Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity.

LAG1 was the first longevity assurance gene discovered in Saccharomyces cerevisiae The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and in vitro assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Δlag1 cells have an increased lifespan while Δlac1 cells do not.

Echocardiographic Changes with Positive Airway Pressure Therapy in Obesity Hypoventilation Syndrome. Long-Term Pickwick Randomized Controlled Clinical Trial.

Rationale: Obesity hypoventilation syndrome (OHS) has been associated with cardiac dysfunction. However, randomized trials assessing the impact of long-term noninvasive ventilation (NIV) or continuous positive airway pressure (CPAP) on cardiac structure and function assessed by echocardiography are lacking.Objectives: In a prespecified secondary analysis of the largest multicenter randomized controlled trial of OHS (Pickwick Project; N = 221 patients with OHS and coexistent severe obstructive sleep apnea), we compared the effectiveness of three years of NIV and CPAP on structural and functional echocardiographic changes.Methods: At baseline and annually during three sequential years, patients underwent transthoracic two-dimensional and Doppler echocardiography. Echocardiographers at each site were blinded to the treatment allocation. Statistical analysis was performed using a linear mixed-effects model with a treatment group and repeated measures interaction to determine the differential effect between CPAP and NIV.Measurements and Main Results: A total of 196 patients were analyzed: 102 were treated with CPAP and 94 were treated with NIV. Systolic pulmonary artery pressure decreased from 40.5 ± 1.47 mm Hg at baseline to 35.3 ± 1.33 mm Hg at three years with CPAP, and from 41.5 ± 1.56 mm Hg to 35.5 ± 1.42 with NIV (P < 0.0001 for longitudinal intragroup changes for both treatment arms). However, there were no significant differences between groups. NIV and CPAP therapies similarly improved left ventricular diastolic dysfunction and reduced left atrial diameter. Both NIV and CPAP improved respiratory function and dyspnea.Conclusions: In patients with OHS who have concomitant severe obstructive sleep apnea, long-term treatment with NIV and CPAP led to similar degrees of improvement in pulmonary hypertension and left ventricular diastolic dysfunction.Clinical trial registered with www.clinicaltrials.gov (NCT01405976).

The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection.

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment.

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.

Long-term clinical effectiveness of continuous positive airway pressure therapy versus non-invasive ventilation therapy in patients with obesity hypoventilation syndrome: a multicentre, open-label, randomised controlled trial.

BACKGROUND: Obesity hypoventilation syndrome is commonly treated with continuous positive airway pressure or non-invasive ventilation during sleep. Non-invasive ventilation is more complex and costly than continuous positive airway pressure but might be advantageous because it provides ventilatory support. To date there have been no long-term trials comparing these treatment modalities. We therefore aimed to determine the long-term comparative effectiveness of both treatment modalities. METHODS: We did a multicentre, open-label, randomised controlled trial at 16 clinical sites in Spain. We included patients aged 15-80 years with untreated obesity hypoventilation syndrome and an apnoea-hypopnoea index of 30 or more events per h. We randomly assigned patients, using simple randomisation through an electronic database, to receive treatment with either non-invasive ventilation or continuous positive airway pressure. Both investigators and patients were aware of the treatment allocation. The research team was not involved in deciding hospital treatment, duration of treatment in the hospital, and adjustment of medications, as well as adjudicating cardiovascular events or cause of mortality. Treating clinicians from the routine care team were not aware of the treatment allocation. The primary outcome was the number of hospitalisation days per year. The analysis was done according to the intention-to-treat principle. This study is registered with ClinicalTrials.gov, number NCT01405976. FINDINGS: From May 4, 2009, to March 25, 2013, 100 patients were randomly assigned to the non-invasive ventilation group and 115 to the continuous positive airway pressure group, of which 97 patients in the non-invasive ventilation group and 107 in the continuous positive airway pressure group were included in the analysis. The median follow-up was 5·44 years (IQR 4·45-6·37) for all patients, 5·37 years (4·36-6·32) in the continuous positive airway pressure group, and 5·55 years (4·53-6·50) in the non-invasive ventilation group. The mean hospitalisation days per patient-year were 1·63 (SD 3·74) in the continuous positive airway pressure group and 1·44 (3·07) in the non-invasive ventilation group (adjusted rate ratio 0·78, 95% CI 0·34-1·77; p=0·561). Adverse events were similar between both groups. INTERPRETATION: In stable patients with obesity hypoventilation syndrome and severe obstructive sleep apnoea, non-invasive ventilation and continuous positive airway pressure have similar long-term effectiveness. Given that continuous positive airway pressure has lower complexity and cost, continuous positive airway pressure might be the preferred first-line positive airway pressure treatment modality until more studies become available. FUNDING: Instituto de Salud Carlos III, Spanish Respiratory Foundation, and Air Liquide Spain.

