Daniela Corda

ARF mediates recruitment of PtdIns-4-OH kinase- β and stimulates synthesis of PtdIns(4,5)P 2 on the Golgi complex

The small GTPase ADP-ribosylation factor (ARF) regulates the structure and function of the Golgi complex through mechanisms that are understood only in part, and which include an ability to control the assembly of coat complexes and phospholipase D (PLD). Here we describe a new property of ARF, the ability to recruit phosphatidylinositol-4-OH kinase-β and a still unidentified phosphatidylinositol-4-phosphate-5-OH kinase to the Golgi complex, resulting in a potent stimulation of synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate; this ability is independent of its activities on coat proteins and PLD. Phosphatidylinositol-4-OH kinase-β is required for the structural integrity of the Golgi complex: transfection of a dominant-negative mutant of the kinase markedly alters the organization of the organelle.

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.

Functional aspects of protein mono-ADP-ribosylation

Mono‐ADP‐ribosylation is the enzymatic transfer of ADP‐ribose from NAD+ to acceptor proteins. It is catalysed by cellular ADP‐ribosyltransferases and certain bacterial toxins. There are two subclasses of cellular enzymes: the ectoenzymes that modify targets such as integrins, defensin and other cell surface molecules; and the intracellular enzymes that act on proteins involved in cell signalling and metabolism, such as the β‐subunit of heterotrimeric G proteins, GRP78/BiP and elongation factor 2. The genes that encode the ectoenzymes have been cloned and their protein products are well characterized, yet little is known about the intracellular ADP‐ribosyltransferases, which may be part of a novel protein family with an important role in regulating cell function. ADP‐ribosylation usually leads to protein inactivation, providing a mechanism to inhibit protein functions in both physiological and pathological conditions.

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.

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.

Human Trop-2 is a tumor-associated calcium signal transducer.

Trop‐2/EGP‐1/GA733‐1 is a recently identified cell surface glycoprotein highly expressed by human carcinomas. The cytoplasmic tail of Trop‐2 possesses potential serine and tyrosine phosphorylation sites and a phosphatidyl‐inositol binding consensus sequence. Thus, we investigated whether Trop‐2 might be a functional signaling molecule. Using the fluorescent probe Fura‐2, we assayed the cytoplasmic calcium levels in human cancer cells stimulated with anti‐Trop‐2 or control antibodies. Three anti‐Trop‐2 MAbs, Rs7‐7G11, MOv16 and 162‐46.2 specifically induced a transient intracellular calcium level increment in up to 40% of the experiments performed. Polyclonal antisera recognizing recombinant Trop‐2 molecules possessed a much lower stimulation efficiency. The average latency of antibody‐induced Ca2+ rise for OvCa‐432 cells was 64 ± 26 sec. Internal Ca2+ concentrations reached peaks of 190 ± 24 nM vs<0R>. basal levels of 61 ± 4 nM and returned to baseline within 193 ± 37 sec. Similar values were obtained in MCF‐7 cells. For comparison, stimulation of P2‐purinergic receptors on MCF‐7 and OvCa‐432 cells induced a Ca2+ rise in most cases, leading to average internal Ca2+ concentrations of 297 ± 41 and 391 ± 39 nM, respectively. Our findings show that Trop‐2 transduces an intracellular calcium signal, are consistent with the hypothesis that it acts as a cell surface receptor and support a search for a physiological ligand. Int. J. Cancer 76:671–676, 1998.© 1998 Wiley‐Liss, Inc.

Phosphoinositides and the Golgi complex

Phosphoinositides act as precursors of second messengers and membrane ligands for protein modules. Specific lipid kinases and phosphatases are located and differentially regulated in cell organelles, generating a non-uniform distribution of phosphoinositides. Although it is not clear whether and how the phosphoinositide pools are integrated, it is certain that they locally control fundamental processes, including membrane trafficking. This applies to the Golgi complex, where a direct, central role of the phosphatidylinositol 4,5-bisphosphate precursor phosphatidylinositol 4-phosphate has recently been reported.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular‐like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl‐coenzyme A (p‐coA) and ARFGAP1, a GTPase‐activating protein (GAP) for ADP‐Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin‐A ADP‐Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p‐coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p‐coA and GAP are linked in this process.

Physiological relevance of the endogenous mono(ADP‐ribosyl)ation of cellular proteins

The mono(ADP‐ribosyl)ation reaction is a post‐translational modification that is catalysed by both bacterial toxins and eukaryotic enzymes, and that results in the transfer of ADP‐ribose from βNAD+ to various acceptor proteins. In mammals, both intracellular and extracellular reactions have been described; the latter are due to glycosylphosphatidylinositol‐anchored or secreted enzymes that are able to modify their targets, which include the purinergic receptor P2X7, the defensins and the integrins. Intracellular mono(ADP‐ribosyl)ation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78/BiP, the β‐subunit of heterotrimeric G‐proteins and glutamate dehydrogenase. The molecular identification of the intracellular enzymes, however, is still missing. A better molecular understanding of this reaction will help in the full definition of its role in cell physiology and pathology.