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Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy

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Abstract

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.

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References

  1. Yu RK, Bieberich E, Xia T, Zeng G (2004) Regulation of ganglioside biosynthesis in the nervous system. J Lipid Res 45(5):783–793

    Article  PubMed  CAS  Google Scholar 

  2. Maxzud MK, Daniotti JL, Maccioni HJ (1995) Functional coupling of glycosyl transfer steps for synthesis of gangliosides in Golgi membranes from neural retina cells. J Biol Chem 270(34):20207–20214

    Article  PubMed  CAS  Google Scholar 

  3. Maccioni HJ (2007) Glycosylation of glycolipids in the Golgi complex. J Neurochem 103(Suppl 1):81–90

    Article  PubMed  CAS  Google Scholar 

  4. de Graffenried CL, Bertozzi CR (2004) The roles of enzyme localisation and complex formation in glycan assembly within the Golgi apparatus. Curr Opin Cell Biol 16(4):356–363

    Article  PubMed  Google Scholar 

  5. Young WW Jr (2004) Organization of Golgi glycosyltransferases in membranes: complexity via complexes. J Membr Biol 198(1):1–13

    Article  PubMed  CAS  Google Scholar 

  6. Giraudo CG, Daniotti JL, Maccioni HJ (2001) Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus. Proc Natl Acad Sci USA 98(4):1625–1630

    Article  PubMed  CAS  Google Scholar 

  7. Giraudo CG, Maccioni HJ (2003) Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells. J Biol Chem 278(41):40262–40271

    Article  PubMed  CAS  Google Scholar 

  8. Hassinen A, Pujol FM, Kokkonen N, Pieters C, Kihlstrom M, Korhonen K, Kellokumpu S (2011) Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells. J Biol Chem 286(44):38329–38340

    Article  PubMed  CAS  Google Scholar 

  9. Kremers GJ, Goedhart J, van Munster EB, Gadella TW Jr (2006) Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius. Biochemistry 45(21):6570–6580

    Article  PubMed  CAS  Google Scholar 

  10. Elangovan M, Wallrabe H, Chen Y, Day RN, Barroso M, Periasamy A (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29(1):58–73

    Article  PubMed  CAS  Google Scholar 

  11. Trenchi A, Gomez GA, Daniotti JL (2009) Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway. Biochem J 421(3):357–369

    Article  PubMed  CAS  Google Scholar 

  12. Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21(11):1387–1395

    Article  PubMed  CAS  Google Scholar 

  13. Lippincott-Schwartz J, Snapp E, Kenworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2(6):444–456

    Article  PubMed  CAS  Google Scholar 

  14. Wallrabe H, Periasamy A (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol 16(1):19–27

    Article  PubMed  CAS  Google Scholar 

  15. Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83(6):3652–3664

    Article  PubMed  CAS  Google Scholar 

  16. Uliana AS, Giraudo CG, Maccioni HJ (2006) Cytoplasmic tails of SialT2 and GalNAcT impose their respective proximal and distal Golgi localization. Traffic 7(5):604–612

    Article  PubMed  CAS  Google Scholar 

  17. Kenworthy AK, Edidin M (1998) Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 Å using imaging fluorescence resonance energy transfer. J Cell Biol 142(1):69–84

    Article  PubMed  CAS  Google Scholar 

  18. Wallrabe H, Elangovan M, Burchard A, Periasamy A, Barroso M (2003) Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. Biophys J 85(1):559–571

    Article  PubMed  CAS  Google Scholar 

  19. Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74(5):2702–2713

    Article  PubMed  CAS  Google Scholar 

  20. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Physik 6(2):55–75

    Article  Google Scholar 

  21. Giraudo CG, Rosales Fritz VM, Maccioni HJ (1999) GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells. Biochem J 342(Pt 3):633–640

    Article  PubMed  CAS  Google Scholar 

  22. Panzetta P, Maccioni HJ, Caputto R (1980) Synthesis of retinal gangliosides during chick embryonic development. J Neurochem 35(1):100–108

    Article  PubMed  CAS  Google Scholar 

  23. Yu RK, Macala LJ, Taki T, Weinfield HM, Yu FS (1988) Developmental changes in ganglioside composition and synthesis in embryonic rat brain. J Neurochem 50(6):1825–1829

    Article  PubMed  CAS  Google Scholar 

  24. Maccioni HJ, Quiroga R, Ferrari ML (2011) Cellular and molecular biology of glycosphingolipid glycosylation. J Neurochem 117(4):589–602

    PubMed  CAS  Google Scholar 

  25. Galperin E, Verkhusha VV, Sorkin A (2004) Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells. Nat Methods 1(3):209–217

    Article  PubMed  CAS  Google Scholar 

  26. Pohlentz G, Klein D, Schwarzmann G, Schmitz D, Sandhoff K (1988) Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver. Proc Natl Acad Sci USA 85(19):7044–7048

    Article  PubMed  CAS  Google Scholar 

  27. Martina JA, Daniotti JL, Maccioni HJ (2000) GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex. Neurochem Res 25(5):725–731

    Article  PubMed  CAS  Google Scholar 

  28. Li J, Yen TY, Allende ML, Joshi RK, Cai J, Pierce WM, Jaskiewicz E, Darling DS, Macher BA, Young WW Jr (2000) Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains. J Biol Chem 275(52):41476–41486

    Article  PubMed  CAS  Google Scholar 

  29. Bieberich E, MacKinnon S, Silva J, Li DD, Tencomnao T, Irwin L, Kapitonov D, Yu RK (2002) Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. Biochemistry 41(38):11479–11487

    Article  PubMed  CAS  Google Scholar 

  30. Hassinen A, Rivinoja A, Kauppila A, Kellokumpu S (2010) Golgi N-glycosyltransferases form both homo- and heterodimeric enzyme complexes in live cells. J Biol Chem 285(23):17771–17777

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by Grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT·2006-01239), Universidad Nacional de Córdoba, y MinCyT Córdoba. We thank the technical assistance of G. Schachner and S. Deza with cell cultures and of C. Mas and M. C. Sampedro with confocal microscopy. M.L.F. and G.A.G. were recipients of CONICET Fellowships and H.J.F.M. is Career Investigator of CONICET (Argentina).

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Author notes

  1. Guillermo A. Gomez

    Present address: Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, QLD, 4072, Australia

Authors and Affiliations

  1. Departamento de Química Biológica, Facultad de Ciencias Químicas, Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Universidad Nacional de Córdoba, Ciudad Universitaria, 5000, Córdoba, Argentina

    Mariana L. Ferrari, Guillermo A. Gomez & Hugo J. F. Maccioni

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  1. Mariana L. Ferrari
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  2. Guillermo A. Gomez
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  3. Hugo J. F. Maccioni
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Correspondence to Hugo J. F. Maccioni.

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Special Issue: In Honor of Bob Ledeen

This work is dedicated to Bob Ledeen, a pioneer in the study of the biochemistry and neurochemistry of glycolipids, great friend and enthusiastic and clever investigator.

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Ferrari, M.L., Gomez, G.A. & Maccioni, H.J.F. Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy. Neurochem Res 37, 1325–1334 (2012). https://doi.org/10.1007/s11064-012-0741-1

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  • DOI: https://doi.org/10.1007/s11064-012-0741-1

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