Interaction of the mitochondrial ATPase complex with phospholipids

The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component.     

Active site ligand stabilization of quaternary structures of glutamine synthetase from Escherichia coli

Auto-inactivated EScherichia coli glutamine synthetase contains 1 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2 eq of Mn2+ tightly bound to the active site of each subunit of the dodecameric enzyme (Maurizi, M. R., and Ginsburg, A. (1982) J. Biol. Chem. 257, 4271-4278). Complete dissociation and unfolding in 6 M guanidine HCl at pH 7.2 and 37 degrees C requires greater than 4 h for the auto-inactivated enzyme complex (less than 1 min for uncomplexed enzyme). Release of ligands and dissociation and unfolding of the protein occur in parallel but follow non-first order kinetics, suggesting stable intermediates and multiple pathways for the dissociation reactions. Treatment of Partially inactivated glutamine synthetase (2-6 autoinactivated subunits/dodecamer) with EDTA and dithiobisnitrobenzoic acid at pH 8 modifies approximately 2 of the 4 sulfhydryl groups of unliganded subunits and causes dissociation of the enzyme to stable oligomeric intermediates with 4, 6, 8, and 10 subunits, containing equal numbers of uncomplexed subunits and autoinactivated subunits. With greater than 70% inactivated enzyme, no dissociation occurs under these conditions. Electron micrographs of oligomers, presented in the appendix (Haschemeyer, R. H., Wall, J. S., Hainfeld, J., and Maurizi, M. R., (1982) J. Biol. Chem. 257, 7252-7253) suggest that dissociation of partially liganded dodecamers occurs by cleavage of intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit interactions are stabilized by active site ligand binding. Isolated tetramers (Mr = 200,000; s20,w = 9.5 S) retain sufficient native structure to express significant enzymatic activity; tetramers reassociate to dodecamers and show a 5-fold increase in activity upon removal of the thionitrobenzoate groups with 2-mercaptoethanol. Thus, the tight binding of ligands to the subunit active site strengthens both intra- and inter-subunit bonding domains in dodecameric glutamine synthetase.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.     

Mitochondrial gene expression in saccharomyces cerevisiae. I. Optimal conditions for protein synthesis in isolated mitochondria

An in vitro mitochondrial protein-synthesizing system, which makes use of intact yeast mitochondria, has been developed in order to study mitochondrial gene expression and its control by nuclear-coded proteins. Studies with this system have revealed that: isolated mitochondria synthesize polypeptide gene products which can be radiolabeled to high specific radioactivities when incubated in a "protein-synthesizing medium" that has been optimized with respect to each of its components; two energy-generating systems, endogenous oxidative phosphorylation and an exogenous ATP-regenerating system, support the highest level of protein synthesis; and the omission of an oxidizable substrate results in the synthesis of two new polypeptides (19.5 and 18 kDa) and a decrease in the amounts of cytochrome c oxidase subunits I and II which are synthesized. They have also revealed that added adenine and guanine nucleotides increase the overall level of protein synthesis and that the added guanine nucleotides facilitate polypeptide chain elongation. Although isolated mitochondria which have been optimized for protein synthesis synthesize normal gene products (McKee, E. E., McEwen, J. E., and Poyton, R. O., (1984) J. Biol. Chem. 259, 9332-9338) they still respond to an added dialyzed S-100 fraction from yeast cells by increasing their level of protein synthesis. This stimulation is observed in the presence of optimal concentrations of GTP, making it unlikely that guanyl nucleotides or enzymes which synthesize them are the sole stimulatory factors present in cellular cytosolic fractions, as suggested by Ohashi and Schatz (Ohashi, A., and Schatz, G. (1980) J. Biol. Chem. 255, 7740-7745).     

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.     

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.     

Identification of an isozyme form of protein synthesis initiation factor 4F in plants.

We showed previously that wheat germ extracts contain two forms of protein synthesis initiation factor 4F that have very similar functional properties (Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232). One form, designated eIF-4F, is a complex containing two subunits, p220 and p26. The other form, designated eIF-(iso)4F, is a complex containing two subunits, p82 and p28, which are antigenically distinct from the subunits of eIF-4F. Both the p26 subunit of eIF-4F and the p28 subunit of eIF-(iso)4F are m7G cap-binding proteins. In this investigation, affinity-purified antibodies to the p220 and p26 subunits of wheat germ eIF-4F and to the p82 and p28 subunits of wheat germ eIF-(iso)4F were used to determine if isozyme forms of eIF-4F are present in maize and cauliflower. Extracts from wheat germ, maize root tips, and cauliflower inflorescences were partially purified by adsorption on m7GTP-Sepharose and elution with m7GTP (MGS eluate). Analysis by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies to the subunits of the wheat germ factors showed that the MGS eluate from maize contains polypeptides that react with antibodies to the p82 and p28 subunits of wheat eIF-(iso)4F, as well as polypeptides that react with antibodies to the p220 and p26 subunits of wheat eIF-4F. The MGS eluate from cauliflower also contains polypeptides that reacted with antibodies to the subunits of wheat eIF-(iso)4F. These results indicate that both maize and cauliflower contain the isozyme form of eIF-4F. In addition, it was found that the factors in the MGS eluate from maize support polypeptide synthesis in a system from wheat deficient in eIF-4F and eIF-(iso)4F, whereas the factors in the MGS eluate from cauliflower support polypeptide synthesis only to a small extent.     

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.     

DNA polymerase III of Escherichia coli. Purification and identification of subunits.

DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83. The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively. The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C. (1975) J. Biol. Chem. 250, 461-469; McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484). It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme. Thus, the DNA polymerase III holoenzyme contains at least six different subunits. Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C. (1975) J. Biol. Chem. 250, 470-478).     

