Glycosylphosphatidylinositol lipid anchoring of plant proteins. Sensitive prediction from sequence- and genome-wide studies for Arabidopsis and rice

Posttranslational glycosylphosphatidylinositol (GPI) lipid anchoring is common not only for animal and fungal but also for plant proteins. The attachment of the GPI moiety to the carboxyl-terminus after proteolytic cleavage of a C-terminal propeptide is performed by the transamidase complex. Its four known subunits also have obvious full-length orthologs in the Arabidopsis and rice (Oryza sativa) genomes; thus, the mechanism of substrate protein processing appears similar for all eukaryotes. A learning set of plant proteins (substrates for the transamidase complex) has been collected both from the literature and plant sequence databases. We find that the plant GPI lipid anchor motif differs in minor aspects from the animal signal (e.g. the plant hydrophobic tail region can contain a higher fraction of aromatic residues). We have developed the "big-Pi plant" program for prediction of compatibility of query protein C-termini with the plant GPI lipid anchor motif requirements. Validation tests show that the sensitivity for transamidase targets is approximately 94%, and the rate of false positive prediction is about 0.1%. Thus, the big-Pi predictor can be applied as unsupervised genome annotation and target selection tool. The program is also suited for the design of modified protein constructs to test their GPI lipid anchoring capacity. The big-Pi plant predictor Web server and lists of potential plant precursor proteins in Swiss-Prot, SPTrEMBL, Arabidopsis, and rice proteomes are available at http://mendel.imp.univie.ac.at/gpi/plants/gpi_plants.html. Arabidopsis and rice protein hits have been functionally classified. Several GPI lipid-anchored arabinogalactan-related proteins have been identified in rice.     

Post-translational GPI lipid anchor modification of proteins in kingdoms of life: analysis of protein sequence data from complete genomes

To investigate the occurrence of glycosylphosphatidylinositol (GPI) lipid anchor modification in various taxonomic ranges, potential substrate proteins have been searched for in completely sequenced genomes. We applied the big-pi predictor for the recognition of propeptide cleavage and anchor attachment sites with a new, generalized analytical form of the extreme-value distribution for evaluating false-positive prediction rates. (i) We find that GPI modification is present among lower and higher Eukaryota (approximately 0.5% of all proteins) but it seems absent in all eubacterial and three archaeobacterial species studied. Four other archaean genomes appear to encode such a fraction of substrate proteins (in the range of eukaryots) that they cannot be explained as false-positive predictions. This result supports the possible existence of GPI anchor modification in an archaean subgroup. (ii) The frequency of GPI-modified proteins on various chromosomes of a given eukaryotic species is different. (iii) Lists of potentially GPI-modified proteins in complete genomes with their predicted cleavage sites are available at http://mendel.imp.univie.ac.at/gpi/gpi_genomes.html. (iv) Orthologues of known transamidase subunits have been found only for EUKARYA: Inconsistencies in domain structure among homologues some of which may indicate sequencing errors are described. We present a refined model of the transamidase complex.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.     

GPI-anchored proteins and lipid rafts

Several proteins are anchored to membranes via a post-translational lipid modification, the glycosylphosphatidylinositol (GPI) anchor. In mammals and other vertebrates, GPI-anchored proteins have been found in almost all tissues and cells examined. Several studies have provided significant insight into the functions of this ubiquitous modification. An intriguing relevant feature of GPI-anchored proteins is their association with lipid rafts, specialized regions of elevated cholesterol and sphingolipid content, that are present within most cell membranes. In addition to the structure and biosynthesis of the GPI-anchor, recent researches have focused on its molecular interaction with lipid rafts and the biological meaning of such interaction. The aim of this review is to examine the emerging evidences of association between lipid rafts and GPI-anchored proteins, and their relationship with the modulation of important cellular functions such as protein/lipid sorting, signaling mechanisms and with human disease.     

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in?vivo assay

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.     

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

Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that have been translocated across the ER membrane and that display a GPI signal anchor sequence at the C terminus. GPI proteins transit the secretory pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but one of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them out, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, we describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.     

Lipid remodeling of GPI-anchored proteins and its function

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.     

The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins

Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.     

Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.     

Stable Cell Surface Expression of GPI‐Anchored Proteins,but not Intracellular Transport,Depends on their Fatty Acid Structure

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI‐APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI‐APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI‐AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi‐to‐plasma membrane transport or selective endocytosis via GPI‐enriched early endosomal compartment (GEEC)/ clathrin‐independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI‐APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI‐APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI‐APs particularly under potential membrane lipid perturbation.      

Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

PER1 is required for GPI-phospholipase A2 activity and involved in lipid remodeling of GPI-anchored proteins

Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway. We found that GPI-anchored proteins could not associate with the detergent-resistant membranes in per1Delta cells. In addition, the mutant cells had a defect in the lipid remodeling from normal phosphatidylinositol (PI) to a C26 fatty acid-containing PI in the GPI anchor. In vitro analysis showed that PER1 is required for the production of lyso-GPI, suggesting that Per1p possesses or regulates the GPI-phospholipase A2 activity. We also found that human PERLD1 is a functional homologue of PER1. Our results demonstrate for the first time that PER1 encodes an evolutionary conserved component of the GPI anchor remodeling pathway, highlighting the close connection between the lipid remodeling of GPI and raft association of GPI-anchored proteins.     

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.     

Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.     

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.     

Structural remodeling of GPI anchors during biosynthesis and after attachment to proteins

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. In mammalian cells, approximately 150 proteins on the plasma membrane are attached to the cell surface by GPI anchors, which confer specific properties on proteins, such as association with membrane microdomains. The structures of lipid and glycan moieties on GPI anchors are remodeled during biosynthesis and after attachment to proteins. The remodeling processes are critical for transport and microdomain-association of GPI-anchored proteins. Here, we describe the structural remodeling of GPI anchors and genes required for the processes in mammals, yeast, and trypanosomes.     

Two subunits of glycosylphosphatidylinositol transamidase,GPI8 and PIG-T,form a functionally important intermolecular disulfide bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.     

MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.