Eckhart Schweizer

Microbial Type I Fatty Acid Synthases (FAS): Major Players in a Network of Cellular FAS Systems

SUMMARY The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits α and β are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell.

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 fas1 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 precurser to lipoic acid.

The pentafunctional FAS1 gene of yeast: its nucleotide sequence and order of the catalytic domains

SummaryFAS1, the structural gene of the pentafunctional fatty acid synthetase subunit β in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intronfree nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5′-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites, 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG-...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1–468) is located proximal to the N-terminus of subunit β, followed by the enoyl reductase (amino acids 480–858), the dehydratase (amino acids 1,134–1,615) and the malonyl/palmityl transferase (amino acids 1,616–1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.

HFA1 Encoding an Organelle-specific Acetyl-CoA Carboxylase Controls Mitochondrial Fatty Acid Synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Δ disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to <10% of its normal cellular concentration. Other than Acc1p, the N-terminal sequence of Hfa1p comprises a canonical mitochondrial targeting signal together with a matrix protease cleavage site. Accordingly, the HFA1-encoded protein was specifically assigned by Western blotting of appropriate cell fractions to the mitochondrial compartment. Removal of the mitochondrial targeting sequence abolished the competence of HFA1 DNA to complement hfal null mutants. Conversely and in contrast to the intact HFA1 sequence, the signal sequence-free HFA1 gene complemented the mutational loss of cytoplasmic acetyl-CoA carboxylase. Expression of HFA1 under the control of the ACC1 promoter restored cellular ACC activity in ACC1-defective yeast mutants to wild type levels. From this finding, it is concluded that HFA1 encodes a specific mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for intraorganellar fatty acid and, in particular, lipoic acid synthesis.

DNA binding site of the yeast heteromeric Ino2p/Ino4p basic helix-loop-helix transcription factor: structural requirements as defined by saturation mutagenesis.

The inositol/choline‐responsive element (ICRE) is an 11 bp cis‐activating sequence motif with central importance for the regulated expression of phospholipid biosynthetic genes in the yeast Saccharomyces cerevisiae. The ICRE containing the CANNTG core binding sequence (E‐box) of basic helix‐loop‐helix (bHLH) regulatory proteins is recognized by the heteromeric bHLH transcription factor Ino2p/Ino4p. In this study, we define the Ino2p/Ino4p consensus binding sequence (5′‐WYTTCAYR‐TGS‐3′) based on the characterization of all possible single nucleotide substitutions. Interestingly, this analysis also identified a single functional deviation (CACATTC) from the CANNTG core recognition element of bHLH proteins. The DNA binding specificities of different yeast bHLH proteins may now be explained by distinct nucleotide preferences especially at two positions immediately preceding the CANNTG core motif.

A Novel Phosphopantetheine:Protein Transferase Activating Yeast Mitochondrial Acyl Carrier Protein

In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4′-phosphopantetheine as a prosthetic group. Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids). In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C. In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation. According to these characteristics, the newly identified gene was designated asPPT2. Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2deletants. ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation ofPPT2 mutants with cloned PPT2 DNA. In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p. The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B. ammoniagenes, Ppt1p. These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.

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 ofYGR065C under the control of the constitutiveADH1 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 thatYGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 forvitamin H transporter1.

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. ybr159Δ 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 ybr159Δ cells, may be postulated.

Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae

SummaryThis study details the characteristics of two temperature-conditional pet mutants of yeast, strains ts1860 and ts379, which at the non-permissive temperature show deficiencies in the formation of three mitochondrially encoded subunits of the ATP synthase complex. By analysis of mitochondrial translation products, and of mitochondrial transcription in temperature shift experiments from the permissive (22°C) to the non-permissive (36°C) temperature, it was concluded that the nuclear mutations in both mutants primarily inhibit synthesis of ATP synthase subunit 9, and that reductions in subunit 8 and 6 synthesis are secondary pleiotropic effects. Following transfer to 36°C, cells of mutant ts379 display a near complete inhibition of subunit 9 synthesis within 1 h, coincident with a marked reduction in the level of the cognate oli1 mRNA. On the other hand, near complete inhibition of subunit 9 synthesis in strain ts1860 occurs after 3 h at 36°C, at which time there is little change in the level of subunit 9 mRNA. In both mutants the mRNA levels for subunits 6 and 8 are not significantly affected at the time of inhibition of subunit 9 synthesis. Provision of an alternative source of subunit 8, translated extra-mitochondrially for import into the organelle, does not overcome the mutant phenotype of either mutant at 36°C, confirming that subunit 8 is not the sole or primary deficiency in each mutant. The mutants indicate that the products of a least two nuclear genes (designated AEP1 and AEP2) are required for the expression of the mitochondrial oli1 gene and the synthesis of subunit 9. The product of the AEP1 gene (defective in mutant ts1860) is required for translation of oli1 mRNA while the AEP2 product (defective in mutant ts379) is essential either for the stability of oli1 mRNA or for the correct processing of precursor transcripts to the mature message.

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

SummaryThe fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS β-subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3′ end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.