A new role for carnitine in yeasts

Font-Sala, Candide (2006-04)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: L-Carnitine (3-0H-4-N-trimethylaminobutanoic acid), also called vitamin Br, is required for the metabolism of fatty acids. Only one specific metabolic activity has been ascribed to L-carnitine in eukaryotic organisms, the transfer of activated acyl residues. In the case of yeast, this process involves the transfer of activated acetyl residues from the peroxisomes or the cytoplasm to the mitochondria. In Saccharomyces cerevisiae, 13-oxidation of fatty acids takes place exclusively in the peroxisomes. The process generates peroxisomal acetyl-CoA, and the activated acetyl-residue has to be transferred to the mitochondria for energy production. Acetyl-CoA and other acyl-CoAs however can not be transferred across intracellular membranes. The activated acetyl residue is therefore transferred to a molecule of carnitine to form acetyl carnitine, which can be shuttled across membranes. The reverse reaction, the transfer of the activated acetyl to free CoA-SH and the liberation of carnitine takes place in the mitochondria. This process is also referred to as the carnitine shuttle. Most organisms, including some yeast, fungi, plants and all mammals, but not S. cerevisiae, can synthesize carnitine from lysine and S-adenosyl-methionine. However, in humans, carnitine synthesis is insufficient to satisfy carnitine requirements, and dietary contributions are essential. Various diseases linked to carnitine deficiencies have been described. Such deficiencies include those found in neonates who, in the absence of carnitine, are unable to assimilate fatty acids from milk, or genetically inborn errors of metabolism, frequently linked to a defective transport of carnitine into cells. More recent literature suggests that carnitine supplementation can have beneficial effects in a number of pathologies, and can also provide some protection against diabetes and liver disease. It has furthermore been suggested that carnitine can contribute to slowing brain aging and to improve conditions of patients suffering from neurodegenerative diseases such as Alzheimer's disease. The accumulation of such data may suggest that carnitine plays additional, as yet unrecognized roles in cellular physiology. In the study reported here, the yeast S. cerevisiae was used to identify possible additional roles for carnitine in cellular metabolism. The study furthermore attempted to identify genes that may be associated with such additional roles. The data show that carnitine supplementation of the growth substrate can protect yeast cells from hyper osmotic and high temperature stress. These protective effects are independent of the metabolic role of carnitine, since deletion of genes that are essential for the carnitine shuttle does not reduce the protective effect. The investigation also suggests that there are no other metabolic roles for carnitine in yeast than the carnitine shuttle, and that it therefore may act as a compatible solute in osmo-protection. The data also indicate a role for PH087, previously identified as a low affinity inorganic phosphate carrier, in the protective effect of carnitine. PH087 overexpression strains accumulate higher concentrations of carnitine, whereas pho87t:. strains contain less carnitine than the corresponding wild type strain. The data therefore suggest either a direct or a regulatory role of the protein in carnitine uptake.

AFRIKAANSE OPSOMMING: L-Karnitien (3-0H-4-N-trimetielaminobutaansuur), ook bekend as vitamien Br, word vir die metabolisme van vetsure benodig. Tot dusver is net een spesifieke metaboliese aktiwiteit in eukariotiese organismes aan L-karnitien toegeskryf, naamlik die oordrag van geaktiveerde asielreste. In die geval van gis behels hierdie proses die oordrag van geaktiveerde asetielreste vanaf die peroksisome of die sitoplasma na die mitochondria. In Saccharomyces cerevisiae vind die P-oksidasie van vetsure uitsluitlik in die peroksisome plaas. Hierdie proses genereer peroksisomale asetiel-KoA, en die geaktiveerde asetielreste moet vir energieproduksie na die mitochondria oorgeplaas word. Asetiel-KoA en ander asiel-KoA's kan egter nie oar intrasellulere membrane vervoer word nie. Die geaktiveerde asetielreste word dus na 'n karnitienmolekuul oorgedra om asetielkarnitien te vorm, wat oor die membrane vervoer kan word. Die omgekeerde reaksie, naamlik die oordrag van die geaktiveerde asetiel na vrye KoA-SH en die vrystelling van karnitien, vind in die mitochondria plaas. Hierdie proses staan ook as die "karnitien heen-en-weerstelsel" bekend. Die meerderheid organismes, insluitende sommige giste, swamme, plante en alle soogdiere, hoewel nie S. cerevisiae nie, kan karnitien vanaf lisien en S-adenosielmetionien sintetiseer. In mense is karnitiensintese egter onvoldoende om in die karnitienbehoeftes te voorsien en voedingsaanvullings is dus noodsaaklik. Verskeie siektes wat met 'n tekort aan karnitien verband hou, is reeds beskryf. Sulke tekorte kom o.a. voor in pasgebore babas wat in die afwesigheid van karnitien nie die vetsure afkomstig van melk kan opneem nie, of in persone met genetiese afwykings in hul metabolisme wat in baie gevalle met die onvoldoende vervoer van karnitien na die selle verband hou. In onlangse literatuur word daar voorgestel dat karnitienaanvulling voordelig vir 'n aantal patologiese toestande kan wees, en dat dit ook 'n mate van beskerming teen diabetes en lewersiektes kan verskaf. Daar is ook beweer dat karnitien die veroudering van die brein kan teewerk en die toestand van pasiente wat aan neurodegeneratiewe siektes soos Alzheimer's ly, kan verbeter. Hierdie data dui dus op die moontlikheid dat karnitien bykomende, en tot dusver onbekende, funksies in sellulere fisiologie kan he. In die studie waaroor hier verslag gedoen word, is die gis S. cerevisiae gebruik om moontlike bykomende funksies vir karnitien in sellulere metabolisme te identifiseer. Die studie poog om gene wat moontlik met sulke addisionele funksies geassosieer kan word, te identifiseer. Die data toon dat karnitienaanvulling van die groeisubstraat die gisselle teen hiperosmotiese en hoe-temperatuur stres kan beskerm. Hierdie beskermende invloed is onafhanklik van die metaboliese funksie van karnitien, aangesien die delesie van die gene wat noodsaaklik is vir die "karnitien heen-en-weerstelsel" nie die beskermende effek verminder nie. Hierdie ondersoek stel ook voor dat, buiten karnitientransport, daar geen ander metaboliese rolle vir karnitien in gis is nie, en dat dit dus moontlik as 'n verenigbare opgeloste stof in osmo-beskerming kan optree. Die data dui ook op 'n funksie vir PH087, wat voorheen as 'n lae-affiniteit anorganiese fosfaatdraer in die beskermende effek van karnitien ge"identifiseer is. PH087-ooruitdrukkingsrasse akkumuleer hoer karnitienkonsentrasies, terwyl pho87!i-rasse minder karnitien as die ooreenkomstige wilde ras bevat. Die data dui dus op 6f 'n direkte 6f 'n regulatoriese funksie vir die proteren in karnitienopname.

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