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Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae . Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo . Bacteria release outer membrane vesicles (OMVs) that play important roles in pathogenesis and intercellular interactions. Here, Roier et al . provide evidence supporting that phospholipid accumulation in the outer leaflet of the outer membrane participates in OMV formation in Gram-negative bacteria.

We are grateful to the Austrian Science Fund FWF (Austria) for grants P2349-B12, P24381-B20, I1000, and DK-MCD to FM, grant ‘Molecular Enzymology’ to KUF, grant ‘SFB Lipotox’ to FM and KUF, grant NFN S93 to PJD, FM and KUF and to the European Commission for project APOSYS (FM). TE is recipient of an APART fellowship of the Austrian Academy of Sciences at the Institute of Molecular Biosciences, University of Graz. This work is supported by grants to GK from the Ligue Nationale contre le Cancer (Equipe labellisée), Agence Nationale pour la Recherche (ANR), Association pour la Recherche sur le Cancer, European Research Council (Advanced Investigator Award), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer, Cancéropôle Ile-de-France, Fondation Bettencourt-Schueller, the LabEx Onco-Immunology, and the Paris Alliance of Cancer Research Institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

During the last decades, lipids have gained much attention due to their involvement in health and disease. Lipids are required for the formation of membranes and contribute to many different processes such as cell signaling, energy supply, and cell death. Various organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, and lipid droplets are involved in lipid metabolism. The yeast Saccharomyces cerevisiae has become a reliable model organism to study biochemistry, molecular biology, and cell biology of lipids. The availability of mutants bearing defects in lipid metabolic pathways and the ease of manipulation by culture conditions facilitated these investigations. Here, we summarize the current knowledge about lipid metabolism in yeast. We grouped this large topic into three sections dealing with (1) fatty acids; (2) membrane lipids; and (3) storage lipids. Fatty acids serve as building blocks for the synthesis of membrane lipids (phospholipids, sphingolipids) and storage lipids (triacylglycerols, steryl esters). Phospholipids, sterols, and sphingolipids are essential components of cellular membranes. Recent investigations addressing lipid synthesis, degradation, and storage as well as regulatory aspects are presented. The role of enzymes governing important steps of the different lipid metabolic pathways is described. Finally, the link between lipid metabolic and dynamic processes is discussed.

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

ABSTRACT The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii. About 2000 intracellular proteins of the methylotrophic yeast Komagataella phaffii were quantified and for most of them the organelle localization was attributed.

Zusammenfassung Hintergrund Das Restless-Legs-Syndrom (RLS) ist eine schlafbezogene Bewegungsstörung mit Beinbewegungen, die durch Missempfindungen verursacht werden. Die Symptome treten vorwiegend abends oder nachts auf und bessern sich durch Bewegung. Im Kindes- und Jugendalter wird das RLS meist spät diagnostiziert, weshalb es häufig zu einer verzögerten Behandlung und einem erschwerten Zugang zu spezialisierter Versorgung kommt. Die Diagnose wird mit Hilfe der Internationalen Restless-Legs-Syndrom-Study-Group(IRLSSG)-Kriterien, unter Berücksichtigung einer alters- und entwicklungsabhängigen Symptombeschreibung, gestellt. Ziel der Arbeit Bewertung von Instrumenten zur Diagnostik des RLS im Kindes- und Jugendalter. Methodik In einer dreiphasigen Studie wurden zur Diagnostik des RLS ein Fragebogen, ein halbstandardisiertes Interview und eine Polysomnographie (PSG) durchgeführt. Ergebnisse Für 1076 Proband/innen wurde anhand des Fragebogens ein individueller RLS-Index-Score (−1,9 ± 9,5 Punkte) zur Vorhersage eines möglichen RLS berechnet. Bei 188 auffälligen Proband/innen (RLS-Index-Score 8,0 ± 7,0 Punkte) erfolgte ein halbstandardisiertes Interview zur detaillierten Anamneseerhebung. Die Diagnose eines „wahrscheinlichen“ oder „unklaren“ RLS erhielten 60 Probanden. Bei 15 Proband/innen (RLS-Index-Score 13,3 ± 2,7 Punkte) erfolgte eine zweinächtige PSG. Fünf Proband/innen zeigten einen auffälligen PLMS-Index (periodische Beinbewegungen im Schlaf). Aus den Studiendaten, klinischer Erfahrung und der Literatur wurde ein Algorithmus erarbeitet. Diskussion Zur Diagnosestellung des RLS im Kindes- und Jugendalter bedarf es einer strukturierten Diagnostik. Der vorgestellte Algorithmus gibt ein aufbauendes Vorgehen vor, welches eine einheitliche Diagnosestellung und Therapieentscheidung ermöglicht.

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a delta gph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a delta gph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.