Glycosylphosphatidylinositol (GPI) Proteins of Saccharomyces cerevisiae Contain Ethanolamine Phosphate Groups on the α1,4-linked Mannose of the GPI Anchor

Abstract

In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7⌬ sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiesterlinked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7⌬ cells by overexpression of PSD1 restore cell growth at 37°C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.

Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

“…These side branches are added during precursor biosynthesis. Protein-bound GPIs of both mammalian and yeast proteins can bear side-branching Etn-Ps on their first mannosyl residue (18,20,24). There is no evidence, however, that side-branching Etn-Ps are involved in linkages to protein (105).…”

“…In yeast, the lipid moiety of many GPIs is remodeled to ceramide (23). Protein-bound GPIs in both yeast and mammals can bear Etn-P on Man-1 (20,24), but it is not clear whether this substituent is invariably retained or whether it can be removed from certain GPIs after anchor transfer to protein. Likewise, the fate of Etn-P moieties added to Man-2 of GPI precursors is unknown.…”

“…In contrast, a conditionally lethal yeast mcd4 allele blocks GPI anchoring (121) and leads to the accumulation of a Man 2 -GPI lacking Etn-P (124). In vitro GPI synthesis by mcd4 membranes also does not proceed beyond Man 2 -GPI (24). The finding that Mcd4p defects lead to the accumulation of Man 2 -GPI lacking Etn-P on Man-1 suggests that Mcd4p normally transfers Etn-P to a dimannosyl GPI, but the accumulation of a Man 1 -GPI with Etn-P on its lone mannose in both yeast and mammalian GPI-Man-T-II mutants (see above) indicates that singly mannosylated GPIs can serve as acceptors for PIG-N/Mcd4p as well.…”

GPI7 Involved in Glycosylphosphatidylinositol Biosynthesis Is Essential for Yeast Cell Separation

“…However, the defective phenotypes of gpi1 and gwt1-20 mutants are dissimilar to those of gpi7 mutant cells. In gpi7 mutants, GPI anchors are still transferred to proteins (5,6,9), whereas the transfer to proteins is defective in other mutants because the biosynthesis of GPI is stopped at the intermediate stage (49 -53). In gwt1-20 cells, Egt2p was not detected at the cell surface after incubation at the non-permissive temperature, consistent with the previous result that Egt2p is modified by the GPI anchor (36) (data not shown).…”

Potential Roles of GPI-Anchor Remodeling in Protein Trafficking and Raft Association in Mammalian Cells

“…The activity and localization of PGAP5 suggest that this reaction is performed in the ER or in the ER-exit sites. The removal of the side chain EtNP from GPI-glycan by PGAP5 is required for the efficient transport of GPI-anchored proteins from the ER to the Golgi, as described in Section D. Also, the 2nd side chain EtNP appears in complete GPI precursors in budding yeast, but is not detected in GPI-anchored proteins (48). There are two PGAP5 homologs (CDC1 and TED1) in yeast.…”