MCD4Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Abstract

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 NH 2 -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 [ 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. † These authors contributed equally to this work. INTRODUCTIONProtein transport through the secretory pathway involves multiple steps that are conserved throughout eukaryotes (Palade, 1975;Schekman, 1985;Rothman, 1994;Schekman and Orci, 1996). Secreted or cell surface proteins are first translocated into the endoplasmic reticulum (ER) and then packaged into COPIIcoated vesicle intermediates for transport to the Golgi complex. Upon reaching the trans-Golgi, these proteins undergo another vesicle packaging event and enter secretory vesicles that ultimately dock and fuse with the plasma membrane. A striking and readily observable example of the secretory process is that of polarized secretion in the budding yeast Saccharomyces cerevisiae, where efficient targeting of secretory vesicles to newly em...

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

“…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.…”

“…GPI11, the yeast homologue of PIG-F, is not required for the addition of EtN-P to Man3 but may be required for the addition of a hydrofluoric acid (HF) labile substituent to Man2 (18). In yeast, deletion of MCD4, GPI11, or GPI13 is lethal, whereas deletion of GPI7 only compromises cell wall integrity (15,18,19,24). This is not unexpected for GPI13, as in all GPI proteins analyzed it invariably is the EtN-P on Man3 that links the GPI to the protein, and even in mutants that cannot add EtN-P to Man3 or cannot add Man3, the GPI proteins were never found to be attached through an EtN-P on Man1 or Man2.…”

“…In this context it was of interest to know the structure of the intermediates accumulating in mcd4 mutants. Several mcd4 mutants had previously been labeled with [ 3 H]inositol but accumulated only very small amounts of Ins-labeled GPI lipids that could not be structurally analyzed (15,19,37). We therefore decided to analyze the in vitro GPI biosynthesis in yeast strains carrying either a temperature-sensitive mutation in MCD4/SSU21 (38) or the wild-type MCD4 gene under control of the GAL1 promoter (19).…”

The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p

“…As shown in Figure 11, Gpi8p remained completely confined within the complexes of 430 -650 kDa when prepro forms of GPI proteins were depleted with the use of cycloheximide, a treatment that was effective since it led to the complete disappearance of the immature 105-kDa form of the GPI protein Gas1p ( Figure 11B). The transamidase complex also persisted after a temperature shift of mutants that, upon a shift to 37°C, block the biosynthesis of GPI lipids at very early stages (gpi1 and mcd4) or interrupt the transfer of GPI lipids onto proteins (gaa1) (Hamburger et al, 1995;Leidich and Orlean, 1996;Gaynor et al, 1999;Packeiser et al, 1999). For gpi1, the efficiency of the block was assessed by following the gradual accumulation of the immature Gas1p ( Figure 2B).…”

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.…”