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

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-local ized proteins that serve many important cellular functions. The pathway med iating synthesis and attachment of the GPI anchor to these proteins in euka ryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein fami ly members. In this article, we demonstrate that MCD4, an essential gene th at was initially identified in a genetic screen to isolate Saccharomyces ce revisiae mutants defective for bud emergence, encodes a previously unidenti fied component of the GPI anchor synthesis pathway. Mcd4p is a multimembran e-spanning protein that localizes to the endoplasmic reticulum (ER) and con tains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryo tes and has two yeast homologues. Mcd4p's lumenal domain contains three con served motifs found in mammalian phosphodiesterases and nucleotide pyrophos phases; notably, the temperature-conditional MCD4 allele used for our studi es (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 m utant (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 a nd releases (potentially up-regulated cell wall) proteins Into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphol ogical defects, most notably the accumulation of distorted, ER- and vesicle -like membranes. mcd4-174 cells synthesize all classes of inositolphosphoce ramides, indicating that the GPI protein transport block is not due to defi cient ceramide synthesis. However, mcd4-174 cells have a severe defect in i ncorporation of [H-3]inositol into proteins and accumulate several previous ly uncharacterized [H-3]inositol-labeled lipids whose properties are consis tent with their being GPI precursors. Together, these studies demonstrate t hat MCD4 encodes a new, conserved component of the GPI anchor synthesis pat hway and highlight the intimate connections between GPI anchoring, bud emer gence, 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 phosphoet hanolamine is also discussed.