Molecular mechanisms of membrane fission at the Golgi complex

Membrane fission is an essential event in cellular processes such as organelle partitioning, cell division and membrane transport [1]. Different types of carriers and fission machineries operate at different stages of the exocytic and endocytic transport pathways. However, the common step for all pathways is the generation of curvature from a flat membrane surface, in order to form a bud, which then protrudes and undergoes fission, to generate free transport carrier. Fission proceeds via intermediates where membrane changes their shape and continuity. The forces for this membrane perturbation event come from the cooperative contributions of lipids and proteins, and from lipid-protein interactions, as well as mechanoenzymes. Membrane bending can be achieved by proteins that induce positive membrane curvature, and/or through local changes in lipid composition and geometry. While several membrane-curving proteins have been identified, relatively little is known about the lipid metabolic enzymes involved in membrane remodelling. Among their possible products, the closely metabolically interrelated lipids lysophospholipids (LPLs), phosphatidic acid (PA), and diacylglycerol (DAG) have high positive or negative intrinsic curvature and have been proposed to mediate rapid lipid-geometry changes [2]. The LPLs might act by facilitating tubulation when in the cytosolic leaflet through their wedge-like shape, while PA and DAG might destabilize tubules through their conical shape. Accordingly, phospholipase A2 and lysophosphatidic acid (LPA) acyltransferases (LPAATs) activities, which generate LPA and PA, respectively, have been shown to have roles in membrane trafficking [3]. We focus on the mechanism of action of CtBP1-S/BARS (BARS), a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. When incorporated into this complex, BARS binds to and activates a trans-Golgi localised LPAAT type δ (LPAATδ) that converts LPA into PA and this reaction is essential for carrier fission [3-6]. To investigate the key proteins and lipids that regulates BARS-mediated fission, we have reconstituted in vitro part of this protein/lipid complex using artificial membranes: giant unilamellar vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) based on Golgi membrane-like lipid composition [7]. The addition of purified BARS to the LPAATδ-associated GUVs and LUVs, promote fission events, similar to those observed during intracellular transport. BARS activates the LPAATδ enzymatic activity, thus increasing the local concentration of PA that is required for constriction and fission of tubules to form vesicles. Of note, once GUVs and LUVs membranes are enriched in PA, BARS per se promotes fission through the insertion of an amphipathic helix that characterizes its structure into the membrane bilayer [8]. This directly indicate that BARSdependent fission requires key lipid and protein components and that, physiologically, the enzymatic conversion of LPA into PA is central for the completion of the fission event [8].