Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

转谷氨酰胺酶（TGs）通过共价交联许多蛋白质在细胞内和细胞外发挥重要作用。果蝇的TG由一个基因编码，具有两种由可变剪接产生的异构体，TG - A和TG - B，它们分别包含不同的N末端46个和38个氨基酸序列。到目前为止所鉴定的TGs没有典型的内质网（ER）信号肽，它们在生理条件下的分泌分子机制尚不清楚。免疫细胞化学显示TG - A定位于多囊泡样结构，而TG - B定位于细胞质。我们还发现TG - A（而非TG - B）同时被N - 豆蔻酰化和S - 棕榈酰化修饰，并且N - 豆蔻酰化是S - 棕榈酰化的先决条件。此外，TG - A（而非TG - B）在由钙离子载体和尿嘧啶（一种病原菌衍生物质）诱导的钙信号作用下被分泌。布雷菲德菌素A和莫能菌素（内质网/高尔基体介导的常规途径的抑制剂）不能抑制TG - A的分泌，然而2 - 溴棕榈酸酯对S - 棕榈酰化的抑制可阻断TG - A的分泌。超速离心、电子显微镜分析以及对多囊泡体形成抑制剂的处理表明，TG - A与共转染的哺乳动物外泌体标记物CD63一起通过外泌体分泌，并且分泌的TG - A可被其他细胞摄取。含有脂肪酰化位点的TG - A的8个残基的N末端片段对于TG - A的外泌体依赖性分泌既是必要的也是充分的。总之，TG - A通过一种涉及两种脂肪酰化和外泌体的非传统的内质网/高尔基体非依赖途径分泌。