An unusual pattern of protein expression and localization of yeast alanyl-tRNA synthetase isoforms

Previous studies have shown that in Saccharomyces cerevisiae the mitochondrial and cytoplasmic forms of alanyl-tRNA synthetase are encoded by a single nuclear gene, ALA1, through alternative use of in-frame successive ACG triplets and a downstream AUG triplet. Here we show that despite the obvious participation of the non-AUG-initiated leader peptide in mitochondrial localization, the leader peptide per se cannot target a cytoplasmic passenger protein into mitochondria under normal conditions. Functional mapping further shows that an efficient targeting signal is composed of the leader peptide and an 18-residue sequence downstream of Met1. Consistent to this observation, overexpression of the cytoplasmic form enables it to overcome the compartmental barrier and function in the mitochondria as well, but deletion of as few as eight amino acid residues from its amino-terminus eliminates such a potential. Thus, the sequence upstream of the first in-frame AUG initiator not only carries an unusual initiation site, but also contributes to a novel pattern of protein expression and localization.     

Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase

It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.     

A single sequence context cannot satisfy all non-AUG initiator codons in yeast<Superscript>†</Superscript>

Background  

Previous studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.     

Cyclic oscillations in rat hepatic heme oxygenase and delta-aminolevulinic acid synthetase following intravenous heme administration.

Heme administration in vivo results in the suppression of synthesis of rat hepatic δ-aminolevulinic acid (ALA) synthetase and induction of rat hepatic heme oxygenase. Intravenous heme administration in vivo results in the appearance of cyclic progressively damped oscillations of both hepatic ALA synthetase activity and hepatic heme oxygenase activity. Heme oxygenase induction precedes in time the induction of ALA synthetase. ALA synthetase oscillations are observed in hepatic cell cytosol and mitochondrial fractions as well as in the total homogenate. Cycloheximide pretreatment abolishes both the ALA synthetase and heme oxygenase oscillations, while actinomycin D pretreatment has only a minimal effect on the induction of heme oxygenase. These results suggest that hepatic heme metabolism is closely regulated by rapid changes in the capacity to synthesize and catabolize heme, and the cyclic oscillations following intravenous heme may be a manifestation of the feedback regulation processes involved. This regulatory capacity is dependent on protein synthesis, and the primary site of regulation may be at the translational level on the endoplasmic reticulum.     

Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.     

Protective Effect of Sclerin on a Lead-acetate Injury Affecting the Growth and Porphyrin Metabolism in Rat

While a continuous ingestion of lead acetate added in drinking water suppressed the rat growth, depressing in some degree the level of hepatic δ-aminolevulinate (ALA) dehydratase, a very small amount of sclerin (SCL) added simultaneously in the water restored the growth and dehydratase level. Moreover, subcutaneous injection of SCL to the rat not only maintained the ALA dehydratase level, but prevented a marked depression of the level of mitochondrial ALA synthetase in liver caused by intraperitoneal injection of lead acetate. Injection of SCL alone increased tolerably (about 1.8 times) the mitochondrial ALA synthetase, but little the extramitochondrial synthetase. The treatment by SCL was attended by a initial decrease, then a gradual increase in the activity of microsomal drug metabolizing enzyme.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.     

Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, δ-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain α-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, α-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and α-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP⁺, and was inhibited by NAD⁺. Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of α-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.     

Altered regulation of hepatic heme metabolism in cadmium exposed chick embryo

An investigation on the process of heme metabolism with special emphasis on ALA synthetase, heme synthetase and heme oxygenase was studied in cadmium exposed chick embryo to enlighten the mechanism of cadmium embryotoxicity. Cadmium chloride injection (2.5-10 mumole/kg) to chick embryo increases the activity of ALA synthetase by 5-7 folds, however, it inhibits the activity of heme synthetase significantly. The activity of heme oxygenase is further shown to be enhanced by cadmium chloride treatment. These changes are accompanied by a marked reduction in hepatic heme content. The induction of ALA synthetase and heme oxygenase was dependent on the initial concentration of exogenous cadmium. Pretreatment with actinomycin D completely blocks the cadmium mediated induction of both ALA synthetase and heme oxygenase. Time course studies on the stimulation of these two enzymes show that cadmium enhances the activity of heme oxygenase to its maximum level after 24 h. of injection, whereas ALA synthetase activity reaches its highest value only by 48 h. and both the enzymes remain elevated at least upto 96 h. This observation can be correlated with the hepatic heme level at different time intervals after cadmium exposure. These observations suggest the presence of regulatory process for heme metabolism which is susceptible to alteration of 'regulatory heme pool' caused by cadmium.     

Biosynthesis of Tetrapyrrole Pigment Precursors : Formation and Utilization of Glutamyl-tRNA for delta-Aminolevulinic Acid Synthesis by Isolated Enzyme Fractions from Chlorella Vulgaris

The universal tetrapyrrole precursor δ-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg²⁺. The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 ± 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process.     

δ-Aminolevulinic acid synthetase: Regulation of activity in various tissues of the aging rat

The basal and ethanol-induced activities of the rate-limiting enzyme of heme biosynthesis, δ-aminolevulinic acid (ALA) synthetase were measured in the liver, heart, kidney, and brain of young, adult, and aged Sprague-Dawley rats. When assayed in whole mitochondria derived from either fed or 24-h fasted animals, the basal levels of hepatic ALA synthetase activity decreased dramatically as a function of age. An equivalent decrease was seen in the ethanol-induced activity although the ratio of induced to basal activities did not change with age. In the heart, ALA synthetase activity also decreased significantly during aging. The activity was not induced by ethanol and was decreased markedly by fasting. By contrast, kidney ALA synthetase activity showed no age-related changes. The activity was unaffected by fasting and showed a variable induction response to ethanol. Brain ALA synthetase activity displayed a significant age-dependent decrease in its activity which was neither affected by fasting nor sensitive to induction by ethanol. The data presented are consistent with the hypothesis that ALA synthetase activity is subject to metabolic regulation. Further, they indicate that while the enzyme activity is regulated in a tissuespecific manner, a time-dependent decrease is a general feature of the aging animal.     

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.     

Discrimination by Escherichia coli initiation factor IF3 against initiation on non-canonical codons relies on complementarity rules.

Translation initiation factor IF3, one of three factors specifically required for translation initiation in Escherichia coli, inhibits initiation on any codon other than the three canonical initiation codons, AUG, GUG, or UUG. This discrimination against initiation on non-canonical codons could be due to either direct recognition of the two last bases of the codon and their cognate bases on the anticodon or to some ability to "feel" codon-anticodon complementarity. To investigate the importance of codon-anticodon complementarity in the discriminatory role of IF3, we constructed a derivative of tRNALeuthat has all the known characteristics of an initiator tRNA except the CAU anticodon. This tRNA is efficiently formylated by methionyl-tRNAfMettransformylase and charged by leucyl-tRNA synthetase irrespective of the sequence of its anticodon. These initiator tRNALeuderivatives (called tRNALI) allow initiation at all the non-canonical codons tested, provided that the complementarity between the codon and the anticodon of the initiator tRNALeuis respected. More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNALIcarrying a complementary anticodon is used for initiation. This suggests that IF3 somehow recognises codon-anticodon complementarity, at least at the second and third position of the codon, rather than some specific bases in either the codon or the anticodon.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

The hardest part of replicating a genome is the beginning. The first step of DNA replication (called "initiation") mobilizes a large number of specialized proteins ("initiators") that recognize specific sequences or structural motifs in the DNA, unwind the double helix, protect the exposed ssDNA, and recruit the enzymatic activities required for DNA synthesis, such as helicases, primases and polymerases. All of these components are orderly assembled before the first nucleotide can be incorporated. On the occasion of the 50th anniversary of the discovery of the DNA structure, we review our current knowledge of the molecular mechanisms that control initiation of DNA replication in eukaryotic cells, with particular emphasis on the recent identification of novel initiator proteins. We speculate how these initiators assemble molecular machines capable of performing specific biochemical tasks, such as loading a ring-shaped helicase onto the DNA double helix.