Gaynor E C, Mondésert G, Grimme S J, Reed S I, Orlean P, Emr S D
Department of Biology, The Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0668, USA.
Mol Biol Cell. 1999 Mar;10(3):627-48. doi: 10.1091/mbc.10.3.627.
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.
糖基磷脂酰肌醇（GPI）锚定蛋白是定位于细胞表面的蛋白质，具有许多重要的细胞功能。在真核细胞中，介导GPI锚合成并将其连接到这些蛋白质的途径复杂且高度保守，在所有GPI锚定蛋白家族成员的正确靶向、运输和功能中起关键作用。在本文中，我们证明MCD4是一个必需基因，最初在遗传筛选中被鉴定出来，用于分离芽出现缺陷的酿酒酵母突变体，它编码GPI锚合成途径中一个先前未被鉴定的组分。Mcd4p是一种多跨膜蛋白，定位于内质网（ER），并含有一个大的NH2末端内质网腔结构域。我们还克隆了人类MCD4基因，发现Mcd4p在整个真核生物中高度保守，并且有两个酵母同源物。Mcd4p的腔结构域包含在哺乳动物磷酸二酯酶和核苷酸焦磷酸酶中发现的三个保守基序；值得注意的是，我们研究中使用的温度条件性MCD4等位基因（mcd4 - 174）在基序2中有一个单氨基酸变化。mcd4 - 174突变体：（1）在GPI锚定蛋白（即Gas1p）从内质网到高尔基体的运输中存在缺陷，而其他蛋白质（即CPY）不受影响；（2）将（潜在上调的细胞壁）蛋白质分泌并释放到培养基中，表明细胞壁完整性存在缺陷；（3）表现出明显的形态缺陷，最显著的是扭曲的内质网和囊泡样膜的积累。mcd4 - 174细胞能合成所有类型的肌醇磷酸神经酰胺，这表明GPI蛋白运输阻滞不是由于神经酰胺合成不足。然而，mcd4 - 174细胞在将[3H]肌醇掺入蛋白质方面存在严重缺陷，并积累了几种先前未表征的[3H]肌醇标记的脂质，其性质与它们作为GPI前体一致。总之，这些研究表明MCD4编码GPI锚合成途径中的一个新的保守组分，并突出了GPI锚定、芽出现、细胞壁功能以及可能参与调节这些基本过程中每一个的反馈机制之间的密切联系。还讨论了Mcd4p参与用侧链磷酸乙醇胺修饰GPI锚的假定作用。
