Show simple item record

dc.date.accessioned2006-05-09T11:39:52Z
dc.date.available2006-05-09T11:39:52Z
dc.date.issued2003-07-02
dc.identifier.uriurn:nbn:de:hebis:34-576
dc.identifier.urihttp://hdl.handle.net/123456789/576
dc.format.extent2975347 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoger
dc.subjectDictyosteliumger
dc.subjectFettsäurenger
dc.subjectEndocytoseger
dc.subjectFettsäureimportger
dc.subjectLC-FACSger
dc.subjectDictyosteliumeng
dc.subjectEndocytosiseng
dc.subjectFatty acid importeng
dc.subjectFatty acideng
dc.subjectLC-FACSeng
dc.subject.ddc570
dc.titleFunktionelle Analyse der LC-FACS in Dictyostelium discoideumger
dc.typeDissertation
dcterms.abstract"Funktionelle Analyse der LC-FACS in Dictyostelium discoideum" Das Dictyostelium discoideum Gen fcsA kodiert für ein 75 kDa großes Protein. Es kann durch Homologieanyalysen der Amino-säuresequenz zu den "long-chain fatty acyl-CoA"-Synthetasen ge-rechnet werden, die lang-kettige Fettsäuren durch die kovalente Bindung von Coenzym A akti-vie-ren und damit für diverse Reak-tionen in Stoffwechsel und Molekül-Synthese der Zelle verfügbar machen. Die hier untersuchte D. discoideum LC-FACS lokalisiert als peripher assoziiertes Protein an der cytosolischen Seite der Membran von Endo-somen und kleiner Vesikel. Bereits kurz nach der Bildung in der frühen sauren Phase kann die Lokalisation der LC-FACS auf Endosomen ge-zeigt werden. Sie dissoziiert im Laufe ihrer Neutra-li-sierung und kann auf späten Endosomen, die vor ihrer Exocytose stehen nicht mehr nach-gewiesen werden. Ein Teil der kleinen die in der gesamte Zelle verteilten kleinen Vesikel zeigt eine Kolokalisation mit lysosomalen Enzymen. Trotz des intrazellulären Verteilungs-mus-ters, das eine Beteiligung dieses Pro-teins an der Endocytose nahe-legt, konnte kein signifikanter Rückgang der Pino- und Phagocytose-Rate in LC-FACS Nullmutanten beobachtet werden. Der endo-cy-to-ti-sche Transit ist in diesen Zellen etwas verlängert, außerdem zeigen die Endosomen einen deutlich erhöhten pH-Wert, was zu einer weniger effektiven Prozessierung eines lysosomalen Enzyms führt (a-Mannosidase). Die Funktion der LC-FACS ist die Aufnahme von langkettigen Fettsäuren aus dem Lumen der Endosomen.ger
dcterms.abstractKatharina von Löhneysen Functional analysis of LC-FACS in Dictyostelium discoideum Vesicles shuttle between the compartments of the secretory pathway and the endosomal system. They originate from a donor-membrane and later fuse with the target-organelle. Many proteins and modified membrane-lipids are involved in budding, fission and fusion of vesicles. When the number of fatty acid resi-dues of a lipid is changed a acyl-CoA is being consumed or synthesized. Simul-taneously, the geometry of the lipid changes and can favour vesicle fission or fusion. Using a monoclonal antibody, we have identified a long-chain-fatty-acyl-CoA-ligase (LC-FACS) in Dictyostelium. The subcellular localisation on vesicles partly over-laps with endosomes. Because we can exclude that the labelled structures represent mitochondria or peroxisomes, it was likely the LCFA-CoA-Ligase is binding to endosomes of different stages of the pathway. To identify these organelles, a fusion of green-fluorescent-protein (GFP) and the full length cDNA of the LCFA-CoA-Ligase was constructed and expressed in D. discoideum. Staining with different endocytic markers in vivo and with a variety of organelle- specific antibodies in vitro identified the GFP-labelled compartments. This analysis was supplemented by cell-fractionation of wild type cells and other biochemical methods. We have identified a subset of Dictyostelium discoideum endosomes that carry the LC-FACS on their surface. Immunfluorescence studies and observation usinf GFP fusion proteins collectively suggest that LC-FACS associates with endo-somes a few minutes after their formation and remain bound through the acidic phase of endocytis maturation and dissociates early in the phase where endosomal content is neutralised prior to exocytosis. The LC-FACS is not a transmembrane but peripheral protein as proofed by membrane-association -assays with a post nuclaer supernatant. To investigate the function of LCFA-CoA-Ligase it was necessary to construct knock-out cells and cells with reduced LCFA-CoA-Ligase expression by gene disruption and expression of antisense RNA, respectively. To analyse whether cells are sensitive to increased amounts of this protein, LC-FACS was over-expressed. These three types of mutants were examined for general pheno-typic changes, and kinetic parameters of endo- and exocytosis were deter-mined. Even though no phenotype in pino- or phagocytosis uptake rates were measured for the LC-FACS Knock-out strain, a slightly prolonged endocytic transit could be shown; in addition endosomens have a increased pH in comparison to those in wildtype. Mutants with different expression rates of the in the fcsA gene encoding the LC-FACS protein (from nullmutant up to overexpression) showed a strong correlation between the uptake of fatty acids and the expression level of the LC-FACS. The nullmutant displayed a decreased uptake level of long-chain fatty acids but this effect was not accompanied by a general defect in endocytosis. It is known that fatty acids are taken up via the plasma membrane (McArthur, M. J. J Lipid Res, 1999), we proofed for our LC-FACS that it acts on the surface of endosomes and that fatty acid uptake is apparently strongly dependent on endocytosis in Dictyostelium discoideum.eng
dcterms.accessRightsopen access
dcterms.creatorLöhneysen, Katharina Elisabeth von
dc.contributor.corporatenameKassel, Universität, FB 19, Biologie/Chemie
dc.contributor.refereeManiak, Markus (Prof. Dr.)
dc.subject.swdDictyostelium discoideumger
dc.subject.swdFettsäurenger
dc.subject.swdEndocytoseger
dc.date.examination2003-06-13


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record