Abstract：
Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.
摘要：
神经酰胺（Cer）在内质网的双层中从头合成，并运输到跨高尔基体的胞浆小叶以进行鞘磷脂（SM）合成。由于SM合酶（SMS）的活性位点位于高尔基体膜的腔侧，Cer通过跨双层运动转移到管腔进行SM合成。然而,跨双层运动的机制尚不完全清楚。由于与Cer相关的易位似乎位于SMS附近，使用邻近依赖生物素鉴定蛋白质组学鉴定蛋白质。磷脂加扰酶1（PLSCR1），它被认为是磷脂酰丝氨酸和磷脂酰乙醇胺的加扰酶，被鉴定为邻近SMS同工型SMS1和SMS2的蛋白质。尽管已经在人类中报道了PLSCR的五种同工型，只有PLSCR1、PLSCR3和PLSCR4在HEK293T细胞中表达。共聚焦显微镜分析表明，PLSCR1和PLSCR4与跨高尔基网络标记p230部分共定位，其中SMS同工型被定位。我们建立了CRISPR/Cas9介导的PLSCR1、PLSCR3和PLSCR4单敲除细胞和PLSCR1、3、4三敲除HEK293T细胞。液相色谱-串联质谱显示，与野生型细胞相比，Cer和SM中具有不同酰基链的物种的水平在单敲除细胞或三敲除细胞中没有显着差异。我们的发现表明PLSCR1位于SMS同工型附近，然而，不参与SM合成的Cer的跨双层运动。
