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A S Lewin

Publications and source records attributed to A S Lewin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein.

The nuclear CBP2 gene encodes a protein essential for the splicing of a mitochondrial group I intron in Saccharomyces cerevisiae. This intron (bI5) is spliced autocatalytically in the presence of high concentrations of magnesium and monovalent salt but requires the Cbp2 protein for splicing under physiological conditions. Addition of Cbp2 during RNA synthesis permitted cotranscriptional splicing. Splicing did not occur in the transcription buffer in the absence of synthesis. The Cbp2 protein appeared to modify the folding of the intron during RNA synthesis: pause sites for RNA polymerase were altered in the presence of the protein, and some mutant transcripts that did not splice after transcription did so during transcription in the presence of Cbp2. Cotranscriptional splicing also reduced hydrolysis at the 3' splice junction. These results suggest that Cbp2 modulates the sequential folding of the ribozyme during its synthesis. In addition, splicing during transcription led to an increase in RNA synthesis with both T7 RNA polymerase and mitochondrial RNA polymerase, implying a functional coupling between transcription and splicing.

Base Sequence↗
PubMed1993

Structure-induced strain determining the internal cyclization site in the yeast cobI5 autocatalytic intron: theory and experimental tests.

The terminal intron of the apocytochrome b gene of yeast is endowed with autocatalytic potential in vitro and has been also shown to be capable of internal circularization at the linkage 236-237 from the 5' extremity. The aim of this work is to identify the secondary interaction responsible for shaping and activating the internal cyclization site (ICS). This is done by simulating the sequential folding of an abnormally large fragment of 237 nucleotides which starts at the 5' cleavage site and contains the internal guide sequence (IGS) starting 220 nucleotides into the intron. The resulting portion of the overall structure features the conserved interaction P1 and is compatible with the complete consensus secondary structure for this intron. The structural motif which emerges from sequential folding and is responsible for shaping the ICS is confirmed by analyzing the localization of structure-induced strain in the RNA backbone. It is shown that after the conserved stem P1 has been dismantled, the highest strain in a phosphodiester linkage is localized internally precisely at the experimentally determined cyclization site. Moreover, it is shown that our dynamical model of folding is compatible with the actual reaction pathways. Thus, internal cyclization becomes feasible only after conserved interaction P1 has been dismantled. This last event, in turn, might take place as a consequence of either of the following events: (i) 5' cleavage caused by nucleophilic attack by the GTP-cosubstrate or (ii) excision of the 3' exon after prior formation of P10. This last event, in turn, requires dismantling helix P1. Moreover, event (ii) is necessary for internal circularization since it habilitates the 3' terminal guanosine as a nucleophilic agent.

Apoproteins↗
PubMed1993

Selective autophagy of peroxisomes in methylotrophic yeasts.

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha respond to a methanol substrate by synthesizing peroxisomal enzymes resulting in the formation of large peroxisomes. When the carbon source was changed from methanol to glucose, we observed a rapid loss of peroxisomes. In this comparative study, we utilized biochemical and morphological techniques to characterize the loss of peroxisomes in these yeasts. We used metabolic labeling and chase procedures to evaluate whether this loss was due to suppressed synthesis or enhanced degradation. The synthesis of alcohol oxidase was depressed 10-fold when cultures grown in methanol attained stationary growth. However, no further reduction of synthesis was observed upon transfer of these cultures to glucose medium. In stationary phase cultures maintained in methanol, two peroxisomal proteins, alcohol oxidase and dihydroxyacetone synthase, were degraded with a half-life of over 3 h. However, within 3 h of glucose repression, as much as 80% of the radiolabeled peroxisomal proteins were lost from both yeasts. This glucose-mediated degradative event appeared to be specific for peroxisomal proteins, since mitochondrial proteins were stable. Ultrastructural examination of both yeasts revealed that glucose induced the sequestration of peroxisomes into the yeast vacuole, the presumed site of degradation. These results suggest that peroxisome loss during glucose repression is due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.

Alcohol Oxidoreductases↗
PubMed1992

A negative regulating element controlling transcription of the gene encoding acyl-CoA oxidase in Saccharomyces cerevisiae.

Peroxisomes are induced in Saccharomyces cerevisiae when this yeast is grown in the presence of oleate, and are repressed when glucose is supplied as the carbon source. Concomitant with this is an induction/repression of peroxisomal beta-oxidation enzymes. We are investigating the transcriptional control of acyl-CoA oxidase, the first and rate-limiting enzyme in the peroxisomal beta-oxidation cycle. The promoter region of POX1 from S. cerevisiae has been analyzed in POX1/lacZ fusions. Expression of the POX1/lacZ fusion protein underwent glucose repression and oleate induction. By deletion, DNA band shift and DNase I footprinting analyses we have identified a region that is involved in transcriptional repression of POX1. Elimination of this DNA sequence results in constitutive expression of POX1 when S. cerevisiae is grown on a fermentable carbon source or glycerol.

Acyl-CoA Oxidase↗
PubMed1992

Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron.

