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The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae
Thesis (MSc)--Stellenbosch University, 2003.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2012
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| _version_ | 1867614068748058624 |
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| access_status_str | Open Access |
| author | Kroppenstedt, Sven |
| author2 | Bauer, Florian |
| author_browse | Bauer, Florian Kroppenstedt, Sven |
| author_facet | Bauer, Florian Kroppenstedt, Sven |
| author_sort | Kroppenstedt, Sven |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2003. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/53678 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:10.315Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/53678 The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae Kroppenstedt, Sven Bauer, Florian Pretorius, I. S. Van der Merwe, M. J. Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. Carnitine Saccharomyces cerevisiae -- Metabolism Carnitine acetyl transferases Thesis (MSc)--Stellenbosch University, 2003. ENGLISH ABSTRACT: L-carnitine is a compound with a long history in biochemistry. It plays an important role in mammals, where many functions have been attributed to it. Those functions include the p-oxidation of long-chain fatty acids, the regulation of the free CoASH/ Acyl-CoA ratio and the translocation of acetyl units into mitochondria. Carnitine is also found in lower eukaryotic organisms. However, in contrast to the multiple roles it plays in mammalian cells, its action appears to be restricted to the transport of activated acyl residues across intracellular membranes in the lower eukaryotes. In the yeast Saccharomyces cere visiae , the role of carnitine consists mainly of the transfer of activated acetyl residues from the peroxisome and cytoplasm to the mitochondria. This process is referred to as the carnitine shuttle. This system involves the transfer of the acetyl moiety of acetyl-CoA, which cannot cross organellar membranes, to a molecule of carnitine. Subsequently, the acetylcarnitine is transported across membranes into the mitochondria, where the reverse transfer of the acetyl group to a molecule of free CoA occurs for further metabolism. Carnitine acetyl transferases (CATs) are the enzymes responsible for catalysing the transfer of the activated acetyl group of acetyl-CoA to carnitine as well as for the reverse reaction. In the yeast S. cerevisiae, three CAT enzymes, encoded by the genes CAT2, YAT1 and YAT2, have been identified. Genetic data suggest, that despite the high sequence similarity, each of the genes encodes for a highly specific activity that is part of the carnitine shuttle. So far, the specific function of any of the three CAT enzymes has been elucidated only partially. The literature review focuses mainly on the importance of the carnitine system in mammals. After discussing the discovery and biosyntheses of carnitine, the enzymatic background of and molecular studies on the carnitine acyltransferases are described. The experimental section focuses on elucidating the physiological roles and cellular localisation of the three carnitine acetyltransferase of S. cere visia e. We developed a novel enzymatic assay to study CAT activity in vivo. By C-terminal tagging with a green fluorescent protein, we localised the three CAT enzymes. However, all our genetic attempts to reveal specific roles for and functions of these enzymes were unsuccessful. The overexpression of any of the CAT genes could not cross-complement the growth defect of other CAT mutant strains. No phenotypical difference could be observed between strains carrying single, double and triple deletions of the CAT genes. Furthermore, the expression of the Schizosaccharomyces pombe dicarboxylic acid transporter can complement the deletion of the peroxisomal citrate synthase, but has no effect on the carnitine shuttle per se. Our data nevertheless suggest that Cat2p is the enzyme mainly responsible for the forward reaction, e.g. the formation of acetylcarnitine and free CoA-SH from acetyl-CoA and carnitine, whereas Yat1 pand Yat2p may be required mainly for the reverse reaction. AFRIKAANSE OPSOMMING: L-karnitien is 'n verbinding met 'n lang geskiedenis in die biochemie-veld. Dit speel 'n