Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers.

Earlier studies (Periyasamy, S. M., Huang, W.-H., and Askari, A. (1983) J. Biol. Chem. 258, 9878-9885) suggested that Cu2+ and o-phenanthroline induced the formation of cross-linked homodimers between alpha-subunits of the erythrocyte (Na+,K+)-ATPase. This was interpreted as indicating that alpha-subunits existed in close proximity in native erythrocyte membranes. The alpha-subunit and band 3 monomers have similar molecular weights (M(r) approximately 100,000) and exist in the membrane in molar ratios of approximately 1:3000 alpha-subunit:band 3. We explored the possibility that alpha-subunit and band 3 could be induced to form heterodimeric structures in the presence of cross-linking reagents. Using methods similar to those employed in the above-cited reference we demonstrated that cross-linked dimers containing phosphorylated alpha-subunits had proteolytic sensitivity that was inconsistent with the formation of alpha-subunit homodimers and fully consistent with heterodimer formation between alpha-subunit and band 3. The data also indicated that alpha-subunit-band 3 heterodimer formation is dependent on the conformational state of the (Na+,K+)-ATPase. Using the appropriate reagents we obtained cross-linked products which were consistent with heterodimer formation between alpha- and beta-subunits of the (Na+,K+)-ATPase. Our data argue against a close association between pairs of (Na+,K+)-ATPase alpha-subunits in the human red cell membrane.     

Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly

Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity. Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source. Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain. Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E. coli, respectively. In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.     

The plant mitochondrial F1-ATPase. The identity of the delta' (20 kDa) subunit

The N-terminal amino acid sequence of the 20 kDa (delta') subunit of the turnip (Brassica napus L.) mitochondrial F1-ATPase has been determined. Comparison of the sequence obtained with those of the epsilon subunits of chloroplast CF1, E. coli F1 and the delta subunit of bovine F1 shows that the turnip delta' subunit is another member of this family of homologous proteins. The delta' subunit of sweet potato F1-ATPase [(1989) J. Biol. Chem. 264, 3183-3186] is very similar to the turnip sequence and thus can also be considered to belong to this family.     

Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the alpha-subunit of an avian sodium pump

cDNA encoding the alpha-subunit of the (Na+ + K+)-ATPase was cloned from a chicken kidney cDNA library and the nucleotide sequence determined. The deduced amino acid sequence showed 92% sequence homology with the alpha-subunit of the sheep kidney (Na+ + K+)-ATPase, and high cross-species homologies were found among nucleotide sequences both in the 5'- and 3'-untranslated regions of the "kidney-type" alpha-subunit mRNAs. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk- cells. Expression of the avian alpha-sub-unit could be activated by culture of the cells in 10 mM butyrate. Cells expressing avian alpha-subunits displayed high-affinity ouabain binding (KD = 2.6 +/- 0.7 x 10(-7) M) and ouabain-sensitive 86Rb+ uptake, characteristic of avian cells.     

Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase

The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain. A recent proposal (Maggio, M. B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A. E. (1987) J. Biol. Chem. 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit. Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide. In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu. The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu). Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants. The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding. The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.     

The properties of hybrid F1-ATPase enzymes suggest that a cyclical catalytic mechanism involving three catalytic sites occurs

Maximal rates of ATP hydrolysis catalyzed by F1-ATPase enzymes are known to involve strong positive catalytic site cooperativity. There are three potential catalytic nucleotide-binding sites on F1. Two important and unanswered questions are (i) whether all three potential catalytic sites must interact cooperatively to yield maximal rates of ATP hydrolysis and (ii) whether a cyclical three-site mechanism operates as suggested by several authors. We have studied these two questions here by measuring the ATPase activities of hybrid enzymes containing normal beta-, gamma-, delta-, and epsilon-subunits together with different combinations of mutant and normal alpha-subunits. The mutant alpha-subunits were derived from uncA401, uncA447, and uncA453 mutant E. coli F1-ATPase, in which positive cooperativity between catalytic sites is strongly attenuated by defined mis-sense mutations. Our data show that three normal catalytic sites are required to interact in order to achieve maximal ATPase rates and suggest that a cyclical mechanism does operate. Hybrid enzyme containing one-third mutant alpha-subunit and two-thirds normal alpha-subunits had substantial but submaximal activity, showing that cooperativity between three sites in a noncyclical fashion, or between pairs of sites, can achieve effective catalysis.     

ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.     

