The cloning of genes involved in carnitine-dependent activities in Saccharomyces cerevisiae

Swiegers, Jan Hendrik (2000-03)

Thesis (MSc)--University of Stellenbosch, 2000.

Thesis

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.

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