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Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha

  • Avanti Gokhale
  • Pearl V. Ryder
  • Stephanie A. Zlatic
  • Victor FaundezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

Abstract

Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KIIα participates in transient, low-affinity interactions that are stabilized by the use of DSP. Using this optimized protocol we have successfully identified actin cytoskeleton regulators—the WASH complex and RhoGEF1, as major novel interactors of PI4KIIα. While this chapter focuses on the PI4KIIα interactome, this protocol can and has been used to generate other membrane interactome networks.

Key words

Phosphatidylinositol Phosphatidylinositol kinase Phosphoinositide Phospholipid Endosome Interactome Mass spectrometry Cross-linking 

Notes

Acknowledgments

This work was supported by grants from the National Institutes of Health (GM077569) and CHOA Children’s Center for Neuroscience to V.F. P.V.R. was supported by National Research Service Award Fellowship F31NS0765.

References

  1. 1.
    De Matteis MA, Di Campli A, Godi A (2005) The role of the phosphoinositides at the Golgi complex. Biochim Biophys Acta 1744(3):396–405. doi: 10.1016/j.bbamcr.2005.04.013 PubMedCrossRefGoogle Scholar
  2. 2.
    Balla A, Balla T (2006) Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol 16(7):351–361PubMedCrossRefGoogle Scholar
  3. 3.
    Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112):651–657PubMedCrossRefGoogle Scholar
  4. 4.
    Tan J, Brill JA (2014) Cinderella story: PI4P goes from precursor to key signaling molecule. Crit Rev Biochem Mol Biol 49(1):33–58. doi: 10.3109/10409238.2013.853024 PubMedCrossRefGoogle Scholar
  5. 5.
    Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93(3):1019–1137. doi: 10.1152/physrev.00028.2012 PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Kim YJ, Hernandez ML, Balla T (2013) Inositol lipid regulation of lipid transfer in specialized membrane domains. Trends Cell Biol 23(6):270–278. doi: 10.1016/j.tcb.2013.01.009 PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Wieffer M, Cibrian Uhalte E, Posor Y, Otten C, Branz K, Schutz I, Mossinger J, Schu P, Abdelilah-Seyfried S, Krauss M, Haucke V (2013) PI4K2beta/AP-1-based TGN-endosomal sorting regulates Wnt signaling. Curr Biol 23(21):2185–2190. doi: 10.1016/j.cub.2013.09.017 PubMedCrossRefGoogle Scholar
  8. 8.
    Waugh MG, Minogue S, Chotai D, Berditchevski F, Hsuan JJ (2006) Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal Rafts. J Biol Chem 281(7):3757–3763. doi: 10.1074/jbc.M506527200 PubMedCrossRefGoogle Scholar
  9. 9.
    Balla A, Tuymetova G, Barshishat M, Geiszt M, Balla T (2002) Characterization of type II phosphatidylinositol 4-kinase isoforms reveals association of the enzymes with endosomal vesicular compartments. J Biol Chem 277(22):20041–20050PubMedCrossRefGoogle Scholar
  10. 10.
    Barylko B, Gerber SH, Binns DD, Grichine N, Khvotchev M, Sudhof TC, Albanesi JP (2001) A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans. J Biol Chem 276(11):7705–7708PubMedCrossRefGoogle Scholar
  11. 11.
    Craige B, Salazar G, Faundez V (2008) Phosphatidylinositol-4-kinase type II alpha contains an AP-3 sorting motif and a kinase domain that are both required for endosome traffic. Mol Biol Cell 19:1415–1426PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Wang YJ, Wang J, Sun HQ, Martinez M, Sun YX, Macia E, Kirchhausen T, Albanesi JP, Roth MG, Yin HL (2003) Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114(3):299–310PubMedCrossRefGoogle Scholar
  13. 13.
