The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents

Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C₂₆ fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C₁₆ and C₁₈ fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C₂₆ fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C₂₆ in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor

Sidedness of Biosynthesis of Glycosylphosphatidylinositol Anchors in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Many surface membrane glycoproteins of eucaryotes are attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthesis of these anchors proceeds through two stages. First, the synthesis of the protein and of a free glycosylphosphatidylinositol (GPI) is achieved separately. In a second step, the protein is hooked onto the preformed free GPI whereby a provisional C-terminal hydrophobic peptide is removed. The GPI-anchored protein is subsequently transported to the cell surface by way of vesicular traffic. It is presumed that the attachment of the preformed free GPI's to proteins occurs on the luminal surface of the endoplasmic reticulum (ER). The stepwise addition of sugars by glycosyltransferases onto phosphatidylinositol to form a free GPY is equally presumed to occur in the ER, but it is unclear whether these reactions take place at the cytosolic or the luminal side of the membrane. Here we tried to get some information on the membrane orientation of free GPYs in Saccharomyces cerevisiae surmising that their orientation might tell us something about the probable location of the biosynthetic process. When using trinitrobenzenesulfonic acid as a probe, we find that 75% of the free GPIs in intact ER-derived microsomes get derivatized, whereas 100% get derivatized in detergent-permeabihzed microsomes. This finding is compatible with the idea that in yeast lipid anchors are built up at the cytosolic surface

Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes

Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [3H]lyso-phosphatidic acid inside liposomes after [3H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [3H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [3H]lyso-phosphatidic acid or of dephosphorylated [3H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required

Automatic glossary term extraction from large-scale requirements specifications

Creating glossaries for large corpora of requirments is an important but expensive task. Glossary term extraction methods often focus on achieving a high recall rate and, therefore, favor linguistic proecssing for extracting glossary term candidates and neglect the benefits from reducing the number of candidates by statistical filter methods. However, especially for large datasets a reduction of the likewise large number of candidates may be crucial. This paper demonstrates how to automatically extract relevant domain-specific glossary term candidates from a large body of requirements, the CrowdRE dataset. Our hybrid approach combines linguistic processing and statistical filtering for extracting and reducing glossary term candidates. In a twofold evaluation, we examine the impact of our approach on the quality and quantity of extracted terms. We provide a ground truth for a subset of the requirements and show that a substantial degree of recall can be achieved. Furthermore, we advocate requirements coverage as an additional quality metric to assess the term reduction that results from our statistical filters. Results indicate that with a careful combination of linguistic and statistical extraction methods, a fair balance between later manual efforts and a high recall rate can be achieved

The N-glycosylation defect of cwh8Δ yeast cells causes a distinct defect in sphingolipid biosynthesis

CWH8/YGR036c of Saccharomyces cerevisiae has been identified as a dolichylpyrophosphate (Dol-PP) phosphatase that removes a phosphate from the Dol-PP generated by the oligosaccharyltransferase (OST), while it adds N‐glycans to nascent glycoproteins in the endoplasmic reticulum (ER). Lack of CWH8 was proposed to interrupt the so called dolichol (Dol) cycle by trapping Dol in the form of Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severe deficiency in N‐glycosylation. We find that cwh8D mutants have strongly reduced levels of inositolphosphorylceramide (IPC), whereas its derivative, mannosyl-(inositol-P)2-ceramide (M(IP)2C) is not affected. Microsomes of cwh8D contain normal ceramide synthase and IPC synthesis activities. Within a large panel of mutants affecting Dol dependent pathways such as N- or O-glycosylation, or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutants having a deficiency of N-glycan addition show the defect in IPC biosynthesis. By mutating genes required for the addition of N-glycans or by treating cells with tunicamycin (Tm) one can similarly reduce the steady state level of IPC and exactly reproduce the phenotype of cwh8D cells. Some potential mechanisms by which the lack of N-glycans could lead to the sphingolipid abnormality were further explore

Functions of ceramide synthase paralogs YPR114w and YJR116w of Saccharomyces cerevisiae

Ceramide is synthesized in yeast by two redundant acyl-CoA dependent synthases, Lag1 and Lac1. In lag1∆ lac1∆ cells, free fatty acids and sphingoid bases are elevated, and ceramides are produced through the redundant alkaline ceramidases Ypc1 and Ydc1, working backwards. Even with all four of these genes deleted, cells are surviving and continue to contain small amounts of complex sphingolipids. Here we show that these residual sphingolipids are not synthesized by YPR114w or YJR116w, proteins of unknown function showing a high degree of homology to Lag1 and Lac1. Indeed, the hextuple lag1∆ lac1∆ ypc1∆ ydc1∆ ypr114w∆ yjr116w∆ mutant still contains ceramides and complex sphingolipids. Yjr116w∆ exhibit an oxygen-dependent hypersensitivity to Cu2+ due to an increased mitochondrial production of reactive oxygen species (ROS) and a mitochondrially orchestrated programmed cell death in presence of copper, but also a general copper hypersensitivity that cannot be counteracted by the antioxidant N-acetyl-cysteine (NAC). Myriocin efficiently represses the synthesis of sphingoid bases of ypr114w∆, but not its growth. Both yjr116w∆ and ypr114w∆ have fragmented vacuoles and produce less ROS than wild type, before and after diauxic shift. Ypr114w∆/ypr114w∆ have an increased chronological life span. Thus, Yjr116w and Ypr114w are related, but not functionally redundant

Retirement from elite sport and self-esteem: a longitudinal study over 12 years

This study examined the complex associations between athletic retirement and self-esteem among former elite athletes. With reference to theoretical and empirical work on the quality of the transition out of sport, information was collected from 290 (junior) elite athletes in a retrospective-prospective design: at the first measurement, active athletes assessed satisfaction with their sporting career, athletic identity, and self-esteem. At the second measurement (12  years later), the now former athletes rated transition characteristics of their career ending, sporting career success, emotional reactions to career termination, extent of necessary adjustment required following athletic retirement, duration and quality of adjustment, and self-esteem. Structural equation modelling revealed that neither sporting career success nor sporting career satisfaction had a direct effect on adjustment. However, athletic identity and retirement planning predicted the extent of adjustment, which in turn predicted the duration and quality of adjustment, and ultimately self-esteem. Voluntariness, timeliness, and perceptions of gain predicted emotional reactions towards career termination, which also predicted the duration of adjustment. Extent of adjustment and emotional reactions mediated between preconditions of career termination and transition characteristics and self-esteem. While self-esteem after career termination was predominantly predicted by self-esteem 12  years earlier, perceived quality of adjustment to career termination had a significant effect on self-esteem in the post-athletic career. These results complement existing literature illustrating that athletic retirement is a complex and dynamic process and the quality of this transition has a small, but still noteworthy effect on self-esteem, a central construct for well-being

“Nothing wrong with prejudice and discrimination:” Omaha newspaper coverage of the Civil Rights Movement in 1968

This thesis discusses Omaha newspaper coverage of the Civil Rights Movement from January to April 1968. As the Vietnam War raged, racial tension continued to build in the United States, including Omaha, Nebraska. Despite its desegregation, a primarily white, male government controlled the city. The visit of Alabama Governor George Wallace, a widely known white-supremacist presidential candidate, and the assassination of Dr. Martin Luther King Jr. exacerbated the anger already felt by black communities throughout the city. This thesis examines the accuracy and contrasting content of the Omaha Star, a newspaper created to serve the black community, and the Omaha World-Herald, which mainly published articles written by white men