Mao C, Xu R, Bielawska A, Obeid L M
Division of General Internal Medicine at the Ralph H. Johnson Veterans Administration Hospital and the Departments of Medicine, Charleston, South Carolina 29425, USA.
J Biol Chem. 2000 Mar 10;275(10):6876-84. doi: 10.1074/jbc.275.10.6876.
Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.
神经酰胺不仅是鞘脂的核心中间体，也是许多细胞事件的重要调节因子，包括细胞凋亡、细胞周期停滞、衰老、分化和应激反应。其周转可能受到严格调控。然而，由于负责其合成和分解的大多数酶尚未被克隆，因此对其代谢调控知之甚少。在这里，我们通过筛选酿酒酵母基因来报告酵母基因YPC1（YBR183w）的克隆和表征，这些基因的过表达赋予对伏马菌素B1的抗性。我们证明酵母基因YPC1编码一种碱性神经酰胺酶活性，负责二氢神经酰胺和植物神经酰胺的分解，但不负责不饱和神经酰胺的分解。使用大肠杆菌表达系统的体外研究证实了YPC1神经酰胺酶活性。重要的是，YPC1p也具有反向活性，催化从棕榈酸和植物鞘氨醇合成植物神经酰胺。这种神经酰胺合酶活性不依赖于辅酶A，并且对伏马菌素B1具有抗性，因此解释了为什么YPC1被克隆为伏马菌素B1抗性基因。
