Deletion of GPI7, a Yeast Gene Required for Addition of a Side Chain to the Glycosylphosphatidylinositol (GPI) Core Structure, Affects GPI Protein Transport, Remodeling, and Cell Wall Integrity

Abstract

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in ⌬gpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9 -11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiesterlinked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the ␣1,6-linked mannose of the GPI core structure. Glycosylphosphatidylinositol (GPI)1 -anchored proteins represent a subclass of surface proteins found in virtually all eukaryotic organisms (1). The genome of Saccharomyces cerevisiae contains more than 70 open reading frames (ORFs) encoding for proteins that, as judged from the deduced primary sequence, can be predicted to be modified by the attachment of a GPI anchor (2, 3). In about 25 of them, the presence of an anchor has been confirmed biochemically. A majority of them lose part of the anchor and become covalently attached to the ␤1,6-glucans of the cell wall (4 -6). A minority of GPI proteins retain the GPI anchor in an intact form and stay at the plasma membrane (PM). For the biosynthesis of GPI anchors, phosphatidylinositol (PI) is modified by the stepwise addition of sugars and ethanolamine phosphate (EtN-P), thus forming a complete precursor lipid (CP) which subsequently is transferred en bloc by a transamidase onto newly synthesized proteins in the ER (7,8). The identification of genes involved in the biosynthesis of the CP and its subsequent attachment to proteins has been possible through the complementation of mammalian and yeast gpi Ϫ mutants, i.e. mutants being deficient in GPI anchoring of membrane proteins (7, 9 -20). In our laboratory, a series of recessive gpi Ϫ mutants (gpi4 to gpi10) has been obtained by screening for yeast mutants that are unable to display the GPI-anchored ␣-agglutinin (Sag1p) at the outer surface of the cell wall, although the synthesis and secretion of soluble proteins is normal (21,22).Here we report on the characterization of...

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

“…Indeed, an EtN-P is invariably found on Man1 of GPI proteins and on the GPI lipids in mammals and in Torpedo californica, but is not found in Trypanosoma brucei, Leishmania major, or Plasmodium falciparum (3)(4)(5)(6)(7)(8)(9)(10). EtN-P attached to Man1 and Man2, respectively, has also been identified on the complete GPI precursor lipid CP2 of Saccharomyces cerevisiae (11)(12)(13) although the identity of the substituent on Man2 has not been formally demonstrated. The presence of these side chains on CP2 came as a surprise in as much as a previous analysis of the pool of the protein-linked GPI anchors of S. cerevisiae had failed to reveal EtN-P or other substituents on Man1 or Man2 (14).…”

“…HF-sensitive Substituents on Man2-We tried to investigate if GPI anchors carried any substituents on Man2 by using limiting HF digestion, a procedure that had previously been used successfully to detect an HF-sensitive substituent on Man2 of [ 3 H]mannose-labeled CP2 of pmi40 cells (12). As can be appreciated from Fig.…”

“…The presence of these side chains on CP2 came as a surprise in as much as a previous analysis of the pool of the protein-linked GPI anchors of S. cerevisiae had failed to reveal EtN-P or other substituents on Man1 or Man2 (14). Candidate EtN-P transferase genes required for the attachment of these substituents have been identified in humans and yeast: PIG-N and MCD4 are involved in the transfer of EtN-P from phosphatidylethanolamine (PE) onto Man1 (13,15,16), GPI7 in the transfer onto Man2 (12), and PIG-O and GPI13 in the transfer of EtN-P from PE onto Man3 (17)(18)(19)(20)(21). These genes are homologous to each other, are found throughout the eukaryotic kingdom, possess an N-terminal globular domain facing the lumen of the ER or the extracellular space and multiple transmembrane domains in their C terminus.…”

Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

“…GPI7 is involved in the addition of ethanolamine phosphate to the second mannose of GPI (Benachour et al, 1999;Fujita et al, 2004). We used a gwt1-20 mutant that has a partial defect in inositol acylation activity at 30°C, and a gpi7⌬ mutant that shows a temperature-sensitive growth phenotype.…”

SETH1andSETH2, Two Components of the Glycosylphosphatidylinositol Anchor Biosynthetic Pathway, Are Required for Pollen Germination and Tube Growth in Arabidopsis [W]

“…Partial deficiency of DPM biosynthesis in human causes the congenital disorder of glycosylation, resulting in seizures, hypotonia, and dysmorphic features (Imbach et al, 2000). Moreover, side chain addition mutations have drastic effects on GPI protein transport, remodeling, and cell wall integrity in yeast (Benachour et al, 1999;Gaynor et al, 1999) but only partially affect the expression of GPI-anchored surface receptors in murine embryonal carcinoma cells (Hong et al, 1999). However, the effects of such mutations on embryogenesis or cell differentiation are unknown.…”