Cells release extracellular vesicles (EVs) that can mediate intercellular communication by the delivery of lipids, proteins and nucleic acids and are therefore able to influence development, immune responses or disease. In previous studies, we showed that the phospholipid flippase TAT-5 (a P4-ATPase) inhibits the budding of EVs in C. elegans embryos and maintains Phosphatidylethanolamine (PE) asymmetry in the plasma membrane (Wehman et al., Curr Biol, 2011). Loss of TAT-5 activity causes increased release of EVs, but it is not known how TAT-5 activity is regulated. To address this, we searched for potential TAT-5 interactors. The large and novel protein PAD-1 is a homolog of the yeast Dop1p, which interacts with the yeast TAT-5 homolog, Neo1p. Therefore, we hypothesized that PAD-1 could also regulate EV budding in C. elegans. We depleted PAD-1 by RNAi and tested for EVs using a fluorescent membrane marker and transmission electron microscopy (TEM). We found excess membrane between cells in
pad-1 mutants and TEM analysis revealed excessive release of EVs similar to
tat-5 mutants. Thus, PAD-1 also prevents EV budding in C. elegans. Whether PAD-1 regulates TAT-5 activity, localization or levels during EV budding remains unclear. To address this, we are currently creating a
pad-1 deletion allele and GFP-tagged alleles using the CRISPR/Cas9 system. We have also generated antibodies against PAD-1 and TAT-5 and are analyzing their localization and expression levels to determine whether PAD-1 regulates TAT-5. We are further checking whether
pad-1 mutants show defects in PE-asymmetry similar to
tat-5 mutants by staining cells with lipid probes. In summary, this approach will give us a better understanding of TAT-5- and PAD-1-mediated EV release and will provide insight into the vital process of cell communication.