    Simons JP, Al-Shawi R, Minogue S, Waugh MG, Wiedemann C, Evangelou S, Loesch A, Sihra TS, King R, Warner TT, Hsuan JJ (2009) Loss of phosphatidylinositol 4-kinase 2alpha activity causes late onset degeneration of spinal cord axons. Proc Natl Acad Sci U S A 106(28):11535–11539. doi: 10.1073/pnas.0903011106, 0903011106 [pii]PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Minogue S, Anderson JS, Waugh MG, dos Santos M, Corless S, Cramer R, Hsuan JJ (2001) Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family. J Biol Chem 276(20):16635–16640PubMedCrossRefGoogle Scholar
  15. 15.
    Minogue S, Waugh MG, De Matteis MA, Stephens DJ, Berditchevski F, Hsuan JJ (2006) Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor. J Cell Sci 119(Pt 3):571–581PubMedCrossRefGoogle Scholar
  16. 16.
    Hammond GR, Machner MP, Balla T (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205(1):113–126. doi: 10.1083/jcb.201312072 PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Li J, Lu Y, Zhang J, Kang H, Qin Z, Chen C (2010) PI4KIIalpha is a novel regulator of tumor growth by its action on angiogenesis and HIF-1alpha regulation. Oncogene 29(17):2550–2559. doi: 10.1038/onc.2010.14 PubMedCrossRefGoogle Scholar
  18. 18.
    Qin Y, Li L, Pan W, Wu D (2009) Regulation of phosphatidylinositol kinases and metabolism by Wnt3a and Dvl. J Biol Chem 284(34):22544–22548. doi: 10.1074/jbc.M109.014399 PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Ryder PV, Vistein R, Gokhale A, Seaman MN, Puthenveedu M, Faundez V (2013) The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type II alpha. Mol Biol Cell. doi: 10.1091/mbc.E13-02-0088 Google Scholar
  20. 20.
    Gokhale A, Perez-Cornejo P, Duran C, Hartzell HC, Faundez V (2012) A comprehensive strategy to identify stoichiometric membrane protein interactomes. Cell Logist 2:1–8, doi:http://dx.doi.org/10.4161/cl.22717 CrossRefGoogle Scholar
  21. 21.
    Gokhale A, Larimore J, Werner E, So L, Moreno-De-Luca A, Lese-Martin C, Lupashin VV, Smith Y, Faundez V (2012) Quantitative proteomic and genetic analyses of the schizophrenia susceptibility factor dysbindin identify novel roles of the biogenesis of lysosome-related organelles complex 1. J Neurosci 32(11):3697–3711. doi: 10.1523/JNEUROSCI.5640-11.2012 PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Perez-Cornejo P, Gokhale A, Duran C, Cui Y, Xiao Q, Hartzell HC, Faundez V (2012) Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network. Proc Natl Acad Sci U S A 109(26):10376–10381. doi: 10.1073/pnas.1200174109 PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Salazar G, Zlatic S, Craige B, Peden AA, Pohl J, Faundez V (2009) Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells. J Biol Chem 284(3):1790–1802PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Zlatic SA, Ryder PV, Salazar G, Faundez V (2010) Isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography. J Vis Exp 37:1855. doi: 10.3791/1855, 1810.3791/1855 [pii]. 1855 [pii]PubMedGoogle Scholar
  25. 25.
    Lomant AJ, Fairbanks G (1976) Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol 104(1):243–261PubMedCrossRefGoogle Scholar
  26. 26.
    Blackstone C, O’Kane CJ, Reid E (2011) Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat Rev Neurosci 12(1):31–42. doi: 10.1038/nrn2946, nrn2946 [pii]PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Avanti Gokhale
    • 1
  • Pearl V. Ryder
    • 1
  • Stephanie A. Zlatic
    • 1
  • Victor Faundez
    • 1
    • 2
    Email author
  1. 1.Department of Cell BiologyEmory UniversityAtlantaUSA
  2. 2.Center for Social Translational NeuroscienceEmory UniversityAtlantaUSA

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