 수행한다고 보고되고 있다. 본 연구실에서도 이전에 바다달팽이인 군소에서 클로닝된 phosphodiesterase 4의 long-form의 경우 N-말단에 존재하는 20개의 아미노산 서열만으로도 충분히 HEK293T세포의 TGN의 세포질막에 타기팅 되게 하며, 이 펩타이드가 sulfatide와 PI4P에 결합성이 있다는 사실을 in vitro에서 확인하였다. 그래서, 본 연구에서는 sulfatide결합성과 TGN막 타기팅과의 연관성을 연구하고자 하였다. 이를 위해 우선 이전 문헌을 통해 sulfatide결합 펩타이드를 찾았고, 이를 GFP단백질과 융합하여 재조합 단백질(mHSBP-EGFP)을 만들어 세포내 타기팅을 실험해 보았다. 이러한 연구를 수행한 결과, mHSBP-EGFP가 HEK293T세포에서 TGN에 타기팅 되고, sulfatide결합이 망가진 돌연변이는 타기팅이 사라짐을 확인하였다. 또한, mHSBP-EGFP가 TGN에 타기팅 되는 것은 억제제인 antimycin A와 PAO와 adenosine에 의해 억제됨을 확인할 수 있었다. 이러한 사실을 통해, PAO와 adenosine에 민감한 인산화효소들, 그중에 PI4KII의 활성이 mHSBP-EGFP를 TGN으로 위치하게 하는데 중요한 역할을 수행한다고 추론할 수 있다.

Many cytoplasmic proteins are targeted to the cytoplasmic membrane of the trans-Golgi network (TGN) via an N-terminal short helix. We previously showed that the 20 N-terminal amino acids of Aplysia phosphodiesterase 4 (ApPDE4) long form are sufficient for its targeting to the plasma membrane and the TGN. The N-terminus of the ApPDE4 long form binds to PI4P and sulfatide in vitro. Therefore, in order to decipher the roles of sulfatide in Golgi complex targeting, we examined the cellular localization of sulfatide-binding peptides. In this study, we found that enhanced green fluorescent protein (EGFP) fused to the C-terminus of modified sulfatide- and heparin-binding peptides (mHSBP-EGFP) was localized to the TGN. On the other hand, its mutant, in which tryptophan was replaced with an alanine, leading to the impairment of heparin and sulfatide binding, was localized to cytosol. We also found that the TGN targeting of mHSBP-EGFP is impaired by the treatment of antimycin A, phenylarsine oxide (PAO), and adenosine but not a high concentration of wortmannin. These results suggest that PAO and adenosine-sensitive kinases, including phosphatidylinositol 4-kinase II, may play key roles in the recruitment of mHSBP-EGFP.

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참고문헌

  1. Baillie, G. S., Huston, E., Scotland, G., Hodgkin, M., Gall, I., Peden, A. H., MacKenzie, C., Houslay, E. S., Currie, R., Pettitt, T. R., Walmsley, A. R., Wakelam, M. J., Warwicker, J. and Houslay, M. D. 2002. TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid. J. Biol. Chem. 277, 28298-28309. https://doi.org/10.1074/jbc.M108353200
  2. Balla, A., Tuymetova, G., Tsiomenko, A., Varnai, P. and Balla, T. 2005. A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1. Mol. Biol. Cell 16, 1282-1295. https://doi.org/10.1091/mbc.E04-07-0578
  3. Cadwallader, K. A., Paterson, H., Macdonald, S. G. and Hancock, J. F. 1994. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization. Mol. Cell Biol. 14, 4722-4730. https://doi.org/10.1128/MCB.14.7.4722
  4. Di Paolo, G. and De Camilli, P. 2006. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651-657. https://doi.org/10.1038/nature05185
  5. Dickson, E. J., Jensen, J. B. and Hille, B. 2014. Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current. Proc. Natl. Acad. Sci. USA. 111, E2281-2290. https://doi.org/10.1073/pnas.1407133111
  6. Godi, A., Di Campli, A., Konstantakopoulos, A., Di Tullio, G., Alessi, D. R., Kular, G. S., Daniele, T., Marra, P., Lucocq, J. M. and De Matteis, M. A. 2004. FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns (4) P. Nat. Cell Biol. 6, 393-404. https://doi.org/10.1038/ncb1119
  7. Guo, N. H., Krutzsch, H. C., Negre, E., Vogel, T., Blake, D. A. and Roberts, D. D. 1992. Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion. Proc. Natl. Acad. Sci. USA. 89, 3040-3044. https://doi.org/10.1073/pnas.89.7.3040
  8. Hanada, K., Kumagai, K., Yasuda, S., Miura, Y., Kawano, M., Fukasawa, M. and Nishijima, M. 2003. Molecular machinery for non-vesicular trafficking of ceramide. Nature 426, 803-809. https://doi.org/10.1038/nature02188
  9. Houslay, M. D. and Adams, D. R. 2003. PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem. J. 370, 1-18. https://doi.org/10.1042/bj20021698
  10. Huston, E., Gall, I., Houslay, T. M. and Houslay, M. D. 2006. Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+. J. Cell Sci. 119, 3799-3810. https://doi.org/10.1242/jcs.03106
  11. Jang, D. J., Park, S. W. and Kaang, B. K. 2009. The role of lipid binding for the targeting of synaptic proteins into synaptic vesicles. BMB Rep. 42, 1-5. https://doi.org/10.5483/BMBRep.2009.42.1.001
  12. Jang, D. J., Park, S. W., Lee, J. A., Lee, C., Chae, Y. S., Park, H., Kim, M. J., Choi, S. L., Lee, N., Kim, H. and Kaang, B. K. 2010. N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes. Learn. Mem. 17, 469-479. https://doi.org/10.1101/lm.1899410
  13. Jun, Y. W., Lee, J. A., Kaang, B. K. and Jang, D. J. 2017. PI4KII activity-dependent Golgi complex targeting of Aplysia phosphodiesterase 4 long-form mutant. Animal Cells Syst. 21, 316-322. https://doi.org/10.1080/19768354.2017.1371073
  14. Kanaani, J., Patterson, G., Schaufele, F., Lippincott-Schwartz, J. and Baekkeskov, S. 2008. A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes. J. Cell Sci. 121, 437-449. https://doi.org/10.1242/jcs.011916
  15. Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N. and Sheng, M. 1995. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378, 85-88. https://doi.org/10.1038/378085a0
  16. Kim, K. H., Jun, Y. W., Park, Y., Lee, J. A., Suh, B. C., Lim, C. S., Lee, Y. S., Kaang, B. K. and Jang, D. J. 2014. Intracellular membrane association of the Aplysia cAMP phosphodiesterase long and short forms via different targeting mechanisms. J. Biol. Chem. 289, 25797-25811. https://doi.org/10.1074/jbc.M114.572222
  17. Lemmon, M. A. 2008. Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 9, 99-111. https://doi.org/10.1038/nrm2328
  18. Levine, T. P. and Munro, S. 2002. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695-704.
  19. Miller, S., Sparacio, S. and Bartenschlager, R. 2006. Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B. J. Biol. Chem. 281, 8854-8863. https://doi.org/10.1074/jbc.M512697200