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

Abstract

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. 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 bymyristoylation and -palmitoylation, andmyristoylation was a pre-requisite for palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca 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 -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.

Comprehensive palmitoyl‐proteomic analysis identifies distinct protein signatures for large and small cancer‐derived extracellular vesicles

“…Interestingly, the loading of STEAP2 and ABCC4 into EVs was reduced by inhibition of palmitoylation, in line with a report showing that palmitoylation of TG-A transglutaminase is required for its secretion in exosomes [57]. In contrast, 2-BP treatment did not alter the localization of other palmitoylated proteins such as Cav-1 in agreement with previous studies reporting that palmitoylation is not necessary for Cav-1 membrane localization [52].…”

Drosophilaas a Genetic Model for Hematopoiesis

“…A similar scenario is observed for larval injuries, where the emphasis thus far is largely on the homing of hemocytes and their role in resolving the wound (Goto et al 2003; Babcock et al 2008; Shibata et al 2017). Here, plasmatocytes are attracted to the wound site, where they phagocytose damaged cells and secrete factors that help form a clot to prevent leakage of the hemolymph (Goto et al 2003; Babcock et al 2008; Shibata et al 2017).…”

“…A similar scenario is observed for larval injuries, where the emphasis thus far is largely on the homing of hemocytes and their role in resolving the wound (Goto et al 2003; Babcock et al 2008; Shibata et al 2017). Here, plasmatocytes are attracted to the wound site, where they phagocytose damaged cells and secrete factors that help form a clot to prevent leakage of the hemolymph (Goto et al 2003; Babcock et al 2008; Shibata et al 2017). Crystal cells rupture and release prophenoloxidase enzymes and proteases such as Mp2/Sp7/PAE1, which also contribute to the formation of melanin (Castillejo-López and Häcker 2005; Bidla et al 2007; Binggeli et al 2014; Dudzic et al 2015).…”

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

“…Another unresolved piece of this puzzle relates to the way in which Xdh, a cytosolic enzyme, is found in pigment granules in the eye (Reaume et al, 1989 , 1991 ). The discovery of exosomes gives a possible answer to this conundrum (Hemler, 2003 ; Gross et al, 2012 ; Gradilla et al, 2014 ; Takeuchi et al, 2015 ; Beer and Wehman, 2017 ; Shibata et al, 2017 ; Tassetto et al, 2017 ). Clearly more experiments are required to explain how Xdh acts in the formation of eye color in flies, but complex non-cell autonomous processes relating to enzyme maturation, regulation, and transport are involved.…”