Mallela Shamroop K, Almeida Reinaldo, Ejsing Christer S, Conzelmann Andreas
Division of Biochemistry, Department of Biology, University of Fribourg, Chemin du Musée 10, Fribourg, CH-1700, Switzerland.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
PLoS One. 2016 Jan 11;11(1):e0145831. doi: 10.1371/journal.pone.0145831. eCollection 2016.
Ceramide is synthesized in yeast by two redundant acyl-CoA dependent synthases, Lag1 and Lac1. In lag1∆ lac1∆ cells, free fatty acids and sphingoid bases are elevated, and ceramides are produced through the redundant alkaline ceramidases Ypc1 and Ydc1, working backwards. Even with all four of these genes deleted, cells are surviving and continue to contain small amounts of complex sphingolipids. Here we show that these residual sphingolipids are not synthesized by YPR114w or YJR116w, proteins of unknown function showing a high degree of homology to Lag1 and Lac1. Indeed, the hextuple lag1∆ lac1∆ ypc1∆ ydc1∆ ypr114w∆ yjr116w∆ mutant still contains ceramides and complex sphingolipids. Yjr116w∆ exhibit an oxygen-dependent hypersensitivity to Cu2+ due to an increased mitochondrial production of reactive oxygen species (ROS) and a mitochondrially orchestrated programmed cell death in presence of copper, but also a general copper hypersensitivity that cannot be counteracted by the antioxidant N-acetyl-cysteine (NAC). Myriocin efficiently represses the synthesis of sphingoid bases of ypr114w∆, but not its growth. Both yjr116w∆ and ypr114w∆ have fragmented vacuoles and produce less ROS than wild type, before and after diauxic shift. Ypr114w∆/ypr114w∆ have an increased chronological life span. Thus, Yjr116w and Ypr114w are related, but not functionally redundant.
在酵母中，神经酰胺由两种冗余的酰基辅酶A依赖性合酶Lag1和Lac1合成。在lag1∆lac1∆细胞中，游离脂肪酸和鞘氨醇碱水平升高，神经酰胺通过冗余的碱性神经酰胺酶Ypc1和Ydc1逆向合成。即使删除了所有这四个基因，细胞仍能存活并继续含有少量复杂的鞘脂。在这里，我们表明这些残留的鞘脂不是由YPR114w或YJR116w合成的，这两种蛋白质功能未知，但与Lag1和Lac1具有高度同源性。事实上，六重突变体lag1∆lac1∆ypc1∆ydc1∆ypr114w∆yjr116w∆仍然含有神经酰胺和复杂的鞘脂。Yjr116w∆对Cu2+表现出氧依赖性超敏反应，这是由于线粒体活性氧（ROS）产生增加以及在铜存在下线粒体介导的程序性细胞死亡，而且还表现出一般的铜超敏反应，抗氧化剂N-乙酰半胱氨酸（NAC）无法抵消这种反应。米里新能有效抑制ypr114w∆鞘氨醇碱的合成，但不影响其生长。在二次生长转换之前和之后，yjr116w∆和ypr114w∆的液泡都有碎片化，并且产生的ROS比野生型少。Ypr114w∆/ypr114w∆的时序寿命增加。因此，Yjr116w和Ypr114w相关，但功能上并非冗余。
