Immunoelectron microscopic localization of thiolases, beta-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis.

The location of acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III), enzymes of the fatty acid beta-oxidation system, was studied in n-alkane-grown Candida tropicalis cells by immunoelectron microscopy using a post-embedding method with colloidal gold conjugated IgG. The deposition of gold particles for T-I was detected in the microbodies and cytoplasm and that of gold particles for T-III specifically in the microbodies. The double labeling technique confirmed that T-I and T-III occurred concurrently in a microbody and T-I also in cytoplasm. These results were consistent with the biochemical data based on subcellular fractionation and indicated that the yeast beta-oxidation system operates efficiently only in the microbodies.     

Isolation of several cDNAs encoding yeast peroxisomal enzymes

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage lambda gt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.     

High-level expression and molecular cloning of genes encoding Candida tropicalis peroxisomal proteins.

The development of peroxisomes in the cells of Candida tropicalis grown on oleic acid was accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, the nuclear DNA library of this yeast was screened by differential hybridization, and 102 clones of oleic acid-inducible sequences were isolated. Seven coding regions were found to form clusters in three stretches of the genomic DNA. Five of the regions were identified as genes for peroxisomal polypeptides (PXPs). The coding sequence for PXP-2 hybrid selected an additional mRNA for PXP-4, the subunit of long-chain acyl coenzyme A oxidase, which was the most abundant PXP. PXP-2 and PXP-4 were close in apparent molecular weight and generated similar peptides when digested with a protease. The gene for PXP-4 was adjacent to that for PXP-2 on the genome and also hybridized to the mRNA coding for PXP-5. These and other similar results suggest that the genes for the peroxisomal proteins of this organism arose by duplication of a few ancestral genes.     

Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid beta-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system.     

The primary structure of a peroxisomal fatty acyl-CoA oxidase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a gene encoding peroxisomal fatty acyl-CoA oxidase (AOx) from the yeast Candida tropicalis pK233. The AOx gene contains no intervening sequences and has a single open reading frame of 2127 nt encoding a protein of 708 amino acids (aa), not including the initiator methionine. The Mr of the protein is 79,155. Codon utilization in the gene is not random, with 87.4% of the aa specified by 25 principal codons. The principal codons used in the expression of AOx in C. tropicalis are similar to those used in highly expressed genes of Saccharomyces cerevisiae. The AOx protein shows a 94.2% homology with POX4 protein of C. tropicalis. One stretch of 36 aa shows no homology between the two proteins.     

Nuclear DNAase of the yeast Candida tropicalis

Using stepwise extraction of chromatin from Candida tropicalis by NaCl (0.1-1.0 M) the protein dissociated by 0.3 and 0.6 M NaCl (fractions 0.3 and 0.6) possessing the DNAase activity were obtained. These DNAases are activated by Mg2+ and cause preferential hydrolysis of heat-denaturated DNA. Fraction 0.3 DNAase has a maximum at neutral values of pH (around 7.0) and causes endonucleolytic hydrolysis of DNA. Fraction 0.6 DNAase causes exonucleolytic hydrolysis of DNA but a maximum at alkaline pH (8.0). The properties of isolated chromatin DNAases of Candida tropicalis differ from those of the known DNAases of the yeast Saccharomyces cerevisiae.     

Genetic evaluation of peroxisomal and cytosolic acetoacetyl-CoA thiolase isozymes in n-alkane-assimilating diploid yeast, Candida tropicalis

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic. Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrated that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.     

Physiological roles of acetoacetyl-CoA thiolase in n-alkane-utilizable yeast, Candida tropicalis: possible contribution to alkane degradation and sterol biosynthesis.

The presence of two types of thiolases, acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase, was demonstrated in peroxisomes of n-alkane-grown Candida tropicalis [Kurihara, T., Ueda, M., & Tanaka, A. (1989) J. Biochem. 106, 474-478], while acetoacetyl-CoA thiolase was also shown to be present in cytosol. The activity of the enzyme in cytosol was constant irrespective of culture conditions, while the peroxisomal enzyme was inducibly synthesized in the alkane-grown yeast cells. These results indicate that peroxisomal acetoacetyl-CoA thiolase participates in alkane degradation, while the cytosolic enzyme is associated with other fundamental metabolic processes, probably sterol biosynthesis, because this enzyme can catalyze the first step of the sterol biosynthesis. 3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulatory enzyme of sterol biosynthesis, was found to be localized exclusively in microsomes of the alkane-grown yeast cells. These results suggest that yeast peroxisomes do not contribute to sterol biosynthesis, unlike the case of mammalian cells.     

cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.     

Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.     

Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis

The gene for the secreted acid protease (ACP), a potential virulence factor of Candida species, was inactivated in Candida tropicalis by gene disruption. The disruption was performed by cotransformation of an ade2 C. tropicalis mutant with a linear DNA fragment carrying a deletion in ACP, and the replicative vector pMK16 which carries a selectable ADE2 gene marker. Few of the transformants exhibited lower protease secretion levels and were shown to have one deleted and one unaffected ACP copy, since C. tropicalis is a diploid yeast. These transformants were rendered homozygotic for this deletion by mild UV-treatment. One of the homozygotic acp deletion mutants obtained was completely devoid of extracellular protease activity and grew poorly on bovine serum albumin-containing medium. This mutant could be complemented by an ACP fragment inserted in pMK16, but also by an acid protease gene isolated from C. parapsilosis.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n-alkane-utilizable yeast, Candida tropicalis, has been isolated by the hybrid-selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m = 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high-expression type. Amino acid residues were all conserved at the active and heme-binding sites. In the N and C-terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.