The P4-ATPase ATP9A is a novel determinant of exosome release

Abstract

Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.

Effects of ATP9A on Extracellular Vesicle Release and Exosomal Lipid Composition

“…ATP9A, a trafficking protein, plays a key role in the initiation of vesicle biogenesis [ 7 , 23 ]. In this study, we found that the release of EVs from HEK293 cells was modulated by P4-ATPase ATP9A.…”

“…ATP9A was detected in the plasma membrane and intracellular compartments. This suggests that the presence of ATP9A in the plasma membrane is important for the purpose of assisting in the recycling of plasma membrane proteins through endocytosis [ 23 ]. EV release was increased in cell lines when ATP9A was knocked out; this implies that ATP9A plays an inhibitory role in EV release.…”

“…In this study, we demonstrated that exosome secretion was inhibited by ATP9A and this could be achieved through glucose transporter 1 (GLUT1) and transferrin receptor, as they are recycled to the plasma membrane through ATP9A endocytic recycling [ 17 ]. The knockdown expression of ATP9A in human hepatoma cells shows that the elevated release of EVs is independent of the activation of caspase-3 [ 23 ]. The quantity of EVs was significantly reduced in ATP9A knockdown cells in which exosome release had been pharmacologically blocked [ 23 ].…”

“…The knockdown expression of ATP9A in human hepatoma cells shows that the elevated release of EVs is independent of the activation of caspase-3 [ 23 ]. The quantity of EVs was significantly reduced in ATP9A knockdown cells in which exosome release had been pharmacologically blocked [ 23 ].…”

Purification and Analysis of <em>Caenorhabditis elegans</em> Extracellular Vesicles

“…C. elegans has been a useful invertebrate model for identifying evolutionarily conserved EV signaling pathways. For instance, a C. elegans flippase was first shown to induce the EV biogenesis in C. elegans embryos, and the human homolog was shown to influence EV release in human cells 10,11 . C. elegans EVs were reported to carry Hedgehog signals necessary for cuticle development.…”