Browsing by Author "Swiegers, Jan Hendrik"

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    Carnitine in yeast and filamentous fungi
    (Stellenbosch : Stellenbosch University, 2003-12) Swiegers, Jan Hendrik; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
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    The cloning of genes involved in carnitine-dependent activities in Saccharomyces cerevisiae
    (Stellenbosch : Stellenbosch University, 2000-03) Swiegers, Jan Hendrik; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science . Dept. of Microbiology.
    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.