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U Hoja

Publications and source records attributed to U Hoja.

8 recordsLinked to original sources
PubMed2003

Functional differentiation and selective inactivation of multiple Saccharomyces cerevisiae genes involved in very-long-chain fatty acid synthesis.

While de novo fatty acid synthesis uses acetyl-CoA, fatty acid elongation uses longer-chain acyl-CoAs as primers. Several mutations that interfere with fatty acid elongation in yeast have already been described, suggesting that there may be different elongases for medium- and long-chain acyl-CoA primers. In the present study, an experimental approach is described that allows differential characterization of the various yeast elongases in vitro. Based on their characteristic primer specificities and product patterns, at least three different yeast elongases are defined. Elongase I extends C12-C16 fatty acyl-CoAs to C16-C18 fatty acids. Elongase II elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and elongase III synthesizes 20-26-carbon fatty acids from C18-CoA primers. Elongases I, II and III are specifically inactivated in, respectively, elo1, elo2 and elo3 mutants. Elongases II and III share the same 3-ketoacyl reductase, which is encoded by the YBR159w gene. Inactivation of YBR159w inhibits in vitro fatty acid elongation after the first condensation reaction. Although in vitro elongase activity is absent, the mutant nevertheless contains 10-30% of normal VLCFA levels. On the basis of this finding, an additional elongating activity is inferred to be present in vivo. ybr159Delta cells show synthetic lethality in the presence of cerulenin, which inactivates fatty acid synthase. An involvement of FAS in VLCFA synthesis may account for these findings, but remains to be demonstrated directly. Alternatively, a vital role for C18 and C20 hydroxyacids, which are dramatically overproduced in ybr159Delta cells, may be postulated.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed2001

A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product.

The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by general transcription factors (such as Reb1, Rap1 and Abf1) and by the phospholipid-specific heterodimeric activator Ino2/Ino4, acting via their corresponding upstream binding sites. Here we provide evidence for a positive autoregulatory influence of FAS1 on FAS2 expression. Even with a constant FAS2 copy number, a 10-fold increase of FAS2 transcript amount was observed in the presence of FAS1 in multi-copy, compared to a fas1 null mutant. Surprisingly, the first 66 nt of the FAS2 coding region turned out as necessary and sufficient for FAS1-dependent gene expression. FAS2-lacZ fusion constructs deleted for this region showed high reporter gene expression even in the absence of FAS1, arguing for a negatively-acting downstream repression site (DRS) responsible for FAS1-dependent expression of FAS2. Our data suggest that the FAS1 gene product, in addition to its catalytic function, is also required for the coordinate biosynthetic control of the yeast FAS complex. An excess of uncomplexed Fas1 may be responsible for the deactivation of an FAS2-specific repressor, acting via the DRS.

Fatty Acid Synthases↗
PubMed1999

Identification of the plasma membrane H+-biotin symporter of Saccharomyces cerevisiae by rescue of a fatty acid-auxotrophic mutant.

Bakers' yeast is auxotrophic for biotin (vitamin H) and depends on the efficient uptake of this compound from the environment. A mutant strain with strongly reduced biotin uptake and with reduced levels of protein biotinylation was identified. The strain was auxotrophic for long-chain fatty acids, and this auxotrophy could be suppressed with high levels of biotin in the medium. After transformation of this mutant with a yeast genomic library, the unassigned open reading frame YGR065C was identified to complement this mutation. This gene codes for a protein with 593 amino acids and 12 putative transmembrane helices. Northern blot analysis revealed that, in wild-type cells, the corresponding mRNA levels were increased at low biotin concentrations. Likewise, cellular biotin uptake was increased with decreasing biotin availability. Expression of YGR065C under the control of the constitutive ADH1 promoter resulted in very high biotin transport rates across the plasma membrane that were no longer regulated by the biotin concentration in the growth medium. We conclude that YGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 for vitamin H transporter 1.

Amino Acid Sequence↗
PubMed1998

Pleiotropic phenotype of acetyl-CoA-carboxylase-defective yeast cells--viability of a BPL1-amber mutation depending on its readthrough by normal tRNA(Gln)(CAG).