Cost-effectiveness of positive airway pressure modalities in obesity hypoventilation syndrome with severe obstructive sleep apnoea.

BACKGROUND: Obesity hypoventilation syndrome (OHS) is treated with either non-invasive ventilation (NIV) or CPAP, but there are no long-term cost-effectiveness studies comparing the two treatment modalities. OBJECTIVES: We performed a large, multicentre, randomised, open-label controlled study to determine the comparative long-term cost and effectiveness of NIV versus CPAP in patients with OHS with severe obstructive sleep apnoea (OSA) using hospitalisation days as the primary outcome measure. METHODS: Hospital resource utilisation and within trial costs were evaluated against the difference in effectiveness based on the primary outcome (hospitalisation days/year, transformed and non-transformed in monetary term). Costs and effectiveness were estimated from a log-normal distribution using a Bayesian approach. A secondary analysis by adherence subgroups was performed. RESULTS: In total, 363 patients were selected, 215 were randomised and 202 were available for the analysis. The median (IQR) follow-up was 3.01 (2.91-3.14) years for NIV group and 3.00 (2.92-3.17) years for CPAP. The mean (SD) Bayesian estimated hospital days was 2.13 (0.73) for CPAP and 1.89 (0.78) for NIV. The mean (SD) Bayesian estimated cost per patient/year in the NIV arm, excluding hospitalisation costs, was €2075.98 (91.6), which was higher than the cost in the CPAP arm of €1219.06 (52.3); mean difference €857.6 (105.5). CPAP was more cost-effective than NIV (99.5% probability) because longer hospital stay in the CPAP arm was compensated for by its lower costs. Similar findings were observed in the high and low adherence subgroups. CONCLUSION: CPAP is more cost-effective than NIV; therefore, CPAP should be the preferred treatment for patients with OHS with severe OSA. TRIAL REGISTRATION NUMBER: NCT01405976.

Echocardiographic changes with non-invasive ventilation and CPAP in obesity hypoventilation syndrome.

RATIONALE: Despite a significant association between obesity hypoventilation syndrome (OHS) and cardiac dysfunction, no randomised trials have assessed the impact of non-invasive ventilation (NIV) or CPAP on cardiac structure and function assessed by echocardiography. OBJECTIVES: We performed a secondary analysis of the data from the largest multicentre randomised controlled trial of OHS (Pickwick project, n=221) to determine the comparative efficacy of 2 months of NIV (n=71), CPAP (n=80) and lifestyle modification (control group, n=70) on structural and functional echocardiographic changes. METHODS: Conventional transthoracic two-dimensional and Doppler echocardiograms were obtained at baseline and after 2 months. Echocardiographers at each site were blinded to the treatment arms. Statistical analysis was performed using intention-to-treat analysis. RESULTS: At baseline, 55% of patients had pulmonary hypertension and 51% had evidence of left ventricular hypertrophy. Treatment with NIV, but not CPAP, lowered systolic pulmonary artery pressure (-3.4 mm Hg, 95% CI -5.3 to -1.5; adjusted P=0.025 vs control and P=0.033 vs CPAP). The degree of improvement in systolic pulmonary artery pressure was greater in patients treated with NIV who had pulmonary hypertension at baseline (-6.4 mm Hg, 95% CI -9 to -3.8). Only NIV therapy decreased left ventricular hypertrophy with a significant reduction in left ventricular mass index (-5.7 g/m2; 95% CI -11.0 to -4.4). After adjusted analysis, NIV was superior to control group in improving left ventricular mass index (P=0.015). Only treatment with NIV led to a significant improvement in 6 min walk distance (32 m; 95% CI 19 to 46). CONCLUSION: In patients with OHS, medium-term treatment with NIV is more effective than CPAP and lifestyle modification in improving pulmonary hypertension, left ventricular hypertrophy and functional outcomes. Long-term studies are needed to confirm these results. TRIAL REGISTRATION NUMBER: Pre-results, NCT01405976 (https://clinicaltrials.gov/).

