Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

C‐terminal‐binding protein/brefeldin A‐ADP ribosylated substrate (CtBP/BARS) plays key roles in development and oncogenesis as a transcription co‐repressor, and in intracellular traffic as a promoter of Golgi membrane fission. Co‐repressor activity is regulated by NAD(H) binding to CtBP/BARS, while membrane fission is associated with its acyl‐CoA‐dependent acyltransferase activity. Here, we report the crystal structures of rat CtBP/BARS in a binary complex with NAD(H), and in a ternary complex with a PIDLSKK peptide mimicking the consensus motif (PXDLS) recognized in CtBP/BARS cellular partners. The structural data show CtBP/BARS in a NAD(H)‐bound dimeric form; the peptide binding maps the recognition site for DNA‐binding proteins and histone deacetylases to an N‐terminal region of the protein. The crystal structure together with the site‐directed mutagenesis data and binding experiments suggest a rationale for the molecular mechanisms underlying the two fundamental co‐existing, but diverse, activities supported by CtBP/BARS in the nucleus and in Golgi membranes.

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.

The Golgi ribbon is a complex structure of many stacks interconnected by tubules that undergo fragmentation during mitosis through a multistage process that allows correct Golgi inheritance. The fissioning protein CtBP1‐S/BARS (BARS) is essential for this, and is itself required for mitotic entry: a block in Golgi fragmentation results in cell‐cycle arrest in G2, defining the ‘Golgi mitotic checkpoint’. Here, we clarify the precise stage of Golgi fragmentation required for mitotic entry and the role of BARS in this process. Thus, during G2, the Golgi ribbon is converted into isolated stacks by fission of interstack connecting tubules. This requires BARS and is sufficient for G2/M transition. Cells without a Golgi ribbon are independent of BARS for Golgi fragmentation and mitotic entrance. Remarkably, fibroblasts from BARS‐knockout embryos have their Golgi complex divided into isolated stacks at all cell‐cycle stages, bypassing the need for BARS for Golgi fragmentation. This identifies the precise stage of Golgi fragmentation and the role of BARS in the Golgi mitotic checkpoint, setting the stage for molecular analysis of this process.

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38