Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

An important 2′-OH group for an RNA–protein interaction

We have investigated the role of 2′-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNACys and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2′-OH by 2′-deoxy or 2′-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2′-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2′-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2′-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2′-OH group instead provides a monitor of the steric environment during the RNA–synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2′-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Lipid monolayer image dipoles.

A simple model is described for calculating the electrostatic energy of lipid domains at the air-water interface, taking account of dipole-dipole repulsions between the lipid molecules themselves, as well as interactions between the molecular dipoles and image dipoles in the subphase. The model assumes that the molecular dipoles within the monolayer arise from the terminal methyl groups of the hydrophobic hydrocarbon chains of the lipid molecules, and that on average they are oriented perpendicular to the plane of the monolayer. With this model the role of the subphase is to enhance rather than suppress the effects of dipole-dipole repulsions.

On the mechanism of the anticlotting action of vitamin E quinone.

Vitamin E in the reduced, alpha-tocopherol form shows very modest anticlotting activity. By contrast, vitamin E quinone is a potent anticoagulant. This observation may have significance for field trials in which vitamin E is observed to exhibit beneficial effects on ischemic heart disease and stroke. Vitamin E quinone is a potent inhibitor of the vitamin K-dependent carboxylase that controls blood clotting. A newly discovered mechanism for the inhibition requires attachment of the active site thiol groups of the carboxylase to one or more methyl groups on vitamin E quinone. The results from a series of model reactions support this interpretation of the anticlotting activity associated with vitamin E.

Muscle protein synthesis by positron-emission tomography with l-[methyl-11C]methionine in adult humans

Existing methods for assessing protein synthetic rates (PSRs) in human skeletal muscle are invasive and do not readily provide information about individual muscle groups. Recent studies in canine skeletal muscle yielded PSRs similar to results of simultaneous stable isotope measurements using l-[1-13C, methyl-2H3]methionine, suggesting that positron-emission tomography (PET) with l-[methyl-11C]methionine could be used along with blood sampling and a kinetic model to provide a less invasive, regional assessment of PSR. We have extended and refined this method in an investigation with healthy volunteers studied in the postabsorptive state. They received ≈25 mCi of l-[methyl-11C]methionine with serial PET imaging of the thighs and arterial blood sampling for a period of 90 min. Tissue and metabolite-corrected arterial blood time activity curves were fitted to a three-compartment model. PSR (nmol methionine⋅min−1⋅g muscle tissue−1) was calculated from the fitted parameter values and the plasma methionine concentrations, assuming equal rates of protein synthesis and degradation. Pooled mean PSR for the anterior and posterior sites was 0.50 ± 0.040. When converted to a fractional synthesis rate for mixed proteins in muscle, assuming a protein-bound methionine content of muscle tissue, the value of 0.125 ± 0.01%⋅h−1 compares well with estimates from direct tracer incorporation studies, which generally range from ≈0.05 to 0.09%⋅h−1. We conclude that PET can be used to estimate skeletal muscle PSR in healthy human subjects and that it holds promise for future in vivo, noninvasive studies of the influences of physiological factors, pharmacological manipulations, and disease states on this important component of muscle protein turnover and balance.

Blockade of N-methyl-d-aspartate receptor activation suppresses learning-induced synaptic elimination

Auditory filial imprinting in the domestic chicken is accompanied by a dramatic loss of spine synapses in two higher associative forebrain areas, the mediorostral neostriatum/hyperstriatum ventrale (MNH) and the dorsocaudal neostriatum (Ndc). The cellular mechanisms that underlie this learning-induced synaptic reorganization are unclear. We found that local pharmacological blockade of N-methyl-d-aspartate (NMDA) receptors in the MNH, a manipulation that has been shown previously to impair auditory imprinting, suppresses the learning-induced spine reduction in this region. Chicks treated with the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) during the behavioral training for imprinting (postnatal day 0–2) displayed similar spine frequencies at postnatal day 7 as naive control animals, which, in both groups, were significantly higher than in imprinted animals. Because the average dendritic length did not differ between the experimental groups, the reduced spine frequency can be interpreted as a reduction of the total number of spine synapses per neuron. In the Ndc, which is reciprocally connected with the MNH and not directly influenced by the injected drug, learning-induced spine elimination was partly suppressed. Spine frequencies of the APV-treated, behaviorally trained but nonimprinted animals were higher than in the imprinted animals but lower than in the naive animals. These results provide evidence that NMDA receptor activation is required for the learning-induced selective reduction of spine synapses, which may serve as a mechanism of information storage specific for juvenile emotional learning events.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.