In vitro reconstitution of the BARS-mediated fission machinery operating in the formation of post-Golgi membrane carriers

Intracellular membrane fission is a crucial cellular event that occurs by constriction and subsequent division of a continuous lipid bilayer into two distinct membranes. Membrane deformation can be achieved by the transient interaction with membrane bending proteins and/or local changes in lipid composition mediated by lipid metabolizing enzymes. In the past, our laboratory has discovered and characterized BARS, a key component of a complex protein machinery that acts at several steps of various membrane fission events, including the formation of post-Golgi carriers. Recently, we have identified a Golgi localized lysophosphatic acid (LPA) acyltransferase (LPAAT?) as component of this BARS-driven machinery. Interestingly, LPAAT? is activated by BARS, and its enzymatic reaction of conversion of LPA into PA is necessary for carrier formation. In order to investigate the role mechanism of action of the key proteins and lipids that regulate the BARS-mediated fission machinery, we have reconstituted in vitro part of this protein complex. As experimental model we used Giant Unilamellar Vescicles (GUVs) characterized by a Golgi mimicking lipid composition, and in parallel we also used purified Golgi membranes from Hela cells analysed by negative-staining electron microscopy. Here, we demonstrate that the addition of the purified BARS and LPAAT? proteins to the GUVs or to pure Golgi membranes, led to several fission events. Further, the modulation of the activation of LPAAT? mediated by BARS as well as the catalytic activity of LPAAT? appear to be essential for these events affects the occurrence of these phenomena. We are presently dissecting the role that each component of the machinery, protein or lipid, plays in the fission process.