Michael Schweizer

The Metabolism and Molecular Physiology of Saccharomyces Cerevisiae

LIFE CYCLE AND MORPHOGENESIS J. Richard Dickinson MOTHER CELL-SPECIFIC AGEING Michael Breitenbach, Peter Laun, Gino Heeren, Stefanie Jarolim, and Alena Pichova CARBON METABOLISM Arthur Kruckeberg and J. Richard Dickinson NITROGEN METABOLISM J. Richard Dickinson MOLECULAR ORGANIZATION AND BIOGENESIS OF THE CELL WALL Frans M. Klis, Piet de Groot, Stanley Brul and Klaas J. Hellingwerf LIPIDS AND MEMBRANES Michael Schweizer PROTEIN TRAFFICKING Jeremy D.Brown PROTEIN PHOSPHORYLATION AND DEPHOSPHORYLATION Michael J.R. Stark STRESS RESPONSES Ian W. Dawes short toc

Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1.

The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites. All three sites have a neighbouring Sp1-binding GC-box. This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken. We have previously shown cooperative binding of NF-Y and Sp1 to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site. Here, we show an in-vivo interaction of NF-YA with Sp1 using the yeast two-hybrid system. The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Sp1. In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Sp1 in extracts of rat hepatoma cells H4IIE. Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Sp1 in-vitro in the absence of DNA. Therefore, NF-Y can be added to the list of transcription factors interacting with Sp1.

Novel Anti-Infective Compounds from Marine Bacteria

As a result of the continuous evolution of microbial pathogens towards antibiotic-resistance, there have been demands for the development of new and effective antimicrobial compounds. Since the 1960s, the scientific literature has accumulated many publications about novel pharmaceutical compounds produced by a diverse range of marine bacteria. Indeed, marine micro-organisms continue to be a productive and successful focus for natural products research, with many newly isolated compounds possessing potentially valuable pharmacological activities. In this regard, the marine environment will undoubtedly prove to be an increasingly important source of novel antimicrobial metabolites, and selective or targeted approaches are already enabling the recovery of a significant number of antibiotic-producing micro-organisms. The aim of this review is to consider advances made in the discovery of new secondary metabolites derived from marine bacteria, and in particular those effective against the so called “superbugs”, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), which are largely responsible for the increase in numbers of hospital acquired, i.e., nosocomial, infections.

NF-Y binds to the inverted CCAAT box, an essential element for cAMP-dependent regulation of the rat fatty acid synthase (FAS) gene

Using EMSA competition experiments together with supershifts and in vitro transcription/translation we show that the basal transcription factor NF-Y or a related factor binds to the cAMP-responsive inverted CCAAT box recently identified in the rat fatty acid synthase (FAS) gene from nucleotide -99 to -92 relative to the transcription start site of the FAS mRNA. This result indicates a putative novel role for NF-Y in the cAMP-dependent gene regulation in a small class of genes such as FAS and tryptophan hydroxylase. Since NF-Y is a constitutively produced factor, not surprisingly, no differences in the specific DNA/protein complex with the CCAAT(FAS) box and nuclear proteins from H4IIE cells treated with cAMP and/or insulin or not could be observed. This implies that NF-Y might be modified in response to cAMP or might interact with another factor whose properties are altered by cAMP.

The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs

While X-ray and NMR structures are now available for most components of the Type II fatty acid synthase (FAS), there are no structures for Type I FAS domains. A region from the mammalian (rat) FAS, including the putative acyl carrier protein (ACP), has been cloned and over-expressed. Here we report multinuclear, multidimensional NMR studies which show that this isolated ACP domain contains four alpha-helices (residues 8-16 [1]; 41-51 [2]; 58-63 [3] and 66-74 [4]) and an overall global fold characteristic of ACPs from both Type II FAS and polyketide synthases (PKSs). The overall length of the structured ACP domain (67 residues) is smaller than the structured regions of the Eschericia coli FAS ACP (75 residues), the actinorhodin PKS ACP (78 residues) and the Bacillus subtilis FAS ACP (76 residues). We further show that the rat FAS ACP is recognised as an efficient substrate by enzymes known to modify Type II ACPs including phosphopantetheinyl and malonyl transferases, but not by the heterologous S. coelicolor minimal polyketide synthase.

SREBP-1c mediates the retinoid-dependent increase in fatty acid synthase promoter activity in HepG2.

Treatment of HepG2 with all‐trans retinoic acid (RA) induces expression of fatty acid synthase (FAS) mRNA and protein. Transfections show that the FAS promoter positively responds to retinoid X receptor (RXR) but not to RA receptor (RAR) agonists. Since RXR alone is capable of mediating the RA response of FAS, the existence of a classical RA‐responsive element in the FAS promoter may be ruled out. Binding sites for NF‐Y and SREBP‐1 proved to be essential for the RA response. Exposure to all‐trans RA increased mRNA and protein levels of SREBP‐1, a transcriptional activator for FAS. Overexpression of a dominant‐negative form of SREBP‐1c diminished the RA‐dependent increase in promoter activity. These data demonstrate that RXR ligands can stimulate the expression of a lipogenic gene solely by inducing transcription and cleavage of membrane‐bound SREBP‐1c.

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

Abstract In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell.

Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV.

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994). Transformants were selected as Geneticin (G418)‐resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418‐resistant marker. We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs. The 5′‐ and 3′‐flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele. The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap‐repaired plasmid by cutting at the inserted flanking restriction sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non‐essential ORFs were subjected to growth test on different media at 15°C, 30°C and 37°C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37°C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate.

Determination of the molecular weight of DNA-binding proteins using UV-crosslinking and SDS-PAGE.

We describe the use of UV-crosslinking in combination with SDS-PAGE to determine the approximate molecular weight of DNA-binding proteins. A 5-bromo-2′-deoxyuridine (5-BrdU)-substituted, radioactively labeled double-stranded oligonucleotide representing the protein binding site is incubated with a crude nuclear extract containing the protein of interest. Following irradiation with a UV light source, the DNA/protein complex is subjected to SDS-PAGE and its molecular weight determined by comparison with appropriate protein standards.

Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems

INTRODUCTIONnFatty acid synthases (FASs) catalyze the de novo biosynthesis of long-chain saturated fatty acids by a process common to eubacteria and eukaryotes, using either a set of monofunctional proteins (Type II FAS) or a polypeptide containing several catalytic functions (Type I FAS). To compare the features of a Type I domain with its Type II counterpart we expressed and characterized an acyl carrier protein (ACP) domain of the Type I rat FAS.nnnRESULTSnAn ACP domain of rat FAS was defined that allows expression of a small percentage of active holo-ACP both in Escherichia coli, increasing fivefold upon co-expression with an E. coli holo-ACP synthase, and in Streptomyces coelicolor. The rat ACP domain functions with some components of the E. coli FAS, and can replace the actinorhodin polyketide synthase (PKS) ACP in S. coelicolorA3(2). Purification of the rat ACP domain from E. coli resulted in loss of its functionality. Purified apo-ACP could be converted to its holo-form upon incubation with purified E. coli holo-ACP synthase in vitro, however, suggesting that the loss of functionality was not due to a conformational change.nnnCONCLUSIONSnFunctionality of the recombinant rat ACP was shown in distantly related and diverse enzyme systems, suggesting that Type I and Type II ACPs have a similar conformation. A procedure was described that might permit the production of rat FAS holo-ACP for structural and further biochemical characterization.