Membrane topology and transient acylation of Toxoplasma gondii glycosylphosphatidylinositols.
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-(3)H]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-(3)H]mannose or UDP-[6-(3)H]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.
Kimmel
Jürgen
J
Institut für Virologie, Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität, Robert-Koch-Strasse 17, 35037 Marburg, Germany.
Smith
Terry K
TK
Azzouz
Nahid
N
Gerold
Peter
P
Seeber
Frank
F
Lingelbach
Klaus
K
Dubremetz
Jean-François
JF
Schwarz
Ralph T
RT
D000215:Acylation; D000818:Animals; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D014122:Toxoplasma / Q000378:metabolism* / Q000648:ultrastructure*
D017261:Glycosylphosphatidylinositols