Reconstitution of yeast microsomal lipid flip-flop using endogenous aminophospholipids

AbstractThe molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown. To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip-flop of endogenous phosphatidylethanolamine in yeast. The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts. Our results demonstrate that flipping is protease sensitive but does not require metabolic energy. Our assay is the first to use the endogenous substrate of the so-called ‘flippase’ to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme

Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery

Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) is proposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P-2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4) P). We investigated the role these phosphatases in regulating PtdIns(4,5) P-2 subcellular distribution. PtdIns(4,5)P-2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Delta sac1 Delta inp54 and sac1(ts) Delta inp54 mutants. Furthermore, sac1(ts) Delta inp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P-2. Under these conditions PtdIns(4,5)P-2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P-2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P-2. Delta sac1 Delta inp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P-2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P-2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects

Refining Critical Structure Contouring in STereotactic Arrhythmia Radioablation (STAR): Benchmark Results and Consensus Guidelines from the STOPSTORM.eu Consortium.

BACKGROUND AND PURPOSE In patients with recurrent ventricular tachycardia (VT), STereotactic Arrhythmia Radioablation (STAR) shows promising results. The STOPSTORM consortium was established to investigate and harmonise STAR treatment in Europe. The primary goals of this benchmark study were to standardise contouring of organs at risk (OAR) for STAR, including detailed substructures of the heart, and accredit each participating centre. MATERIALS AND METHODS Centres within the STOPSTORM consortium were asked to delineate 31 OAR in three STAR cases. Delineation was reviewed by the consortium expert panel and after a dedicated workshop feedback and accreditation was provided to all participants. Further quantitative analysis was performed by calculating DICE similarity coefficients (DSC), median distance to agreement (MDA), and 95th percentile distance to agreement (HD95). RESULTS Twenty centres participated in this study. Based on DSC, MDA and HD95, the delineations of well-known OAR in radiotherapy were similar, such as lungs (median DSC=0.96, median MDA=0.1mm and median HD95=1.1mm) and aorta (median DSC=0.90, median MDA=0.1mm and median HD95=1.5mm). Some centres did not include the gastro-oesophageal junction, leading to differences in stomach and oesophagus delineations. For cardiac substructures, such as chambers (median DSC=0.83, median MDA=0.2mm and median HD95=0.5mm), valves (median DSC=0.16, median MDA=4.6mm and median HD95=16.0mm), coronary arteries (median DSC=0.4, median MDA=0.7mm and median HD95=8.3mm) and the sinoatrial and atrioventricular nodes (median DSC=0.29, median MDA=4.4mm and median HD95=11.4mm), deviations between centres occurred more frequently. After the dedicated workshop all centres were accredited and contouring consensus guidelines for STAR were established. CONCLUSION This STOPSTORM multi-centre critical structure contouring benchmark study showed high agreement for standard radiotherapy OAR. However, for cardiac substructures larger disagreement in contouring occurred, which may have significant impact on STAR treatment planning and dosimetry evaluation. To standardize OAR contouring, consensus guidelines for critical structure contouring in STAR were established

Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate

<p>Abstract</p> <p>Background</p> <p>Phosphoinositides play a central role in regulating processes at intracellular membranes. In yeast, a large number of phospholipid biosynthetic enzymes use a common mechanism for transcriptional regulation. Yet, how the expression of genes encoding lipid kinases and phosphatases is regulated remains unknown.</p> <p>Results</p> <p>Here we show that the expression of lipid phosphatase Sac1p in the yeast <it>Saccharomyces cerevisiae </it>is regulated in response to changes in phosphatidylinositol-4-phosphate (PI(4)P) concentrations. Unlike genes encoding enzymes involved in phospholipid biosynthesis, expression of the <it>SAC1 </it>gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of <it>SAC1 </it>that is responsible for PI(4)P-mediated regulation. Upregulation of <it>SAC1 </it>promoter activity correlates with elevated levels of Sac1 protein levels.</p> <p>Conclusion</p> <p>Regulation of Sac1p expression via the concentration of its major substrate PI(4)P ensures proper maintenance of compartment-specific pools of PI(4)P.</p

Activation of Hog1 MAPK during glucose starvation is dependent of Ssk1.

<p>(A) Schematic diagram of the yeast Hog1 pathway—Osmotic stress can be transduced by Sho1 and Sln1 activation branches. Proteins important for Hog1 phosphorylation during glucose starvation are highlighted. (B) Wild-type (wt), <i>ssk1</i>Δ and <i>sho1</i>Δ strains were exponentially grown and subjected to 0.4 M NaCl for 5 min (+NaCl) or starved for glucose for 45 min (-Glc). Cells were collected at the indicated times and equal amounts of cell extracts were analyzed by SDS-PAGE and by immunoblotting using anti-phospho-p38 antibody. Glucose-6-phosphate dehydrogenase (G6pd) was probed as the loading control. Levels of phosphorylated Hog1 were normalized using G6pd levels and then the relative quantification was normalized by time 0.</p