Membrane Topology and Transient Acylation ofToxoplasma gondiiGlycosylphosphatidylinositols

Abstract

Using hypotonically permeabilized

The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway

“…This clearly shows for the first time that the previously observed differences in the R F of T. gondii GPI intermediates formed by a cell-free system and those formed by an in vivo labeling (40,44,49) are not due to a difference in the lipid moiety, leaving the obvious conclusion that in an in vivo labeling there is an extra, as yet uncharacterized labile component attached to the glycan core that is causing the observed lower R F . The identification of this elusive component is presently being investigated.…”

“…7) is to ensure full mannosylation of GPI intermediates before the addition of the GalNAc side chain on the first mannose. Together with the recent T. gondii topology study (49) and similar topology models for GPI biosynthetic pathways in other organisms (Ref. 42; for review, see Refs.…”

“…4 Using hypotonically permeabilized T. gondii tachyzoites, the topology of the 4 M. Hyams and R. T. Schwarz, unpublished information. free GPIs within the endoplasmic reticulum membrane was recently investigated (49). They demonstrate that the higher mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are preferentially localized on the cytoplasmic leaflet of the ER (49).…”

“…free GPIs within the endoplasmic reticulum membrane was recently investigated (49). They demonstrate that the higher mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are preferentially localized on the cytoplasmic leaflet of the ER (49). A new early intermediate with an acyl modification on the inositol was identified indicating that inositol acylation also occurs in T. gondii.…”

“…These early events occur on the cytoplasmic face of the endoplasmic reticulum, whereas the subsequent steps of mannosylation by three separate Dol-P-Man-dependent mannosyltransferases (23) occurs on the luminal face (10), this followed by the transfer of ethanolamine phosphate to the third mannose from phosphatidylethanolamine (48). A recent study on the topology of the Toxoplasma GPI biosynthesis suggests the transfer of GalNAc and Glc to the first mannose takes place on the cytoplasmic face of the ER (49). The pre-assembled glycolipid GPI precursor is then flipped inside the ER where it is transferred en bloc to a protein via a transamidase reaction involving the cleavage of a hydrophobic C-terminal GPI signal sequence (for review, see Refs.…”