Antonino Colanzi

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.

Protein Kinase D Regulates the Fission of Cell Surface Destined Transport Carriers from the Trans-Golgi Network

When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.

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.

The closure of Pak1‐dependent macropinosomes requires the phosphorylation of CtBP1/BARS

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C‐terminal‐binding protein‐1/brefeldinA‐ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin‐independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21‐activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS‐induced fission to human viral infection.

Cell-cycle-specific Golgi fragmentation: how and why?

The Golgi membranes, in the form of stacks of cisternae, are contained in the pericentriolar region of mammalian cells. During mitosis, these membranes are fragmented and dispersed throughout the cell. Recent studies are revealing the significance of pericentriolar position of the Golgi apparatus and mechanism by which these membranes are fragmented during mitosis.

The Golgi and the centrosome: building a functional partnership

The mammalian Golgi apparatus is characterized by a ribbon-like organization adjacent to the centrosome during interphase and extensive fragmentation and dispersal away from the centrosome during mitosis. It is not clear whether this dynamic association between the Golgi and centrosome is of functional significance. We discuss recent findings indicating that the Golgi–centrosome relationship may be important for directional protein transport and centrosome positioning, which are both required for cell polarization. We also summarize our current knowledge of the link between Golgi organization and cell cycle progression.

Molecular Cloning and Functional Characterization of Brefeldin A-ADP-ribosylated Substrate A NOVEL PROTEIN INVOLVED IN THE MAINTENANCE OF THE GOLGI STRUCTURE

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.

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.

RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis.

Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells. We show that activated MEK1 is found on the Golgi apparatus in late prophase. The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1. MEK1-dependent Golgi complex fragmentation is through activation by RAF1 and not MEK1 kinase 1. We propose that a RAF1-dependent activation of MEK1 and its presence on the Golgi apparatus in late prophase is required for Golgi complex fragmentation.

A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation

Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.