DNA polymerase alpha from Drosophila melanogaster embryos. Subunit structure

The homogeneous DNA polymerase alpha from early embryos of Drosophila melanogaster contains four polypeptides designated alpha, beta, gamma, and delta, with molecular weights of 148,000, 58,000, 46,000, and 43,000, respectively (Banks, G. R., Boezi, J. A., and Lehman, I. R. (1979) J. Biol. Chem. 254, 9886-9892). The four polypeptides are structurally distinct from one another, as indicated by their different peptide patterns following limited proteolysis with Staphylococcus aureus protease. Furthermore, the inclusion of the protease inhibitors, leupeptin and pepstatin, in addition to phenpylmethylsulfonyl fluoride and sodium metabisulfite, which are used routinely during the purification, does not alter the pattern of polypeptides in the purified polymerase, suggesting that the four polypeptides are not a consequence of nonspecific proteolysis during purification. Thus, the alpha, beta, gamma, and delta polypeptides appear to be distinct subunits of the alpha-DNA polymerase of D. melanogaster. The alpha subunit is required for DNA polymerase activity. However, the specific activity of the isolated subunit is substantially lower than when it is associated with the beta, gamma, and delta subunits.     

Synthesis of diadenosine 5',5' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.     

The Fo subunits of the Escherichia coli F1Fo-ATP synthase are sufficient to form a functional proton pore

The assembly of the Fo sector of the Escherichia coli ATP synthase has been studied using both structural and functional criteria for assembly. Cross-linking E. coli minicell membranes containing only the Fo subunits a, b, and c with dithiobis(succinimidyl propionate) (DSP) produces b2 and c2 dimers that are generated by cross-linking membranes containing the assembled holoenzyme. Five plasmids carrying the genes specifying the Fo polypeptides in a bacterial strain lacking all of the unc (ATP synthase) genes show a good correlation between Fo function and the amount of the membrane-bound Fo polypeptides. In this report we revise a conclusion reached previously (Klionsky, D.J., Brusilow, W.S.A., and Simoni, R.D. (1983) J. Biol. Chem. 258, 10136-10143) and present evidence that the Fo subunits alone are sufficient to assemble a functional proton pore.     

Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.     

Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes

Two new allophycocyanin-containing complexes were found among the products of partial dissociation of the phycobilisomes of Synechococcus 6301 strain AN112. These complexes were purified to homogeneity and characterized with respect to composition, stability, and spectroscopic properties. The structures of the complexes were established to be (alpha AP beta AP)3 . 10.5K and (alpha 1APB alpha 2AP beta 3AP) . 10.5 K, where alpha AP and beta AP are subunits of allophycocyanin, and alpha APB is the subunit of allophycocyanin B (see Lundell, D. J., and Glazer, A. N. (1981) J. Biol. Chem. 256, 12600-12606), and 10.5K is an uncolored polypeptide of 10.5-kilodaltons. These complexes are derived from the core substructure of the phycobilisome. Electron microscopic studies of the morphology of the core of strain AN112 phycobilisomes (Yamanaka, G., Glazer, A. N., and Williams, R. C. (1980) J. Biol. Chem. 255, 11004-11010) as well as structural studies of an 18 S subassembly derived from the phycobilisomes by partial dissociation (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086) indicated that the core assembly consisted of two cylindrical elements each made up of the same four distinct "trimeric" biliprotein-containing complexes. Two such core components, (alpha AP beta AP)3 and alpha 2AP beta 2AP. 18.3K . 75K (where 18.3K and 75K are polypeptides of 18.3- and 75-kilodaltons), were shown to be contained within the 18 S subassembly (Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901). The isolation of the two allophycocyanin-containing complexes described here completes the characterization of the four types of components in the Synechococcus 6301 phycobilisome core. Two lines of evidence indicate that each of the four complexes is present twice in the core: comparison of the compositions (and yields) of the complexes with that of the intact AN112 phycobilisome, and near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes taken in appropriate molar proportions.     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.     

Protein kinase C phosphorylates glutamyl-tRNA synthetase in rabbit reticulocytes stimulated by tumor promoting phorbol esters

A high Mr synthetase core complex isolated from higher eukaryotes contains aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. Previously, five of the synthetases were shown to be phosphorylated in reticulocytes, and the glutaminyl- and aspartyl-tRNA synthetases were shown to be selectively phosphorylated in response to 8-bromo cAMP (Pendergast, A. M., Venema, R. C., and Traugh, J. A. (1987) J. Biol. Chem. 262, 5939-5942). Exposure of reticulocytes to phorbol 12-myristate 13-acetate stimulates the selective phosphorylation of one synthetase in the complex, glutamyl-tRNA synthetase. Only the glutamyl-tRNA synthetase is modified to a significant extent when the purified complex is phosphorylated in vitro by protein kinase C; up to 0.7 mol of phosphate is incorporated per mol of synthetase. Two-dimensional phosphopeptide mapping shows a single tryptic phosphopeptide, which is identical for the enzyme modified in vitro by protein kinase C or in phorbol 12-myristate 13-acetate-stimulated cells. Phosphorylation in vivo is reproducibly accompanied by a 38 +/- 10% reduction in aminoacylation activity of partially purified glutamyl-tRNA synthetase assayed in vitro. Phosphorylation in vitro has no detectable effect on aminoacylation. This difference may be due to the absence of a required effector molecule which alters activity by interaction with the phosphorylated synthetase. Glutamyl-tRNA synthetase is one of a growing number of translational components, including initiation factors, which are coordinately modified by protein kinase C in response to phorbol 12-myristate 13-acetate.