The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

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.

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31 P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA)7 is a minor but important bioactive lipid involved in at least three essential and likely interrelated processes in a typical eukaryotic cell. PA is a key intermediate in the biosynthetic route of the main membrane phospholipids and triglycerides (1); it is involved in membrane dynamics, i.e. fission and fusion (2-4), and has important signaling functions (5-7). The role of PA in membrane dynamics and signaling is most likely 2-fold, either via an effect on the packing properties of the membrane lipids or via the specific binding of effector proteins (4, 7-9). The origin of the specific binding of effector proteins to PA is not known.The PA binding domains thus far identified (for review, see Ref. 10) and more recently (7)) are diverse and share no apparent sequence homology, in contrast to other lipid binding domains, such as e.g. the PH, PX, FYVE, and C2 domains (11-15). One general feature that the PA binding domains do have in common is the presence of basic amino acids (7). Where examined in detail, these basic amino acids were shown to be essential for the interaction with PA, which underscores the importance of electrostatic interactions. Indeed, the negatively charged phosphomonoester head group of PA would be expected to interact electrostatically with basic amino acids in a lipid binding domain. However, such a simple electrostatic interaction cannot explain the strong preference of PA-binding proteins for PA over other, often more abundant, negatively charged phospholipids.In a recent study we showed that the phosphomonoester head group of PA has remarkable properties (8). The phosphomonoester head group is able to form an intramolecular hydrogen bond upon initial deprotonation (when its charge is Ϫ1), which stabilizes the second proton against dissociation. We provided evidence that competing hydrogen bonds, e.g. from the primary amine of the head group of phosphatidylet...

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37°C, pH 7.2, 0.5 mM free Mg 2+ ), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca 2+ (pH 6.6±5.9, 0.3 mM Ca 2+ ). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cationlysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.