Isolation of the ATP synthase subunit 6 and sequence of the mitochondrial ATP6 gene of the yeast Candida parapsilosis.

The mitochondrially translated product called subunit 6 was extracted from the yeast Candida parapsilosis mitochondria using an organic solvent mixture and purified by reverse-phase HPLC. The partial N-terminal sequence of subunit 6 reveals a post-translational cleavage site as in Saccharomyces cerevisiae. The structural mitochondrial gene ATP6 was isolated form a mitochondrial DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13tg130 and M13tg131 phage vectors. The insert contained an open reading frame 738-bp encoding a 246-amino-acid polypeptide. Mature subunit 6 contains 243 amino acid residues and the predicted molecular mass is 26,511 Da. The subunit shows 52% similarity with ATP synthase subunit 6 of the yeast S. cerevisiae. Comparison between protein and DNA sequences shows that the CUN codon family codes for a leucine in C. parapsilosis mitochondria.     

ATP synthase from bovine mitochondria. The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an alpha-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F1, and the intrinsic membrane domain, FO. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.

We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.     

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II.

Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193. The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer. The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities. Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA. These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable. We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks. The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.     

Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae

Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H. (1978) Biochim. Biophys. Acta 527, 31-41). A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced. The DNA sequence encodes a precursor protein containing 514 amino acid residues. The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids. A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix. This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria. It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways. Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.     

The yeast ATP synthase subunit 4: structure and function

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.     

Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites

We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

Two overlapping genes in bovine mitochondrial DNA encode membrane components of ATP synthase.

Two hydrophobic proteins have been purified to homogeneity from a mixture of about 13 proteins that are extracted from bovine mitochondria with a chloroform:methanol mixture. Sequence analysis shows that the smaller is a protein of 66 amino acids and is the product of a mitochondrial gene, A6L. The larger, a protein of 226 amino acids, is ATPase-6, a membrane component of ATP synthase, also encoded in mitochondrial DNA. The protein sequences determined establish that the genes for the two proteins overlap by 40 bases and indicate that translation of the second gene, ATPase-6, is initiated within the coding region of A6L. The A6L and the ATPase-6 proteins have also been isolated from the ATP synthase complex and so appear to be bona fide components of the enzyme. The function of A6L is unknown. However, weak structural homology suggests a functional similarity to the yeast mitochondrial protein, aapI, which is required for assembly of the fungal ATP synthase complex. Homologies between ATPase-6 and subunit a of the Escherichia coli ATP synthase complex indicate that the ATPase-6 protein has a similar role in the mitochondrial complex to its bacterial counterpart, being essential for the formation of an active proton channel.     

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.     

Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.     

Molecular cloning and sequencing of cDNA for yeast porin, an outer mitochondrial membrane protein: a search for targeting signal in the primary structure.

We have cloned a full-length cDNA for yeast porin, the major outer mitochondrial membrane protein from Saccharomyces cerevisiae, and determined its nucleotide sequence. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 283 amino acid residues and its NH2-terminal sequence, Met-Ser-Pro-Pro-Val-Tyr-Ser, coincided with that determined by Edman degradation for yeast porin, except that the initiator methionine was missing in the mature protein. The deduced sequence had an overall polarity index of 46.3%, a value which falls in the normal range for soluble proteins. An evaluation of hydropathy of the protein indicated that the NH2-terminal one third was relatively hydrophilic and the rest of the molecule was rather hydrophobic. An interesting finding was that the NH2-terminal region of yeast porin (consisting of some 50 amino acid residues) shows structural features that resemble those of the corresponding portion of 70-kd protein, which is also a yeast outer mitochondrial membrane protein. We postulate that this NH2-terminal sequence, like that of 70-kd protein, is required for targeting the porin to the outer mitochondrial membrane.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

The epsilon-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat.

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3' non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.     

A simple, rapid method for purification of epsilon-subunit, coupling factor 6, subunit d, and subunit e from rat liver H(+)-ATP synthase and determination of the complete amino acid sequence of epsilon-subunit.

The rat liver mitochondrial epsilon-subunit, coupling factor 6, subunit d, and subunit e of H(+)-ATP synthase, which are all extra subunits with no counterparts in Escherichia coli, were purified by reverse-phase high performance liquid chromatography. The complete amino acid sequence of the rat epsilon-subunit was determined by automated Edman degradation of the whole protein and derived peptides. The protein contains 50 amino acids and has a molecular mass of 5635 kDa. It is a basic hydrophilic protein with an isoelectric point of 10.5. The sequence of the rat epsilon-subunit is highly homologous with that of the epsilon-subunit of bovine heart and slightly similar to those of the epsilon-subunit of the yeast and sweet potato mitochondria. However, it has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase.