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

[摘要] ENGLISH ABSTRACT:L-Carnitine is a unique and important compound in eukaryotic cells. In Saccharomycescerevisiae, L-carnitine plays a role in the transfer of acetyl groups from the peroxisomes tothe mitochondria. This takes place with the help of the carnitine acetylcarnitine shuttle. Theactivated acyl group of acetyl-CoA in the peroxisome is transferred to carnitine with thehelp of a peroxisomal carnitine acetyltransferase to form an acetylcarnitine ester, releasingthe CoA-SH. This ester is then transported through the peroxisomal membrane to thecytosol from where it is transported to the mitochondrion. After transport of theacetylcarnitine through the mitochondrial membranes, the reverse reaction takes place inthe matrix with the help of a mitochondrial carnitine acetyltransferase, releasing carnitineand 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 theperoxisomal 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 acylgroup needs to be transported in the form of intermediate compounds. Acetyl-CoA isformed 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 theperoxisome where they are oxidized by the p-oxidation cycle to form two-carbon acylgroups in the form of acetyl-CoA. These two-carbon acyl groups are then transferred fromthe peroxisome to the rest of the cell for gluconeogenesis and other anabolic pathways, orused 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 inthe peroxisome and that is the glyoxylate cycle. The glyoxylate cycle is a modified TCAcycle, which results in the synthesis of C4 succinate from two molecules of acetyl-CoA. InS. cerevisiae, all of the enzymes of the glyoxylate cycle are located in the peroxisomeexcept for one, whereas in other yeasts studied, all of the glyoxylate enzymes areperoxisomal. As a result of the glyoxylate cycle, the two carbons of acetyl-CoA can leavethe peroxisome in the form of succinate or other TCA intermediates like malate and citrate.These compounds are transferred through dicarboxylic acid carriers present in theperoxisomal 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 involvedin carnitine-dependent activities in S. cerevisiae was developed. The disruption of thecitrate synthetase gene, CIT2, of the glyoxylate cycle yielded a strain that was dependenton carnitine when grown on the fatty acid oleic acid. This allowed for a mutagenesisstrategy based on negative selection of mutants affected in carnitine-dependent activities.The ~cit2 strain was mutagenized and plated on minimal media. After replica plating onoleic acid media, mutant strains were selected that were unable to grow even in thepresence of carnitine. In order to eliminate strains with defects in peroxisome biogenesisand ~-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 affectedin activities independent of carnitine, were retained for further analysis. Transforming oneof these mutants with a S. cerevisiae genomic library for functional complementation,yielded a clone carrying the YAT1 gene, coding for the carnitine acetyltransferase of theouter-mitochondrial membrane. No phenotype had previously been assigned to a mutantallele of this gene.

[发布机构] Stellenbosch University