Ceramide sorting into non-vesicular transport is independent of acyl chain length in budding yeast.

The transport of ceramide from the endoplasmic reticulum (ER) to the Golgi is a key step in the synthesis of complex sphingolipids, the main building blocks of the plasma membrane. In yeast, ceramide is transported to the Golgi either through ATP-dependent COPII vesicles of the secretory pathway or by ATP-independent non-vesicular transport that involves tethering proteins at ER-Golgi membrane contact sites. Studies in both mammalian and yeast cells reported that vesicular transport mainly carries ceramide containing very long chain fatty acids, while the main mammalian non-vesicular ceramide transport protein CERT only transports ceramides containing short chain fatty acids. However, if non-vesicular ceramide transport in yeast similarly favors short chain ceramides remained unanswered. Here we employed a yeast GhLag1 strain in which the endogenous ceramide synthase is replaced by the cotton-derived GhLag1 gene, resulting in the production of short chain C18 rather than C26 ceramides. We show that block of vesicular transport through ATP-depletion or the use of temperature-sensitive sec mutants caused a reduction in inositolphosphorylceramide (IPC) synthesis to similar extent in WT and GhLag1 backgrounds. Since the remaining IPC synthesis is a readout for non-vesicular ceramide transport, our results indicate that non-vesicular ceramide transport is neither blocked nor facilitated when only short chain ceramides are present. Therefore, we propose that the sorting of ceramide into non-vesicular transport is independent of acyl chain length in budding yeast.

Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.

Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.

Clinical Profiling and Biomarkers for Post-Operative Atrial Fibrillation Prediction in Patients Undergoing Cardiac Surgery.

Post-operative atrial fibrillation (POAF) is the most common arrhythmia in the post-operative period after cardiac surgery. We aim to investigate the main clinical, local, and/or peripheral biochemical and molecular predictors for POAF in patients undergoing coronary and/or valve surgery. Between August 2020 and September 2022, consecutive patients undergoing cardiac surgery without previous history of AF were studied. Clinical variables, plasma, and biological tissues (epicardial and subcutaneous fat) were obtained before surgery. Pre-operative markers associated with inflammation, adiposity, atrial stretch, and fibrosis were analyzed on peripheral and local samples with multiplex assay and real-time PCR. Univariate and multivariate logistic regression analyses were performed in order to identify the main predictors for POAF. Patients were followed-up until hospital discharge. Out of 123 consecutive patients without prior AF, 43 (34.9%) developed POAF during hospitalization. The main predictors were cardiopulmonary bypass time (odds ratio (OR) 1.008 (95% confidence interval (CI), 1.002-1.013), p = 0.005), and plasma pre-operative orosomucoid levels (OR 1.008 (1.206-5.761). After studying differences regarding sex, orosomucoid was the best predictor for POAF in women (OR 2.639 (95% CI, 1.455-4.788), p = 0.027) but not in men. The results support the pre-operative inflammation pathway as a factor involved in the risk of POAF, mainly in women.

Impact of sphingolipids on protein membrane trafficking.

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.

GPI anchors: Regulated as needed.

GPI anchoring is an essential post-translational modification in eukaryotes that links proteins to the plasma membrane. In this issue, Liu et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208159) suggest, for the first time, a regulation on demand of the GPI glycolipid precursor biosynthesis.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

CD4+ and CD8+ T-cell responses in bone marrow to fatty acids in high-fat diets.

Obesity is associated with disruptions in the adaptive immune system; however, dietary fatty acids in high-fat diets (HFDs) that induce obesity have consequences that are currently unclear regarding T-cell maintenance in bone marrow (BM). C57BL/6J mice were randomly assigned to isocaloric HFDs formulated with dietary fats rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), or MUFAs supplemented with eicosapentaenoic and docosahexaenoic acids for 20 weeks, followed by an analysis of the immunophenotypic feature of lymphocytes (CD3+) T and their subsets CD4+ and CD8+ T cells in spleen and BM, identification of fatty acids in BM extracellular fluid and analysis of the correspondence between fatty acids with the frequency of T-cell subsets in BM. Splenic CD3+ T cells were reduced irrespective of HFDs. In BM, CD3+ T cells were reduced after HFD-SFAs, while CD4+ T cells were increased after HFDs enriched in MUFAs and CD8+ T cells were reduced irrespective of HFDs. In BM extracellular fluid, the content of palmitic and myristic acids increased after HFD-SFAs and that of oleic acid increased after HFDs enriched in MUFAs. There was a statistical correspondence between HFD-induced changes in fatty acids in BM extracellular fluid and HFD-induced changes in the frequency of CD3+ and CD4+ T cells in BM. These findings reveal an undervalued critical role for dietary fatty acids in the selective acquisition of T-cell subsets in BM, highlighting that oleic acid existing in the surroundings of T-cell niches during HFD-induced obesity could be instrumental in the maintenance of CD4+ T cells.

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway.

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein crosslinking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

[Positioning Document of Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) on Tobacco Harm Reduction Strategies]. / Documento de posicionamiento de la Sociedad Española de Neumología y Cirugía Torácica (SEPAR) ante las estrategias de reducción del daño del tabaco.

In the last decade, the appearance of electronic cigarettes and new tobacco products (heated tobacco or smokeless tobacco) has generated a growing interest in harm reduction methods that are defined as mechanisms that seek to reduce the harmful consequences of tobacco without give up the pleasant effects of it by replacing it with these new electronic devices. However, these products are addictive and not safe as they contain nicotine. Harm reduction is a false solution as it represents a commercial strategy of the tobacco industry to increase its sales, making it difficult to control smoking, since it keeps smokers from consuming tobacco and prevents them from making serious attempts to quit. In addition, these products are a gateway to adolescents in tobacco.

A Role for Lipids in Protein Sorting?

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

Structural analysis of the GPI glycan.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.

Determination of the lipid composition of the GPI anchor.

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.

Is the motivation to quit smoking a predictor of abstinence maintenance?

INTRODUCTION: The aim of this study was to explore the utility of measuring motivation to quit smoking as a predictor of abstinence maintenance among smokers who wanted to quit and who were included in a multicenter study conducted in daily clinical practice. METHODS: This observational, longitudinal (prospective cohort), multicenter study was conducted in smoking clinics in Spain and the Argentine Republic in daily clinical practice. Motivation was assessed using three quantitative motivation tests and a Visual Analogue Scale. Statistical analysis included descriptive, association measures and logistic regression models. RESULTS: Of a total of 404 subjects, 273 were ultimately included for analysis (147 women; 53.8%), mean age 51±11 years). In one year, 53.5% (36.13% by intention to treat) of subjects (146) were successful in quitting smoking [men: 45.2% (66) and women: 54.8% (80)], with no differences between sexes. None of the scales utilized was associated, in an unquestionable or direct way, with long-term abstinence, although three of them, in a very complex model, with additional variables and added interactions, were associated with the 'result' variable, when other variables intervened in certain circumstances. CONCLUSIONS: None of the analyzed motivational scales alone demonstrated an association with success or failure in quitting smoking; thus, their use in isolation is of no value. Some of the scales analyzed might be related to the maintenance of abstinence but in complex models where other variables intervene, which makes interpretation considerably difficult. Therefore, the predictive capacity of the tests analyzed, based on the models, was low.