A guanosine to cytosine transversion at position 2 of the fifth intron of the mitochondrial gene COB blocks the ligation step of splicing. This mutation prevents the formation of a base pair within the P1 helix of this group I intron--the RNA duplex formed between the 3' end of the upstream exon and the internal guide sequence. The mutation also reduces the rate of the first step of splicing (guanosine addition at the 5' splice junction) while stimulating hydrolysis at the 3' intron-exon boundary. Consequently, the ligation of exons is blocked because the 3' exon is removed prior to cleavage at the 5' splice junction. The lesion can be suppressed by second-site mutations that preserve the potential for base-pairing at this position. Because the P1 duplex and the P10 duplex (between the guide sequence and the 3' exon) overlap at the affected pairings represent alternative structures that do not, indeed cannot, form simultaneously.

Base Composition↗
PubMed1992

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

Amino Acid Sequence↗
PubMed1991

The rate and specificity of a group I ribozyme are inversely affected by choice of monovalent salt.

The fifth intron of the COB gene of yeast mitochondria splices autocatalytically. The rate of splicing is increased by high concentrations of monovalent salts, but the choice of both cation and anion is significant: The smaller the cation in solution, the faster the reaction (the rate in K+ greater than NH4+ greater than Na+ greater than Li+). Chloride, bromide, iodide and acetate salts enhance autocatalytic processing, but sulfate salts do not and fluoride salts are inhibitory. The choice of monovalent salt affects the KM of the intron for guanosine nucleotide, implying an alteration in the affinity of the RNA for that substrate. Under optimal conditions (1M KCl, 50 mM MgCl2) the catalytic efficiency of this intron exceeds that reported for the ribosomal intron from Tetrahymena, but several side reactions occur, including guanosine-addition within the downstream exon. The site of addition resembles the 5' splice junction, but selection of this site does not involve the internal guide sequence of the intron.

Cations, Monovalent↗
PubMed1990

Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

Base Composition↗
PubMed1990

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

Cell Fractionation↗
PubMed1988

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria.

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.

Base Sequence↗
PubMed1987

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex.

Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20 degrees C). At 28 degrees C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20 degrees C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.

Amino Acid Sequence↗
PubMed1987

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase.

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.

Adenine↗
PubMed1986

The rate of import and assembly of F1-ATPase in Saccharomyces cerevisiae.

Subunit specific antiserum can be employed to study the course of ATPase assembly in mitochondria isolated from bakers' yeast. Comparing rates of subunit import with rates of enzyme assembly indicated that no substantial pool of unassembled subunits exists for the three largest ATPase peptides (alpha, beta, and gamma). Blocking import of specific ATPase subunits, however, did reveal a possible accumulation of unassembled alpha and gamma subunits in isolated mitochondria. The kinetic experiments also revealed a lag in the import of beta subunit relative to the uptake of alpha and gamma precursors. Experiments conducted in yeast cells confirmed that beta subunit is assembled soon after it is imported, but did not indicate a delay in import relative to the other subunits of F1.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Extramitochondrial citrate synthase activity in bakers' yeast.

We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements.

Citrate (si)-Synthase↗
PubMed1984

Hypothesis--a chemical mechanism for the biosynthesis of ATP involving ion-exchange reactions.

Dissociation constants for Mg . ATP were determined by displacing ATP from Dowex-1 resin with magnesium. These constants were then used to analyze the kinetics of yeast mitochondrial ATPase, in terms of the concentrations of free magnesium and free ATP, at a series of pH values. Both Mg . ATP and hydroxide ions were found to compete with the binding of ATP to the enzyme. These results were interpreted, in terms of an ion-exchange model, to mean that the synthesis of ATP may require the utilization of both magnesium and hydroxide ions for the dissociation of ATP from the enzyme as Mg . ATP. The concentrations of Mg and hydroxide required to compete with ATP were both found to be about three orders of magnitude greater than those required to form products, indicating that magnesium and hydroxide ions can contribute about 8 kcal of energy when ATP is synthesized.

Adenosine Triphosphatases↗
PubMed1984

Derepression of citrate synthase in Saccharomyces cerevisiae may occur at the level of transcription.

Pulse-chase labeling in whole cells and cell-free protein synthesis were used to establish that the mitochondrial enzyme citrate synthase is made as a larger precursor in Saccharomyces cerevisiae. A 54,000 Mr precursor form appeared to be a primary translation product since it could be labeled with N-[35S]formylmethionine in vitro. The induction of citrate synthase was monitored in S. cerevisiae cells grown on fermentable (glucose) and nonfermentable (ethanol and glycerol) carbon sources. The amount of citrate synthase activity and immune-reactive protein increased more than 15-fold as S. cerevisiae cells entered the stationary growth phase on glucose-containing medium. This increase was paralleled by an increase in translatable RNA for the enzyme. When cells were grown on a nonfermentable carbon source, no increase in either citrate synthase or its mRNA was detected. The results suggest that the release of citrate synthase from catabolite repression may occur at the level of transcription.

Citrate (si)-Synthase↗
PubMed1983

Assembly of F1-ATPase in isolated mitochondria.

The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system. Following such an incubation, the ATPase complex (F1F0) was isolated. Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive. Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria. Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors. These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria.

Antigen-Antibody Complex↗