ATP synthase that lacks F0a-subunit: isolation, properties, and indication of F0b2-subunits as an anchor rail of a rotating c-ring

In a rotary motor F1F0-ATP synthase, F0 works as a proton motor; the oligomer ring of F0c-subunits (c-ring) rotates relative to the F0ab2 domain as protons pass through F0 down the gradient. F0ab2 must exert dual functions during rotation, that is, sliding the c-ring (motor drive) while keeping the association with the c-ring (anchor rail). Here we have isolated thermophilic F1F0(-a) which lacks F0a. F1F0(-a) has no proton transport activity, and F0(-a) does not work as a proton channel. Interestingly, ATPase activity of F1F0(-a) is greatly suppressed, even though its F1 sector is intact. Most likely, F0b2 associates with the c-ring as an anchor rail in the intact F1F0; without F0a, this association prevents rotation of the c-ring (and hence the gamma-subunit), which disables ATP hydrolysis at F1. Functional F1F0 is easily reconstituted from purified F0a and F1F0(-a), and thus F0a can bind to its proper location on F1F0(-a) without a large rearrangement of other-subunits.     

Solution structure of the N-terminal F1 module pair from human fibronectin.

Multiple sites within the N-terminal domain (1-5F1) of fibronectin have been implicated previously in fibronectin matrix assembly, heparin binding, and binding to cell surface proteins of pathogenic bacteria. The solution structure of 1F1(2)F1, the N-terminal F1 module pair from human fibronectin, has been determined using NMR spectroscopy. Both modules in the pair conform to the F1 consensus fold. In 4F1(5)F1, the only other F1 module pair structure available, there is a well-defined intermodule interface; in 1F1(2)F1, however, there is no detectable interface between the modules. Comparison of the backbone 15N-{1H} NOE values for both module pairs confirms that the longer intermodule sequence in 1F1(2)F1 is flexible and that the stabilization of the 4F1 C-D loop observed in 4F1(5)F1, as a result of the intermodule interface, is not observed in 1F1(2)F1.     

Orosomucoid (ORM 1) subtyping and formal genetics

Summary Orosomucoid (ORM) phenotyping has been performed in 141 families with 407 children from southwest Germany. Eight families were observed in which duplicated ORM1 genes, F1F2, F1F3, segregated. The family data gave no information about the presence of tandemly duplicated ORM1 F1F4 and ORM1 F1F5 genes. To date, the segregation of the phenotypes of the children agrees with the extended formal model: two ORM1 loci with two common (^*F1, ^*S) and several rare (^*F1F2, ^*F1F3, ^*F4, ^*F5) alleles. The parental allele frequencies were calculated by gene counting as ORM1 ^*F1 = 0.5781, ^*S = 0.3901, ^*F1F2 = 0.0195, ^*F4 = 0.0053, ^*F1F3 = 00.0035, ^*F5 = 0.0035.     

Specificity of acidic phospholipids (CL & PA) in the activation of mitochondrial F0F1 ATPase by Mg2+

The interaction of Mg2+ with native F0F1 ATPase was studied. The hydrolytic activity of F0F1 ATPase could be competitively activated by Mg2+, but the preincubation of F0F1 ATPase with cholate eliminated the Mg2+ effect. The result from the comparison of the effect of Mg2+ on F0F1 ATPase with that on soluble F1 ATPase, and the fact that the activation of Mg2+ on cholate-treated F0F1 ATPase could be reconstituted only by divalent acidic phospholipid cardiolipin, indicate that there exists a specificity between the acidic phospholipids of the mitochondrial inner membrane and Mg2+ enhancement of ATP-hydrolyzing activity of F0F1 ATPase.     

Methylation-mediated regulation of E2F1 in DNA damage-induced cell death

E2F1 promotes DNA damage-induced apoptosis and the post-translational modifications of E2F1 play an important role in the regulation of E2F1-mediated cell death. Here, we found that Set9 and LSD1 regulate E2F1-mediated apoptosis upon DNA damage. Set9 methylates E2F1 at lysine 185, a conserved residue in the DNA-binding domain of E2F family proteins. The methylation of E2F1 by Set9 leads to the stabilization of E2F1 and up-regulation of its proapoptotic target genes p73 and Bim, and thereby induces E2F1-mediated apoptosis in response to genotoxic agents. We also found that LSD1 demethylates E2F1 at lysine 185 and reduces E2F1-mediated cell death. The identification of the methylation/demethylation of E2F1 by Set9/LSD1 suggests that E2F1 is dynamically regulated by epigenetic enzymes in response to DNA damage.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.     

Methylation-mediated regulation of E2F1 in DNA damage-induced cell death

E2F1 promotes DNA damage-induced apoptosis and the post-translational modifications of E2F1 play an important role in the regulation of E2F1-mediated cell death. Here, we found that Set9 and LSD1 regulate E2F1-mediated apoptosis upon DNA damage. Set9 methylates E2F1 at lysine 185, a conserved residue in the DNA-binding domain of E2F family proteins. The methylation of E2F1 by Set9 leads to the stabilization of E2F1 and up-regulation of its proapoptotic target genes p73 and Bim, and thereby induces E2F1-mediated apoptosis in response to genotoxic agents. We also found that LSD1 demethylates E2F1 at lysine 185 and reduces E2F1-mediated cell death. The identification of the methylation/demethylation of E2F1 by Set9/LSD1 suggests that E2F1 is dynamically regulated by epigenetic enzymes in response to DNA damage.     

Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.     

The effect of Mg2+ on mitochondrial F0.F1 ATPase and characteristics of the nucleotide binding sites

The interaction of Mg2+ with nucleotide-washed F0.F1 ATPase from pig heart was studied. Mg2+ had no effect on nucleotide-washed F0.F1 ATPase, but it competitively inhibited the hydrolytic activity of washed F0.F1 ATPase preincubated with ADP and slightly activated the hydrolytic activity of washed F0.F1 ATPase preincubated with ATP. In the last two cases, it revealed negative cooperativity. The effect of Mg2+ on F0.F1 ATPase is therefore closely related to the characteristics of the nucleotide binding sites on mitochondrial F0.F1 ATPase.     

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.     

Associated and independent comparisons between the two largest follicles preceding follicle deviation in cattle

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4- to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

The quantitative and qualitative difference between a F1 hybrid of maize and its F2 generation

The majority of modern maize varieties are single F1 hybrids. The yield performance of the F2 generation is known to be inferior to the F1 yield performance. We crossed several F1 hybrids and compared these crossings, together with true F2 generations, with the original F1s. Compared to the F1 generation, biomass yield in the F2 generation dropped with -26.7%, and with -8.7% in the crossings. Ear yield dropped with -35.3% and -10.7% respectively. F2 generations had a reduced early vigour and the ear filling startedlater. The yield of some F1 diallel crosses was not significantly different from the yield of the parental F1s.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein,Inh1

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.