Saccharomyces cerevisiae depend on vesicular traffic between Golgi and vacuole when Inositolphosphorylceramide synthase Aur1 is inactivated

Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells under this condition. We established hydroxylated C(26) fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance

Chemogenetic E-MAP in Saccharomyces cerevisiae for identification of membrane transporters operating lipid flip flop

While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data

mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1

mTORC1 is a master regulator of cell growth with well-known functions in inhibiting autophagic vesicle formation. Here, the authors show that mTORC1 also affects Golgi architecture and vesicle secretion by phosphorylating the scaffold protein SCYL1. The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0–4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response

CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, the fatty acid in sn-2 of the diacylglycerol moiety can be replaced by a C26:0 fatty acid by a deacylation–reacylation cycle catalysed by Per1p and Gup1p. Furthermore the diacylglycerol moiety of the yeast GPI anchor can also be replaced by ceramides. CWH43 of yeast is homologous to PGAP2, a gene that recently was implicated in a similar deacylation reacylation cycle of GPI proteins in mammalian cells, where PGAP2 is required for the reacylation of monoradylglycerol-type GPI anchors. Here we show that mutants lacking CWH43 are unable to synthesize ceramide-containing GPI anchors, while the replacement of C18 by C26 fatty acids on the primary diacylglycerol anchor by Per1p and Gup1p is still intact. CWH43 contains the COG3568 metal hydrolase motif, which is found in many eukaryotic and prokaryotic enzymes. The conserved His 802 residue of this motif was identified as being essential for ceramide remodelling. Ceramide remodelling is not required for the normal integration of GPI proteins into the cell wall. All remodelling reactions are dependent on prior removal of the inositol-linked fatty acid by Bst1p

Étude sur le TDAH et les différents types de familles

Le Trouble du Déficit de l’Attention avec ou sans Hyperactivité (TDAH) fait partie des troubles neurodéveloppementaux et représente la pathologie pédopsychiatrique la plus fréquente. Le TDAH entraîne des répercussions importantes sur la qualité de vie de l’enfant ayant un TDAH, mais également sur celle de la famille qui comportent des constellations très variées dans les sociétés occidentales. Cette recherche cherche à comprendre si les familles ayant un enfant souffrant d’un TDAH sont touchées différemment en fonction de leur structure. Cent-vingt-six parents ont répondu à un questionnaire anonyme en ligne recueillant des données sur les difficultés auxquelles sont confrontées les familles comportant un enfant avec un TDAH ainsi que les processus diagnostiques et les différentes prises en charge. En effet, dans une société où les décisions se prennent de manière centrée autour du patient, les parents ont un rôle important dans le choix des prises en charge. Les résultats de l’analyse du questionnaire démontrent qu’il existe des divergences dépendantes du type de famille. Les monoparentales et recomposées semblent souffrir d’un impact plus fortement négatif au niveau de leur couple parental. Cependant, ce sont les enfants issus de ce type de foyer qui ont le plus tendance à consulter un thérapeute de manière régulière. Il existe donc un moyen privilégié pour aborder ce sujet avec les parents, en effet ces derniers ont un rôle important dans le bien-être de leur enfant. Il est donc crucial, pour soigner le patient TDAH, de prendre soin de toute la famille

Incorporation of ceramides into yeast glycosylphosphatidylinositol-anchored proteins can be monitored in vitro

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors the thus modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction, which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells having been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide based anchors. Such microsomes, when incubated with [³H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, unable to make ceramide-based anchors in vivo, do not incorporate [³H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [³H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [³H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A or C26:0-Coenzyme A, particularly if microsomes are depleted of acyl-CoA. Thus, [³H]DHS cannot be incorporated into proteins in the absence of acyl-CoA

Electronic Evidence in Swiss Criminal Procedure

Bertrand Perrin, Marc Rémy and Romain Roubaty outline how the Federal Government has amended the rules of the Penal Code and the Law on International Mutual Assistance in Criminal Matters to bring them into line with the Convention on Cybercrime, together with the Swiss Code of Criminal Procedure, which entered into effect on 1 January 2011

SLC1 and SLC4 encode partially redundant acyl-Coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

Phosphatidic acid (PA) is the intermediate, from which all glycerophospholipids are synthesized. In yeast it is generated from lyso-phosphatidic acid, which is acylated by Slc1p, a sn-2-specific, acyl-Coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane bound O-acyl transferase (MBOAT) motif, and down regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Δ cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Δ and slc4Δ cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Δ and slc4Δ cells incubated with [¹⁴C]-oleoyl-Coenzyme A in the absence of lyso-phosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lyso-glycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Δ and slc4Δ cells are different and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg²⁺-dependent acyltransferase activity not only towards lyso-phosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases, but also be involved in fatty acid exchange at the sn-2 position of mature glycerophospholipids