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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53678 |
| Date | 03 1900 |
| Creators | Kroppenstedt, Sven |
| Contributors | Bauer, F. F., Pretorius, I. S., Van der Merwe, M. J., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 91 pages : illustrations |
| Rights | Stellenbosch University |
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