The Saccharomyces cerevisiae gene BPL1 encodes the enzyme biotin:protein ligase (BPL), which is required for acetyl-CoA carboxylase (ACC) holoenzyme formation. Disruption of one of the two BPL1 alleles present in diploid cells results, upon sporulation, in a 2+:2(0) segregation of cell viability, with none of the two viable spores being BPL1 negative. In contrast to BPL1 deletants, BPL1 base-substitution mutants are potentially viable and may be isolated as long-chain-fatty-acid-requiring auxotrophs. In addition to ACC pyruvate carboxylase and an additional biotin-containing protein of unknown function fail to be biotinylated in BPL1-defective yeast mutants. In this study, one of these mutants, bpl1-C25/17, is shown to contain an amber stop codon at position 151 of the 689-amino-acid BPL sequence. In bpl1-C25/17 cells, de novo fatty acid synthesis is almost absent (< 2% of the wild type), while very-long-chain fatty acid (VLCFA) synthesis and, to some extent, medium-long-chain fatty acid elongation are still active. Hence, endogenous malonyl-CoA synthesis is reduced but not abolished by the translational stop mutation. A low rate of intact-BPL synthesis is accomplished in the mutant by occasional readthrough of the bpl1-C25/17 UAG nonsense triplet by normal yeast tRNA(Gln)(CAG). Correspondingly, ACC biotinylation is severely reduced though not completely absent in the two bpl1 mutants studied in this work. Residual BPL1 expression in bpl1-C25/17 cells is increased to a level allowing wild-type-like growth by transformation with high copy numbers of either the wild-type tRNA(Gln)(CAG) or the mutant bpl1-C25/17 genes. It is concluded that the lethality of BPL1 deletants is due to the lack of malonyl-CoA-dependent VLCFA synthesis and that the viability of distinct ACC-defective point mutants is due to their maintenance of a critical level of malonyl-CoA and, hence, VLCFA production. The residual capacity of malonyl-CoA synthesis, though, is inadequate to allow cytoplasmic bulk de novo fatty acid synthesis, nor does it support mutant growth on 13:0 as the only dietary fatty acid. ACC-defective mutants are respiratory deficient, which is attributed to the failure of mitochondrial fatty acid synthesis. Since lipoic acid levels of ACC1 and BPL1 mutants are essentially normal, an unknown product of mitochondrial fatty acid synthesis appears to be critically reduced in malonyl-CoA-deficient yeast cells.

Acetyl-CoA Carboxylase↗
PubMed1998

Fatty acid elongation in yeast--biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.

Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA were almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inactive. In particular, acetyl-CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl-CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmitoyl-CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (> or = 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoyl-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation products are converted in part, by alpha-oxidation, to their odd-chain-length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell homogenate. In addition, the in vivo process appears to be protected against side reactions such as hydrolysis or alpha-oxidation. Yeast mutants defective in 12:0 or 13:0 elongation were derived from fas-mutant strains according to their failure to grow on 13:0-supplemented media. In vivo, 12:0 elongation was reduced to 0-10% of the normal level, while 16:0 elongation and VLCFA synthesis were unimpaired. It is concluded that yeast contains either two different elongation systems, or that the respective mutation interferes differentially with medium-chain and long-chain fatty acid elongation. The yeast gene affected in the elongation-defective mutants was isolated and, upon sequencing, identified as the known ELO1 sequence. It encodes a putative membrane protein of 32-kDa molecular mass with no obvious similarity to any of the known FAS component enzymes.

Acyl Coenzyme A↗
PubMed1997

Mitochondrial acyl carrier protein is involved in lipoic acid synthesis in Saccharomyces cerevisiae.

The yeast gene, ACP1, encoding the mitochondrial acyl carrier protein, was deleted by gene replacement. The resulting acp1-deficient mutants had only 5-10% of the wild-type lipoic acid content remaining, and exhibited a respiratory-deficient phenotype. Upon meiosis, the lipoate deficiency co-segregated with the acp1 deletion. The role of ACP1 in long-chain fatty acid synthesis was studied in fast and fas2 null mutants completely lacking cytoplasmic fatty acid synthase. When grown on odd-chain (13:0 and 15:0) fatty acids, these cells showed less than 1% of C-16 and C-18 acids in their total lipids. Mitochondrial ACP is therefore suggested to be involved with the biosynthesis of octanoate, a precursor to lipoic acid.

Acyl Carrier Protein↗
PubMed1993

Nucleotide sequence of a Dictyostelium discoideum gene encoding a protein homologous to the yeast ribosomal protein S31.

A cDNA clone has been isolated whose coding potential is significantly homologous to the yeast ribosomal protein S31. The single copy genomic gene contains a 271 bp intron immediately downstream from the ATG translation initiation codon and is flanked by cannonical exon/intron junctions. The intron carries a CAATCAAT motif which has been described as inducer element for discoidin I gamma expression and which has also been found within the intron of the rp29 gene form D. discoideum. The deduced protein contains 110 amino acids and is slightly basic.

Amino Acid Sequence↗