Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Basis for prokaryotic specificity of action of aminoglycoside antibiotics.

The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 (Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid-encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6' amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside-rRNA complexes.     

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.     

Mitochondrial ATP synthase. cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver alpha subunit

The predicted amino acid sequence of the alpha subunit of the rat liver mitochondrial ATP synthase has been obtained by sequencing a cDNA for the alpha subunit. Analysis of the sequence shows that it contains the A and B consensus sequences found in many nucleotide-binding proteins. Twelve amino acids of the rat liver alpha subunit differ from the sequence of the bovine heart alpha subunit; four of these involve differences in charge. The rat liver alpha subunit, from arginine 15 to the C-terminal proline 510, has been overexpressed in Escherichia coli using the alkaline phosphatase promoter (phoA) and leader peptide to direct the export of the expressed protein to the bacterial periplasm. By treating the cells with lysozyme, osmotic shock, and alkaline pH washes, the alpha subunit can be extracted in high yield (greater than 25 mg/liter) and in a high state of purity. The expressed alpha subunit remains soluble at pH 9.5 or greater and precipitates when treated with Mg2+ ions at low millimolar concentration. The bacterially expressed alpha subunit interacts with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), resulting in a marked fluorescence enhancement upon binding. An enhancement of fluorescence is also observed upon the interaction of the alpha subunit with TNP-ADP. Preincubating the alpha subunit with 1.5 mM ATP significantly reduces the fluorescence enhancement seen with TNP-ATP. The alpha subunit binds TNP-ATP with an apparent Kd in the low micromolar range (1-5 microM) and binds TNP-ADP with an affinity at least 10-fold lower. This work shows that the rat liver alpha subunit can be overexpressed in E. coli to yield a large amount of functional protein. With the acquisition of the overexpressed alpha subunit, it is now possible to test the reconstitution of ATPase activity from a mixture of recombinant and rat liver-derived subunits and to test the formation of complexes by the overexpressed alpha and beta subunits of the rat liver F1-ATPase.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes.

A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L. Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA. Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs. To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.     

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.     

Molecular characterization of two point mutants in the chloroplast atpB gene of the green alga Chlamydomonas reinhardtii defective in assembly of the ATP synthase complex

Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the beta subunit of the CF1/CF0 ATP synthase, are here shown to be missense alterations near the 5' end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the beta subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-u-c-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of beta subunit associated with mutant thylakoids is only approximately 3% of that seen in wild type and that the CF1 alpha and gamma subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli beta subunit gene point mutants, which retain significant amounts of alpha and beta subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that beta must be present for assembly of the alpha and gamma subunits of CF1 onto chloroplast membranes.     

Complete DNA sequence coding for the large ribosomal RNA of yeast mitochondria.

The mitochondrial gene coding for the large ribosomal RNA (21S) has been isolated from a rho- clone of Saccharomyces cerevisiae. A DNA segment of about 5500 base pairs has been sequenced which included the totality of the sequence coding for the mature ribosomal RNA and the intron. The mature RNA sequence corresponds to a length of 3273 nucleotides. Despite the very low guanine-cytosine content (20.5%), many stretches of sequence are homologous to the corresponding Escherichia coli 23S ribosomal RNA. The sequence can be folded into a secondary structure according to the general models for prokaryotic and eukaryotic large ribosomal RNAs. Like the E.coli gene, the mitochondrial gene contains the sequences that look like the eukaryotic 5.8S and the chloroplastic 4.5S ribosomal RNAs. The 5' and 3' end regions show a complementarity over fourteen nucleotides.     

Homology of the 3'' terminal sequences of the 18S rRNA of Bombyx mori and the 16S rRNA of Escherchia coli.

The terminal 220 base pairs (bp) of the gene for 18S rRNA and 18 bp of the adjoining spacer rDNA of the silkworm Bombyx mori have been sequenced. Comparison with the sequence of the 16S rRNA gene of Escherichia coli has shown that a region including 45 bp of the B. mori sequence at the 3' end is remarkably homologous with the 3' terminal E. coli sequence. Other homologies occur in the terminal regions of the 18S and 16S rRNAs, including a perfectly conserved stretch of 13 bp within a longer homology located 150--200 bp from the 3' termini. These homologies are the most extensive so far reported between prokaryotic and eukaryotic genomic DNA.     

Expression and functional assembly into bacterial ribosomes of a nuclear-encoded chloroplast ribosomal protein with a long NH2-terminal extension

Chloroplast ribosomal protein L13 is encoded in the plant nucleus and is considerably larger than its eubacterial homologue by having NH2- and COOH-terminal extensions with no homology to any known sequences (Phua et al., J Biol. Chem. 264, 1968-1971, 1989). We made two gene constructs of L13 cDNA using the polymerase chain reaction (PCR) and expressed them in Escherichia coli. Analysis of the ribosomes and polysomes from these cells, using an antiserum specific to chloroplast L13, shows that the expressed proteins are incorporated, in the presence of the homologous E. coli L13, into functional ribosomes which participate in protein synthesis (i.e. polysomes). Evidence is obtained that the large NH2-terminal extension probably lies on the surface of these 'mosaic ribosomes.' This first report of the assembly into E. coli ribosomes of nuclear-coded chloroplast ribosomal protein with terminal extensions thus suggest an extraordinary conservation in the function of eubacterial type ribosomal proteins, despite the many changes in protein structure during their evolution inside a eukaryotic system.     

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A⁵¹ residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.     

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.