Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: L-Carnitine is a unique and important compound in eukaryotic cells. In Saccharomyces
cerevisiae, L-carnitine plays a role in the transfer of acetyl groups from the peroxisomes to
the mitochondria. This takes place with the help of the carnitine acetylcarnitine shuttle. The
activated acyl group of acetyl-CoA in the peroxisome is transferred to carnitine with the
help of a peroxisomal carnitine acetyltransferase to form an acetylcarnitine ester, releasing
the CoA-SH. This ester is then transported through the peroxisomal membrane to the
cytosol from where it is transported to the mitochondrion. After transport of the
acetylcarnitine through the mitochondrial membranes, the reverse reaction takes place in
the matrix with the help of a mitochondrial carnitine acetyltransferase, releasing carnitine
and the acyl group. In S. cerevisiae, the main carnitine acetyltransferase contributing to
>95% of the total carnitine acetyltransferase activity, is encoded by a single gene, CAT2.
Cat2p has a peroxisomal and mitochondrial targeting signal and is located to the
peroxisomal membrane and the inner-mitochondrial membrane, respectively.
The reason for the activated acyl group to be transferred in the form of an acetylcarnitine,
is that the peroxisomal membrane is impermeable to acetyl-CoA. This means that the acyl
group needs to be transported in the form of intermediate compounds. Acetyl-CoA is
formed in the peroxisome of S. cerevisiae as a result of p-oxidation of fatty acids. In yeast,
the peroxisome is the sole site for p-oxidation. Fatty acids are transported to the
peroxisome where they are oxidized by the p-oxidation cycle to form two-carbon acyl
groups in the form of acetyl-CoA. These two-carbon acyl groups are then transferred from
the peroxisome to the rest of the cell for gluconeogenesis and other anabolic pathways, or
used in the tricarboxylic acid cycle (TCA) of the mitochondia to generate ATP. In this way,
it is possible for the cell to use fatty acid as a sole carbon source.
There is a second pathway allowing for the utilization of activated acyl groups produced in
the peroxisome and that is the glyoxylate cycle. The glyoxylate cycle is a modified TCA
cycle, which results in the synthesis of C4 succinate from two molecules of acetyl-CoA. In
S. cerevisiae, all of the enzymes of the glyoxylate cycle are located in the peroxisome
except for one, whereas in other yeasts studied, all of the glyoxylate enzymes are
peroxisomal. As a result of the glyoxylate cycle, the two carbons of acetyl-CoA can leave
the peroxisome in the form of succinate or other TCA intermediates like malate and citrate.
These compounds are transferred through dicarboxylic acid carriers present in the
peroxisomal membrane and used in further metabolic needs of the cell.
To understand the role of carnitine in the cell, a strategy for the cloning of genes involved
in carnitine-dependent activities in S. cerevisiae was developed. The disruption of the
citrate synthetase gene, CIT2, of the glyoxylate cycle yielded a strain that was dependent on carnitine when grown on the fatty acid oleic acid. This allowed for a mutagenesis
strategy based on negative selection of mutants affected in carnitine-dependent activities.
The ~cit2 strain was mutagenized and plated on minimal media. After replica plating on
oleic acid media, mutant strains were selected that were unable to grow even in the
presence of carnitine. In order to eliminate strains with defects in peroxisome biogenesis
and ~-oxidation, and only select for strains with defects in carnitine-dependent activities,
the mutant strains were transformed with the CIT2 gene to restore the glyoxylate cycle.
Mutants that grew on oleic acid after transformation, and which are therefore not affected
in activities independent of carnitine, were retained for further analysis. Transforming one
of these mutants with a S. cerevisiae genomic library for functional complementation,
yielded a clone carrying the YAT1 gene, coding for the carnitine acetyltransferase of the
outer-mitochondrial membrane. No phenotype had previously been assigned to a mutant
allele of this gene. / AFRIKAANSE OPSOMMING: L-Karnitien is 'n unieke en belangrike verbinding in eukariotiese selle. In Saccharomyces
cerevisiae speel L-karnitien In rol in die oordrag van asielgroepe van die peroksisoom na
die mitochondrion. Dit vind plaas met behulp van die karnitien-asetielkarnitien-weg. Die
geaktiveerde asiel groep van asetiel-KoA in die peroksisoom word na karnitien oorgedra
met behulp van 'n peroksisomale karnitien-asetielkarnitien-transferase-ensiem om 'n
asetielkarnitien ester te vorm, waarna die KoA-SH vrygestel word. Hierdie ester word dan
deur die peroksisomale membraan na die sitoplasma vervoer waarna dit na die
mitochondrion vervoer word. Nadat die asetielkarnitien deur die mitochondriale membrane
vervoer is, vind die omgekeerde reaksie in die matriks plaas met behulp van die
mitochondriale karnitien-asetielkarnitien-transferase-ensiem, waarna die karnitien en die
asielgroep vrygestel word. In S. cerevisiae word die hoof karnitien-asetielkarnitien
transferase wat tot >95% van die totale karnitien-asetielkarnitien-transferase-aktiwiteit
bydra, deur 'n enkele geen, CA T2 gekodeer. CAT2p het 'n peroksisomale en
mitochondriale teikensein en dit word onderskeidelik na die peroksisomale en binnemitochondriale
membrane gelokaliseer.