A mutation in the alpha-subunit of F1-ATPase from Escherichia coli affects the binding of F1 to the membrane

The mutation Gly-29----Asp in the alpha-subunit of the F1-ATPase from Escherichia coli was characterized and shown to cause the following effects. 1) Oxidative phosphorylation was markedly impaired in vivo 2) Membrane ATPase and ATP-driven proton-pumping activities were decreased markedly. 3) Membranes were proton-permeable, and membrane-bound ATPase was dicyclohexylcarbodiimide-insensitive. Therefore, it appeared that integration between F1 and F0 was abnormal. This was confirmed directly by the demonstration that the mutant F1 bound poorly to stripped membranes from a normal strain. Purified, soluble mutant F1 had normal ATPase activity. These results suggest that residue Gly-29, which is strongly conserved in alpha-subunits of F1-ATPases, lies in a region of the alpha-subunit important for membrane binding. Thus, three regions of the F1-alpha-subunit have now been recognized, specialized for membrane binding, nucleotide binding, and alpha/beta intersubunit signal transmission, respectively. The approximate locations of the three regions are described.     

A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.     

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.

Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of alpha-subunit folding.     

Core I protein of bovine ubiquinol-cytochrome-c reductase; an additional member of the mitochondrial-protein-processing family. Cloning of bovine core I and core II cDNAs and primary structure of the proteins.

Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain-reaction-amplified fragment. The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W. & Weiss, H. (1989) Nature 339, 147 - 149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase.     

ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphibian lutropin and follitropin from the bullfrog Rana catesbeiana. Complete amino acid sequence of the alpha subunit.

The amino acid sequence of the alpha-subunit of the gonadotropins, lutropin and follitropin from bullfrog, Rana catesbeiana, has been determined. The alpha-subunit was identified in both hormones by the amino acid composition and ovulation activity of lutropin in the Xenopus ovary, by means of reconstituted hormones in various combinations. The amino acid sequences of two identical alpha-subunits from lutropin and follitropin were determined or deduced by different strategies. The alpha-subunit of those gonadotropins have 97 amino acid residues, the longest among the known alpha-subunits of gonadotropins, and one arginine insertion at position 29. Ten cysteine residues and two sugar-chain binding sites at Asn57 and Asn83 are completely conserved among the species. The molecular mass of this subunit is 11,026 Da not including the sugar chains. The bullfrog alpha-subunit has approximately 70% sequence identity with mammalian alpha-subunits.     

Structure of the human ornithine transcarbamylase gene

Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.     

The 'lysine cluster' in the N-terminal region of Na+/K(+)-ATPase alpha-subunit is not involved in ATPase activity.

The alpha-subunit of the Na+/K(+)-ATPases from several animal species have markedly similar amino acid sequences. However, the N-terminal sequences of the alpha-subunit are rather divergent except for lysine-rich sequences, the 'lysine cluster'. Here we report that the alpha-subunit from frog (Rana catesbeiana) has an N-terminal sequence with the 29 amino acid residues shorter than that of the Xenopus alpha-subunit deduced from its cDNA and hence lacks the 'lysine cluster'. Nevertheless, the Rana enzyme still exhibits ATPase activity. The ATP-dependent Na+ transport activity of the Rana enzyme was similar to that of the dog enzyme, which contains the 'lysine cluster'. Moreover, the Torpedo alpha-subunits deprived of the 'lysine cluster' by means of two gene deletions showed the same Na+/K(+)-ATPase activities as that of the wild type when expressed in Xenopus oocytes from their mRNAs. These results strongly suggest that the 'lysine cluster' in the N-terminal region of the alpha-subunit is not involved in the ATPase and ion transport activities. Since an active alpha-subunit was translated in Xenopus oocytes from mRNA lacking the N-terminal region including the 'lysine cluster', these regions were proved not to function as a membrane insertion signal sequence.     

Identification of the amino acids comprising a surface-exposed epitope within the nucleotide-binding domain of the Na+,K(+)-ATPase using a random peptide library.

Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8-P1-A3 has been shown to bind to the catalytic (alpha) subunit of the Na+,K(+)-ATPase holoenzyme and the synthetic peptide sequence 496-HLLVMK*GAPER-506, which includes Lys 501 (K*), the major site for fluorescein-5'-isothiocyanate labeling of the Na+,K(+)-ATPase. This sequence region of alpha is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W. R. & Green, N. W., 1989, Eur. J. Biochem. 179, 241-248). In this study we have determined M8-P1-A3's ability to recognize the alpha-subunit or homologous E1E2-ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or "epitope" library, recently developed by Scott and Smith (1990, Science 249, 386-390) and Devlin et al. (Devlin, J. J., Panganiban, L. C., & Devlin, P. E., 1990, Science 249, 404-406), has served as a convenient technique to confirm the species-specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8-P1-A3 epitope was found to consist of the five amino acid 494-PRHLL-498 sequence stretch of alpha, with residues PRxLx being critical for antibody recognition.     

The defective proton-ATPase of uncA mutants of Escherichia coli. Identification by DNA sequencing of residues in the alpha-subunit which are essential for catalysis or normal assembly

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.