Juan M. Durán

Unconventional secretion of Acb1 is mediated by autophagosomes

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Manjithaya et al. in this issue).

Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion

A novel membrane structure called CUPS is assembled during the secretion of unconventional cargo such as Acb1.

Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum‐to‐Golgi transport monitored with the vesicular stomatitis virus‐G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A‐induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi‐to‐endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI‐coated and uncoated vesicles contain β/γ‐actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi‐to‐endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.

Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network

Sorting of both soluble and integral membrane proteins is disrupted by loss of ADF/cofilin, suggesting that actin severing controls expansion of a sorting domain within the TGN.

The Role of GRASP55 in Golgi Fragmentation and Entry of Cells into Mitosis

GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D‐ceramide‐C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D‐ceramide‐C6‐treated HeLa cells revealed an increase in the levels of C6‐sphingomyelin, C6‐glucosylceramide, and diacylglycerol. D‐ceramide‐C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D‐ceramide‐C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6‐sphingomyelin prevented liquid‐ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

Protective effects of lysophosphatidic acid (LPA) on chronic ethanol-induced injuries to the cytoskeleton and on glucose uptake in rat astrocytes

Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol‐treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2‐deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol‐treated astrocytes cultured without fetal calf serum, it induced the re‐appearance of actin stress fibres and the normalization of 2‐deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule‐disrupting agent nocodazole. Again, pre‐treatment with LPA prevented this alteration. Ethanol‐treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP‐bound form) were insensitive to ethanol, as they showed no alteration either in actin stress‐fibre organization or in 2‐deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol‐induced damages. The latter opens the possibility that LPA or a similar non‐hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.

Remodeling of secretory compartments creates CUPS during nutrient starvation

Starvation triggers relocation of membranes of the secretory compartments and endosomes to create a compartment called CUPS, which may be involved in processing and secretion of proteins that cannot enter the ER–Golgi pathway.

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes; Harmful effects of chronic ethanol exposure on sphingolipid trafficking and metabolism.

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent‐labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C6‐NBD‐glucosylceramide (NBD‐GlcCer) and C6‐NBD‐sphingomyelin (NBD‐SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD‐GlcCer and NBD‐SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD‐sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD‐ceramide to NBD‐GlcCer and NBD‐SM. We conclude that in rat astrocytes internalised plasma membrane NBD‐sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD‐sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Calculation of the gastric absorption rate constants of 5-substituted barbiturates through theRm values or substituent ΔRm constants in reversed-phase partition chromatography

A linear correlation between the logarithm of the in situgastric absorption rate constants (ka)of 5-substituted barbiturates and their Rmvalues in selected reversed-phase partition chromatographic systems is demonstrated, as well as between the Δlog kaand the ΔRmderived parameters. On the basis of the chromatographic behavior of 14 closely related compounds of this series, the substituent ΔRmconstants for the main functional groups are calculated, so that the gastric absorption constants of nontested barbiturates can be predicted with close approximation. Predicted Rmand log kavalues show an excellent correlation with log 1/C values taken from pharmacological literature data. The migration mechanisms involved in reversed-phase partition chromatography are discussed in connection with the results obtained with other types of partition systems and with bulk-phase solvents. The possible relationships between chromatographic parameters and absorption rate constants found from absorption sites other than the stomach are outlined; as well as the advantages and limitations of the procedure.