OPSOMMING
Die geaktiveerde asielgroep word in die vorm van 'n asetielkarnitien vervoer omdat die
peroksisomale membraan ondeurlaatbaar vir asetiel-KoA is. Dit beteken dat die
asielgroepe slegs in die vorm van intermediêre verbindings vervoer kan word. Asetiel-KoA
word weens p-oksidasie van vetsure in die peroksisoom van S. cerevisiae gevorm. In gis
is die peroksisoom die enigste plek waar p-oksidasie plaasvind. Vetsure word na die
peroksisoom vervoer waar dit deur die p-oksidasiesiklus geoksideer word om tweekoolstof
asielgroepe in die vorm van asetiel-KoA te vorm. Hierdie twee-koolstof
asielgroepe word dan vanaf die peroksisoom na die res van die sel vervoer vir
glukoneogenese en ander metaboliese paaie, of dit word in die trikarboksielsuursiklus
(TKS) van die mitochondrion gebruik om ATP te genereer. Op hierdie wyse is dit moontlik
vir die sel om vetsure as enigste koolstofbron te benut.
Die glioksilaatsiklus is 'n tweede weg wat die benutting van asielgroepe, wat in die
peroksisoom geproduseer is, toelaat. Die glioksilaatsiklus is 'n gemodifiseerde TKS-siklus
wat die sintese van C4 suksinaat van uit twee molekules asetiel-KoA bewerkstellig. In
teenstelling met ander giste waar al die glioksilaatsiklus ensieme in die peroksisoom geleë
is, kom een van S. cerevisiae se ensieme buite die peroksisoom voor. Die resultaat van
die glioksilaatsiklus is dat die twee koolstowwe van asetiel-KoA die peroksisoom in die
vorm van suksinaat of ander TKS-intermediêre verbindings soos malaat en sitraat, kan
verlaat. Hierdie verbindings word deur middel van dikarboksielsuur-transporters in die
peroksisomale membraan vervoer en word dan vir verdere metaboliese behoeftes in die
sel gebruik. Om die rol van karnitien in die sel te verstaan, is 'n strategie ontwikkel om gene wat by
karnitien-afhanklike aktiwiteite in S. cerevisiae betrokke is, te kloneer. Die disrupsie van
die sitraatsintesegeen, CIT2, van die glioksilaatsiklus het 'n ras gelewer wat van karnitien
vir groei op die vetsuur oleiensuur afhanklik was. Die fl.cit2-ras is gemuteer en op minimale
media uitgeplaat. Na replika-platering op oleiensuur media, is mutante rasse geselekteer
wat nie gegroei het nie, selfs nie in die teenwoordigheid van karnitien nie. Om mutantrasse
uit te skakel wat defekte in peroksisoom-biogenese en p-oksidasie het en net mutantrasse
te selekteer wat defekte in karnitien-afhanklike aktiwiteite het, is die rasse met die CIT2-
geen getransformeer om die glioksilaatsiklus te herstel. Mutante wat na transformasie op
oleiensuur gegroei het, en dus nie in aktiwiteite onafhanklik van karnitien geaffekteer is
nie, is behou en aan verdere analise blootgestel. Komplimentering van een van hierdie
mutante met 'n S. cerevisiae genomiese biblioteek, het 'n kloon wat die geen YAT1 bevat,
gelewer. YAT1 is 'n geen wat die karnitienasetieltransferase van die buite-mitochondriale
membraan kodeer. Geen fenotipe is ooit voorheen aan 'n mutant alleel in hierdie geen
toegeskryf nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51947 |
| Date | 03 1900 |
| Creators | Swiegers, Jan Hendrik |
| Contributors | Bauer, F. F., Pretorius, I. S., Stellenbosch University. Faculty of Science . Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 82 pages : illustrations |
| Rights | Stellenbosch University |
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