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast.

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

Non-invasive ventilation in obesity hypoventilation syndrome without severe obstructive sleep apnoea.

BACKGROUND: Non-invasive ventilation (NIV) is an effective form of treatment in patients with obesity hypoventilation syndrome (OHS) who have concomitant severe obstructive sleep apnoea (OSA). However, there is a paucity of evidence on the efficacy of NIV in patients with OHS without severe OSA. We performed a multicentre randomised clinical trial to determine the comparative efficacy of NIV versus lifestyle modification (control group) using daytime arterial carbon dioxide tension (PaCO2) as the main outcome measure. METHODS: Between May 2009 and December 2014 we sequentially screened patients with OHS without severe OSA. Participants were randomised to NIV versus lifestyle modification and were followed for 2 months. Arterial blood gas parameters, clinical symptoms, health-related quality of life assessments, polysomnography, spirometry, 6-min walk distance test, blood pressure measurements and healthcare resource utilisation were evaluated. Statistical analysis was performed using intention-to-treat analysis. RESULTS: A total of 365 patients were screened of whom 58 were excluded. Severe OSA was present in 221 and the remaining 86 patients without severe OSA were randomised. NIV led to a significantly larger improvement in PaCO2 of -6 (95% CI -7.7 to -4.2) mm Hg versus -2.8 (95% CI -4.3 to -1.3) mm Hg, (p<0.001) and serum bicarbonate of -3.4 (95% CI -4.5 to -2.3) versus -1 (95% CI -1.7 to -0.2 95% CI)  mmol/L (p<0.001). PaCO2 change adjusted for NIV compliance did not further improve the inter-group statistical significance. Sleepiness, some health-related quality of life assessments and polysomnographic parameters improved significantly more with NIV than with lifestyle modification. Additionally, there was a tendency towards lower healthcare resource utilisation in the NIV group. CONCLUSIONS: NIV is more effective than lifestyle modification in improving daytime PaCO2, sleepiness and polysomnographic parameters. Long-term prospective studies are necessary to determine whether NIV reduces healthcare resource utilisation, cardiovascular events and mortality. TRIAL REGISTRATION NUMBER: NCT01405976; results.

Sphingolipid homeostasis in the web of metabolic routes.

Sphingolipids play a key role in cells as structural components of membrane lipid bilayers and signaling molecules implicated in important physiological and pathological processes. Their metabolism is tightly regulated. Mechanisms controlling sphingolipid metabolism are far from being completely understood. However, they already reveal the integration of sphingolipids in the whole metabolic network as signaling devices that coordinate different metabolic pathways. A picture of sphingolipids integrated into metabolic networks might help to understand sphingolipid homeostasis. This review describes recent advances in the regulation of de novo sphingolipid synthesis with a focus on the bridges that exist with other metabolic pathways and the importance of this crosstalk in the control of sphingolipid homeostasis. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

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.

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.

