Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

EXTRACELLULAR VESICLE BUDDING AND LIPID ASYMMETRY

Lauren R Pitts 1 ; Alexander T Nguyen 1 ; Julia Frondoni 1 ; Katharina B Beer 2 ; Seungmee Park 3 ; Yishi Jin 3 ; Andrew Chisholm 3 ; Dinah Loerke 4 ; Ann M. Wehman 1,2 ; 1 University of Denver, Biological Sciences, Denver, CO, USA

2 Universität Würzburg, Rudolf Virchow Zentrum, Würzburg, Germany 3 University of California San Diego, Neurobiology, San Diego, CA, USA 4 University of Denver, Physics & Astronomy, Denver, CO, USA

The P4-ATPase TAT-5 acts as a lipid flippase to maintain phosphatidylethanolamine (PE) asymmetry in the plasma membrane and inhibit extracellular vesicle (EV) release from the surface of C. elegans cells. TAT-5 is required for embryonic development and fertility, but how flippase activity regulates these processes was unclear. Furthermore, its interactor PAD-1 shares essential roles with TAT-5 in PE asymmetry, EV release, morphogenesis, and fertility. However, PAD-1 is a cytosolic protein, making it unclear how PAD-1 could regulate PE asymmetry in the plasma membrane. As mutations in the DGET motif of a mammalian P4-ATPase lead to a 3-fold (D to T) to complete (E to Q) loss in lipid transport (Coleman et al., PNAS 2012), we generated similar mutations in the DGET motif of TAT-5. We discovered that tat-5(E246Q) mutants were sterile, while tat-5(D244T) mutants produced embryos that arrested during development. Using degron-based reporters and an automated EV detection algorithm, we found that EV release was increased in both mutants and that phagocytosis was also disrupted. These data suggest that a low level of flippase activity can support fertility, while a higher level of flippase activity is required to inhibit EV release, allow phagocytosis, and carry out embryonic development. We discovered that conserved domains of PAD-1 are required for fertility and to inhibit EV release from the plasma membrane and we predict that these N- and C-terminal domains could interact directly with TAT-5. Using a gain-of-function PAD-1 allele, we also revealed that PAD-1 regulates TAT-5 flippase activity to modulate EV release, suggesting that the bilayer localization of the lipid PE influences membrane budding. In the future, we plan to determine the physical characteristics of PE that promote EV budding from the plasma membrane. This work was funded in part by DFG WE5719/2-1.microstates influence site redox potentials, proton affinities of proton loading sites and the connectivity of extended proton transfer pathways.

22