belangrike rol in soogdiere, waar verskeie funksies daaraan toegeskryf word. Dié funksies sluit in die p-oksidasie van lang-ketting-vetsure, die regulering van die vrye KoA-SH-tot-asiel-KoA-verhouding en die oordrag van asetieleenhede na die mitochondria. Karnitien word ook in laer eukariotiese organismes gevind. In teenstelling met die verskeidenheid rolle wat dit in soogdierselle vervul, is die funksie in laer eukariote tot die transport van geaktiveerde asetielderivate oor intrasellulêre membrane beperk. In die gis Saccharomyces cerevisiae is die funksie van karnitien meestal beperk tot die vervoer van geaktiveerde asetielresidu's vanaf die sitoplasma en piroksisome na mitochondria, 'n proses wat as die "karnitiensiklus" bekend staan. Die proses behels die oordrag van die asetielgedeelte van asetiel-KoA, wat nie oor organelmembrane kan beweeg nie, na 'n molekuul van karnitien. Gevolglik word die asetielkarnitien oor die membraan na die mitochondria vervoer, waar - met die oog op verdere metabolisme - die omgekeerde oordrag van die asetielgroep na 'n vrye molekuul van KoA plaasvind. Karnitienasetiel-transferases (KAT's) is die ensieme wat verantwoordelik is vir die katalisering van die oordrag van die geaktiveerde asetielgroepe van asetiel-KoA na karnitien, sowel as vir die omgekeerde reaksie. In die gis S. cerevisiae is drie KAT-ensieme geïdentifiseer wat deur die gene CAT2, YAT1 en YAT2 gekodeer word. Genetiese data dui daarop dat, ten spyte van die hoë mate van homologie van die DNA-volgordes, elke geen vir 'n hoogs spesifieke aktiwiteit, wat deel van die karnitiensiklus is, kodeer. Tot dusver is die spesifieke funksie van die drie individuele KAT-ensieme net gedeeltelik ontrafel. Die literatuurstudie fokus hoofsaaklik op die belangrikheid van karnitiensisteme in soogdiere. Na 'n bespreking van die ontdekking en biosintese van karnitien, word die ensimatiese agtergrond en molekulêre studies van KAT's beskryf. Die eksperimentele deel konsentreer op die ontrafelling van die fisiologiese rol en intrasellulêre lokalisering van die drie KAT-ensieme van S. cerevisiae. Eerstens is 'n nuwe ensimatiese toets ontwikkel om KAT-aktiwiteit in vivo te bestudeer. Deur C-terminale aanhegting van 'n groen fluoreserende proteïen kon die drie KATensieme gelokaliseer word. Daar kon egter nie met behulp van genetiese studies verder lig gewerp word op die spesifieke rolle en funksies van hierdie KAT-ensieme nie. Die ooruitdrukking van enige van die KAT-gene kon nie die groeidefek van ander KAT-mutantrasse kruiskomplementeer nie. Geen fenotipiese verskil tussen rasse wat 'n enkel, dubbel of trippel delesie van die KAT-gene bevat, kon waargeneem word nie. Verder kon die uitdrukking van Schizosaccharomyces pombe se dikarboksielsuurtransporter die delesie van die peroksisomale sitraatsintetase komplementeer, maar het dit as sulks geen effek op die karnitiensiklus gehad nie. Die data wat deur hierdie studie verkry is, dui nogtans daarop dat Cat2p die ensiem is wat hoofsaaklik verantwoordelik is vir die voorwaartse reaksie, met ander woorde die vorming van asetielkarnitien en vrye KoH-SH van asetiel-KoA en karnitien, terwyl Yat1 p en Yat2p hoofsaaklik vir die omgekeerde reaksie benodig word. Masters 2012-08-27T11:35:35Z 2012-08-27T11:35:35Z 2003-03 Thesis http://hdl.handle.net/10019.1/53678 en_ZA Stellenbosch University 91 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Carnitine Saccharomyces cerevisiae -- Metabolism Carnitine acetyl transferases Kroppenstedt, Sven The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title | The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title_full | The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title_fullStr | The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title_full_unstemmed | The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title_short | The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiae |
| title_sort | role of carnitine acetyltransferases in the metabolism of saccharomyces cerevisiae |
| topic | Carnitine Saccharomyces cerevisiae -- Metabolism Carnitine acetyl transferases |
| url | http://hdl.handle.net/10019.1/53678 |
| work_keys_str_mv | AT kroppenstedtsven theroleofcarnitineacetyltransferasesinthemetabolismofsaccharomycescerevisiae AT kroppenstedtsven roleofcarnitineacetyltransferasesinthemetabolismofsaccharomycescerevisiae |