Claudia Cericola

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid

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.

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

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.

Ctbp/Bars: A Dual-Function Protein Involved in Transcription Co-Repression and Golgi 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.

The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2

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.

The C‐terminal domain of the transcriptional corepressor CtBP is intrinsically unstructured

C‐terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate‐binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide‐binding domain, involved in NAD(H)‐binding and dimerization. On the contrary, little is known about the structure of CtBP C‐terminal region (∼90 residues), hosting sites for post‐translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small‐angle X‐ray scattering, and we show that the CtBP C‐terminal region is intrinsically unstructured in the full‐length CtBP and in constructs lacking the substrate‐ and/or the nucleotide‐binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners.

Characterization of Chemical Inhibitors of Brefeldin A-activated Mono-ADP-ribosylation

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 ± 3.1 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing theV max of the reaction, whereas it does not influence the K m of the enzyme for NAD+(154 ± 13 μm). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

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.

Crystallization and preliminary X-ray diffraction analysis of brefeldin A-ADP ribosylated substrate (BARS).

Brefeldin A-ADP ribosylated substrate (BARS) is a newly discovered enzyme involved in membrane fission, catalyzing the formation of phosphatidic acid by transfer of an acyl group from acyl-CoA to lysophosphatidic acid. A truncated form of BARS, lacking the C-terminal segment expected to interact with the Golgi membrane, has been expressed in soluble form in Escherichia coli, purified and crystallized. BARS crystals diffract up to 2.5 A resolution using synchrotron radiation and belong to space group P6(2)22/P6(4)22, with unit-cell parameters a = b = 89.2, c = 162.6 A, alpha = beta = 90, gamma = 120 degrees and one molecule (39.5 kDa) per asymmetric unit. SeMet-substituted BARS has been crystallized under growth conditions very similar to those of the native protein.

Brefeldin A-Induced ADP-Ribosylation in the Structure and Function of the Golgi Complex

Brefeldin A (BFA) is a fungal metabolite that exerts generally inhibitory actions on membrane transport and induces the disappearance of the Golgi complex. Previously we have shown that BFA stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 KD. The BFA-binding components mediating the BFA-sensitive ADP-ribosylation (BAR) and the effect of BFA on ARF binding to Golgi membranes have similar specificities and affinities for BFA and its analogues, suggesting that BAR may have a role in the cellular effects of BFA. To investigate this we used the approach to impair BAR activity by the use of BAR inhibitors. A series of BAR inhibitors was developed and their effects were studied in RBL cells treated with BFA. In addition to the common ADP-ribosylation inhibitors (nicotinamide and aminobenzamide), compounds belonging to the cumarin (novobiocin, cumermycin, dicumarol) class were active BAR inhibitors. All BAR inhibitors were able to prevent the BFA-induced redistribution of a Golgi marker (Helix pomatia lectin) into the endoplasmic reticulum, as assessed in immunofluorescence experiments. At the ultrastructural level, BAR inhibitors prevented the tubular-vesicular transformation of the Golgi complex caused by BFA. The potencies of these compounds in preventing the BFA effects on the Golgi complex were similar to those at which they inhibited BAR. Altogether these data support the hypothesis that BAR mediates at least some of the effects of BFA on the Golgi structure and function.

Characterization of the Endogenous Mono-ADP-Ribosylation Stimulated by Brefeldin A

We have recently described a novel enzymatic mono-ADP-ribosyl transfer reaction induced by brefeldin A, a well characterized inhibitor of vesicular traffic, which selectively modifies two cytosolic proteins of 38 kDa (p38) and 50 kDa (BARS-50). p38 was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme and a multifunctional protein involved in several cellular processes; BARS-50 might be a novel G protein, since it is able to bind GTP and the beta gamma subunit of G proteins. We have characterized this enzymatic activity and screened in vitro the effects of different drugs belonging to the coumarine (dicumarol, coumermicin A1 and novobiocin) and quinone (ilimaquinones, benzoquinones and naphtoquinones) class. These drugs blocked the BFA-dependent mono-ADP-ribosylation, showed remarkable effects on Golgi morphology in control cells, and antagonized the tubular reticular redistribution of the Golgi complex in brefeldin A treated cells (see papers of Corda and Colanzi in this issue) suggesting a possible role for ADP-ribosylation in both the cellular effects of brefeldin A and the maintenance of the structure/function of the Golgi complex.