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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Å resolution using synchrotron radiation and belong to space group P6222/P6422, with unit-cell parameters a = b = 89.2, c = 162.6 A, α = β = 90, γ = 120° 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. [ABSTRACT FROM AUTHOR]

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.