Mutations in the CDP-Choline Pathway for Phospholipid Biosynthesis Bypass the Requirement for an Essential Phospholipid Transfer Protein

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo

Biochemical analyses of the Golgi and its associated membrane proteins in Saccharomyces cerevisiae

Secretory trafficking in eukaryotic cells occurs via membrane-enclosed organelles and vesicular intermediates which deliver proteins and lipids to their cellular destinations. Throughout this process, the unique nature of organelles is likely to be maintained through: (i) membrane recycling events that function in the retrieval of protein and lipid components from later stages in the pathway, and (ii) compartment-specific scaffolding factors that act in securing the structural and spatial integrity of the organelle. The Golgi apparatus of the yeast Saccharomyces cerevisiae has been defined through the use of conditional mutants which accumulate aberrant Golgi structures under restrictive conditions. This manuscript defines a novel enrichment scheme for the purification of these aberrant structures and identifies two proteins as steady-state residents of the yeast Golgi. These resident proteins have been used for the enrichment of Golgi from wild-type cells. Further, a series of monoclonal antibodies were generated to carbonate insensitive proteins from this membrane population in an attempt to identify additional compartment-specific factors. Four of these monoclonal antibodies recognized protein species of 51 kDa, 36 kDa, 95 kDa and 77 kDa. Each of these proteins displayed carbonate insensitivity and only the 95 kDa species was found to be glycosylated. The Golgi-specific residence of each of these proteins was established by indirect immunofluorescence and quantitative biochemical fractionation. In addition, analyses of conditional secretory (sec) mutants blocked at various stages in the secretory process suggested that the 51 kDa species progressed to the Golgi but was not incorporated into post-Golgi secretory vesicles. These data were consistent with the proposed intracellular localization of these proteins to the yeast Golgi.U of I OnlyETDs are only available to UIUC Users without author permissio

Cross-reactive carbohydrate determinant interference in cellulose-based IgE allergy tests utilizing recombinant allergen components.

BackgroundCross-reactive carbohydrate determinant (CCD) structures found in plant and insect glycoproteins are commonly recognized by IgE antibodies as epitopes that can lead to extensive cross-reactivity and obscure in vitro diagnostic (IVD) serology results. With the introduction of component resolved diagnosis (CRD), recombinant non-glycosylated components have been utilized to mitigate the risk of CCD-specific IgE (sIgE) detection. However, a recent study has shown that CCD-sIgE may bind directly to the cellulose solid phase matrix used in certain in vitro diagnostic assays, eliminating the advantage of CRD over traditional extract-based testing. The aim of this study is to further investigate the prevalence of CCD-sIgE interference on a commonly-used in vitro sIgE automated platform which employs a cellulose-based matrix to immobilize CCD-free recombinant components.MethodsSera from patients sensitized to peanut, silver birch, and/or timothy grass were analyzed for CCD-sIgE reactivity on ImmunoCAP/Phadia and NOVEOS autoanalyzers against the MUXF3 carbohydrate component. Positive CCD-sIgE sera were further analyzed against non-glycosylated recombinant components bound to the ImmunoCAP solid phase in the absence and presence of a soluble CCD inhibitor. For comparison, sera were then analyzed on NOVEOS, a non-cellulose based automated sIgE assay.ResultsSera from 35% of the sensitized population tested in this study were positive (≥0.35 kU/L) for CCD-sIgE. Of those positives, 17% resulted in CCD-sIgE-positive (false positive) results on ImmunoCAP using non-glycosylated allergosorbents that were negative on NOVEOS. Sera producing false-positive results on ImmunoCAP had varying levels of CCD-sIgE from 0.67 kU/L to 36.52 kU/L. The incidence of CCD interference was predominantly delimited to low-positive IgE results (0.35 kUA/L- 3.00 kUA/L).ConclusionFalsely elevated diagnostic allergen-sIgE results can commonly occur due to the presence of CCD-sIgE using assays that employ a carbohydrate matrix-based allergosorbent. Even the use of non-glycosylated recombinant allergenic components coupled to cellulose matrices do not reduce their risk of detection. The risk of CCD interference that compromises quantitative IgE results can be mitigated by the addition of a soluble CCD inhibitor to positive CCD-sIgE containing sera or by alternatively using a non-cellulose based sIgE assay, such as the NOVEOS assay

Polythiophenes inhibit prion propagation by stabilizing PrP aggregates

LCPs interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here we show that a variety of anionic, cationic and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices COCS, and decreased the amount of PrP(Sc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, and triggered the compaction and enhanced the resistance to proteolysis of recombinant mPrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on COCS and in vitro conversion assays with mPrP(23-231) indicated that PTAA may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits