Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).     

In vitro synthesis of precursor forms of pig heart aspartate aminotransferase isozymes

Precursor forms of the isozymes of aspartate aminotransferase from pig heart were synthesized in vitro and purified by binding to specific antibodies. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that the precursor of the cytosolic enzyme has a similar molecular weight to that of the mature protein whereas the precursor of the mitochondrial isozyme has a molecular weight greater than that of the corresponding mature protein (ΔMW ? 2500). Preliminary sequence studies seem to suggest that the precursor of the mitochondrial isozyme has an extra N-terminal peptide sequence while that of the cytosolic protein has only an extra N-terminal methionine residue.     

Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.     

Synthesis of glutamic oxaloacetic transaminase isozymes in rat liver cells

The synthesis of glutamic oxaloacetic transaminase isozymes in rat liver explants was studied using specific antisera against the cytosolic and mitochondrial isozymes. The pulse-labeled cytosolic isozyme was detected in the cytosolic fraction and remained there in pulse-chase experiments. On the other hand, the pulse-labeled mitochondrial isozyme was detected as a larger precursor in the cytosolic fraction. During chase, the amount of pulse-labeled precursor of the mitochondrial isozyme decreased and labeled mature mitochondrial isozyme appeared in the mitochondrial fraction.     

The N-terminal sequence directs import of mitochondrial alanine aminotransferase into mitochondria

Herein, we report cloning and subcellular localization of two alanine aminotransferase (ALT) isozymes, cALT and mALT, from liver of gilthead sea bream (Sparus aurata). CHO cells transfected with constructs expressing cALT or mALT as C- or N-terminal fusion with the enhanced green fluorescent protein (EGFP) showed that cALT is cytosolic, whereas mALT localized to mitochondria. Fusion of EGFP to mALT N-terminus or removal of amino acids 1-83 of mALT avoided import into mitochondria, supporting evidence that the mALT N-terminus contains a mitochondrial targeting signal. The amino acid sequence of mALT is the first reported for a mitochondrial ALT in animals.     

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X₅-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.     

Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol.

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.     

Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: Comparison with that of the pig heart isozyme

The complete amino acid sequence of the mitochondrial glutamic oxaloacetic transaminase isozyme from rat liver is presented. The sequence contained 401 amino acid residues, 10 of which are methionine. Cyanogen bromide cleavage of mitochondrial glutamic oxaloacetic transaminase produced 12 peptides, one of which contained an internal homoserine residue resulting from incomplete cleavage by cyanogen bromide. The calculated molecular weight was 44,358. The sequence showed 94% homology with that of the corresponding isozyme from pig heart. These findings support the conclusion that the rate of evolution of the mitochondrial isozymes is lower than that of their cytosolic isozymes.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

Phytochrome-mediated activation of the gene for cytosolic glutamine-synthetase (GS1) during imbibition of photosensitive lettuce seeds

A full-length cDNA encoding glutamine synthetase was isolated from a gt11 library constructed from the poly(A)⁺ RNA isolated from lettuce seeds incubated under red light. The nucleotide sequence of the cDNA and the deduced sequence of amino acids showed a high degree of homology to those of the cytosol-type glutamine synthetase from other plants. Northern and dot-blot analyses of poly(A)⁺ RNA extracted from the seeds incubated under various light conditions showed that the activation of the gene for cytosolic glutamine-synthetase during imbibition of lettuce seeds is directly or indirectly regulated by phytochrome.Abbreviations GS glutamine synthetase - GA gibberellin     

Glutamine synthetase isozymes in elasmobranch brain and liver tissues

Glutamine synthetase is present as isozymic forms in the elasmobranchs Squalus acanthias (dogfish shark) and Dasyatis sabina (stingray). Subcellular fractionation of elasmobranch brain and liver tissue shows the enzyme to be predominantly cytosolic in the former tissue and mitochondrial in the latter. For the cytosolic brain enzyme, the subunit Mr equals 42,000 in the stingray and 45,000 in the shark, as determined by sodium dodecyl sulfate-gel electrophoresis/Western blotting. The subunit Mr = 45,000 and 47,000, respectively, for stingray and dogfish mitochondrial liver enzymes. Translation of total brain RNA from both species gives immunoprecipitable nascent peptides of the same size as their respective mature enzymes. However, in liver tissue, translation of glutamine synthetase mRNA yields peptides of higher Mr than that of the mature enzymes. In dogfish liver, Mr = 50,000 for the translation product and, in stingray liver, Mr = 48,000. This suggests that the translocation of the enzyme into liver mitochondria may be via a signal or leader sequence mechanism. The larger liver isozyme of elasmobranch glutamine synthetase is found in kidney where it is also known to be mitochondrial. The smaller cytosolic isozyme occurs in retina, heart, gill, and rectal gland tissue as well as in brain.     

Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver

1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.