Effectiveness of CPAP vs. Noninvasive Ventilation Based on Disease Severity in Obesity Hypoventilation Syndrome and Concomitant Severe Obstructive Sleep Apnea.

RATIONALE: Obesity hypoventilation syndrome (OHS) with concomitant severe obstructive sleep apnea (OSA) is treated with CPAP or noninvasive ventilation (NIV) during sleep. NIV is costlier, but may be advantageous because it provides ventilatory support. However, there are no long-term trials comparing these treatment modalities based on OHS severity. OBJECTIVE: To determine if CPAP have similar effectiveness when compared to NIV according to OHS severity subgroups. METHODS: Post hoc analysis of the Pickwick randomized clinical trial in which 215 ambulatory patients with untreated OHS and concomitant severe OSA, defined as apnoea-hypopnea index (AHI)≥30events/h, were allocated to NIV or CPAP. In the present analysis, the Pickwick cohort was divided in severity subgroups based on the degree of baseline daytime hypercapnia (PaCO2 of 45-49.9 or ≥50mmHg). Repeated measures of PaCO2 and PaO2 during the subsequent 3 years were compared between CPAP and NIV in the two severity subgroups. Statistical analysis was performed using linear mixed-effects model. RESULTS: 204 patients, 97 in the NIV group and 107 in the CPAP group were analyzed. The longitudinal improvements of PaCO2 and PaO2 were similar between CPAP and NIV based on the PaCO2 severity subgroups. CONCLUSION: In ambulatory patients with OHS and concomitant severe OSA who were treated with NIV or CPAP, long-term NIV therapy was similar to CPAP in improving awake hypercapnia, regardless of the severity of baseline hypercapnia. Therefore, in this patient population, the decision to prescribe CPAP or NIV cannot be solely based on the presenting level of PaCO2.

Risk factors associated with pulmonary hypertension in obesity hypoventilation syndrome.

STUDY OBJECTIVES: Pulmonary hypertension (PH) is prevalent in obesity hypoventilation syndrome (OHS). However, there is a paucity of data assessing pathogenic factors associated with PH. Our objective is to assess risk factors that may be involved in the pathogenesis of PH in untreated OHS. METHODS: In a post hoc analysis of the Pickwick trial, we performed a bivariate analysis of baseline characteristics between patients with and without PH. Variables with a P value ≤ .10 were defined as potential risk factors and were grouped by theoretical pathogenic mechanisms in several adjusted models. Similar analysis was carried out for the 2 OHS phenotypes, with and without severe concomitant obstructive sleep apnea. RESULTS: Of 246 patients with OHS, 122 (50%) had echocardiographic evidence of PH defined as systolic pulmonary artery pressure ≥ 40 mm Hg. Lower levels of awake PaO2 and higher body mass index were independent risk factors in the multivariate model, with a negative and positive adjusted linear association, respectively (adjusted odds ratio 0.96; 95% confidence interval 0.93 to 0.98; P = .003 for PaO2, and 1.07; 95% confidence interval 1.03 to 1.12; P = .001 for body mass index). In separate analyses, body mass index and PaO2 were independent risk factors in the severe obstructive sleep apnea phenotype, whereas body mass index and peak in-flow velocity in early/late diastole ratio were independent risk factors in the nonsevere obstructive sleep apnea phenotype. CONCLUSIONS: This study identifies obesity per se as a major independent risk factor for PH, regardless of OHS phenotype. Therapeutic interventions targeting weight loss may play a critical role in improving PH in this patient population. CLINICAL TRIAL REGISTRATION: Registry: Clinicaltrial.gov; Name: Alternative of Treatment in Obesity Hypoventilation Syndrome; URL: https://clinicaltrials.gov/ct2/show/NCT01405976; Identifier: NCT01405976. CITATION: Masa JF, Benítez ID, Javaheri S, et al. Risk factors associated with pulmonary hypertension in obesity hypoventilation syndrome. J Clin Sleep Med. 2022;18(4):983-992.

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.

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.

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.

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.