Department of Molecular Physiology, and the WSU Molecular Medicine Research Group, School of Medicine, Western Sydney University, Campbelltown, NSW, 2560, Australia.
Department of Health Sciences, Faculty of Applied Health Sciences, Department of Biology, Faculty of Mathematics and Science, Brock University, St. Catharines, Ontario, Canada.
Int J Biochem Cell Biol. 2018 Nov;104:43-54. doi: 10.1016/j.biocel.2018.09.001. Epub 2018 Sep 5.
Docking, priming, and membrane fusion of secretory vesicles (i.e. regulated exocytosis) requires lipids and proteins. Sphingolipids, in particular, sphingosine and sphingosine-1-phosphate, have been implicated in the modulation of exocytosis. However, the specific exocytotic steps that sphingolipids modulate and the enzymes that regulate sphingolipid concentrations on native secretory vesicle membranes remain unknown. Here we use tightly coupled functional and molecular analyses of fusion-ready cell surface complexes and cortical vesicles isolated from oocytes to assess the role of sphingolipids in the late, Ca-triggered steps of exocytosis. The molecular changes resulting from treatments with sphingolipid modifying compounds coupled with immunoblotting analysis revealed the presence of sphingosine kinase on native vesicles; the presence of a sphingosine-1-phosphate phosphatase is also indicated. Changes in sphingolipid concentrations on vesicles altered their docking/priming, Ca-sensitivity, and ability to fuse, indicating that sphingolipid concentrations are tightly regulated and maintained at optimal levels and ratios to ensure efficient exocytosis.
分泌囊泡（即调节型胞吐作用）的 docking、priming 和膜融合需要脂质和蛋白质。鞘脂，特别是神经酰胺和 1-磷酸鞘氨醇，已被牵涉到胞吐作用的调节中。然而，鞘脂调节的特定胞吐作用步骤以及调节天然分泌囊泡膜上鞘脂浓度的酶仍然未知。在这里，我们使用紧密偶联的融合准备好的细胞表面复合物和从卵母细胞中分离的皮质小泡的功能和分子分析，来评估鞘脂在胞吐作用的后期 Ca 触发步骤中的作用。用鞘脂修饰化合物进行处理的分子变化与免疫印迹分析相结合，揭示了天然囊泡上存在鞘氨醇激酶；也表明存在鞘氨醇-1-磷酸磷酸酶。囊泡上鞘脂浓度的变化改变了它们的 docking/priming、Ca 敏感性和融合能力，表明鞘脂浓度受到严格调控，并保持在最佳水平和比例，以确保有效的胞吐作用。
