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Eukaryotic Cell, August 2006, p. 1420-1429, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00078-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

Jürgen Kimmel,¹ Terry K. Smith,² Nahid Azzouz,¹ Peter Gerold,¹ Frank Seeber,³ Klaus Lingelbach,³ Jean-François Dubremetz,⁴ and Ralph T. Schwarz^1,5*

Institut für Virologie, Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität, Robert-Koch Strasse 17, 35037 Marburg, Germany,¹ Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, University of Dundee, Dundee, Scotland, United Kingdom,² FB Biologie/Parasitologie, Philipps-Universität Marburg, Karl-von-Frisch-Str., 35032 Marburg, Germany,³ CNRS UMR 5539, Université de Montpellier, 2 Place Eugène Bataillon, 34095 Montpellier Cedex 05, France,⁴ Unité de Glycobiologie Structurale et Fonctionnelle UMR CNRS/USTL no. 8576, Université des Sciences et Technologies de Lille, Bâtiment C9, 59655 Villeneuve D’Ascq Cedex, France⁵

Received 17 March 2006/ Accepted 2 June 2006

Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-³H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-³H]mannose or UDP-[6-³H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER.

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* Corresponding author. Mailing address: Institut für Virologie, Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität, Robert-Koch-Strasse 17, 35037 Marburg, Germany. Phone: 49 6 42 12 86 51 49. Fax: 49 6 42 12 86 89 76. E-mail: schwarz{at}staff.uni-marburg.de.

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Eukaryotic Cell, August 2006, p. 1420-1429, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00078-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Smith, T. K., Kimmel, J., Azzouz, N., Shams-Eldin, H., Schwarz, R. T. (2007). The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway. J. Biol. Chem. 282: 32032-32042 [Abstract] [Full Text]