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L K Peters

Publications and source records attributed to L K Peters.

6 recordsLinked to original sources
PubMed1984

Mutations altering aspartyl-61 of the omega subunit (uncE protein) of Escherichia coli H+ -ATPase differ in effect on coupled ATP hydrolysis.

Mutations in the H+-translocating ATPase complex (F1F0) of Escherichia coli have been described in which aspartyl-61 of the omega subunit ( uncE protein) is substituted by either glycine ( uncE105 ) or asparagine ( uncE107 ). Either substitution blocks the H+-translocation activity of the F0 sector of the complex. Here we report a difference in the effects of the two substitutions on the coupled ATPase activity of F1 bound to F0. Wild-type F1 was bound to the F0 of either mutant with affinities comparable to wild-type. The ATPase activity of F1 bound to uncE107 F0 was inhibited by 50%, whereas that bound to uncE105 F0 was not inhibited. Complementation studies with a pBR322-derived plasmid that carried the E gene of the unc operon only indicated that a single mutation in the host strain was responsible for the respective phenotypes. In mutants complemented by the uncE + plasmid, restoration of wild-type biochemical properties was only partial and may be attributed to a mixing of wild-type and mutant omega subunits in a hybrid F0 complex. The activity of membrane-bound F1 was less inhibited in the uncE +/ uncE107 hybrid. Paradoxically, complementation of uncE105 by the uncE + plasmid resulted in substantial inhibition of the activity of membrane-bound F1. The results indicate that a glycine-versus-asparagine substitution for aspartyl-61 must lead to altered conformations of omega and that these differences in conformation are important in the coupling between the F0 and F1 sectors of the complex.

Adenosine Triphosphate↗
PubMed1983

H+-ATPase of Escherichia coli uncB402 mutation leads to loss of chi subunit of subunit of F0 sector.

The uncB402 mutation in Escherichia coli results in formation of an H+-ATPase complex that is defective in energy-transducing capacity. The mutation, originally described by Butlin et al. (Butlin, J.D., Cox, G.B., and Gibson, F. (1973) Biochim. Biophys. Acta 292, 366-375), alters the F0 sector of the H+-ATPase complex. Here, we show that uncB402 is an amber-suppressible, chain-terminating mutation that results in loss of the chi subunit from F0. This was demonstrated in crude membrane fractions after overproduction of the ATPase complex by heat induction of a lambda transducing phage carrying the unc operon of uncB402. The lambda-uncB402 DNA was used as a template in an in vitro transcription-translation system. A synthesis product that may correspond to the truncated form of the chi subunit was observed. Despite the absence of chi, the F1-ATPase was still bound to the membrane, although more weakly than in wild type. The omega subunit of F0 ("dicyclohexylcarbodiimide-binding protein") shows normal reactivity with dicyclohexylcarbodiimide, indicating that at least this portion of F0 integrates properly in the membrane in the absence of the chi subunit. The F0 of uncB402 was not functional in H+ translocation activity. This was shown by direct H+ flux measurements with crude membrane vesicles that were treated with guanidine to disrupt the binding of F1 to F0. Secondly, a method was developed for isolation of F0 from F1-depleted membranes. The F0 from uncB402 was shown to have less than 5% the proton-translocase activity of wild type F0 when reconstituted into liposomes. Although the uncB402 mutant shows these defects, the question of whether the chi subunit plays a direct role in F1-binding or H+ translocation remains open, since the loss of chi may lead to subtle changes in the assembly of the other F0 subunits. Analysis of other mutants should permit a more definitive assignment of function.

Adenosine Triphosphatases↗
PubMed1983

Use of lambda unc transducing bacteriophages in genetic and biochemical characterization of H+-ATPase mutants of Escherichia coli.

The eight subunits of the H+-ATPase of Escherichia coli are coded by the genes of the unc operon, which maps between bglB and asnA. A collection of unc mutations were transferred via P1 transduction into a strain in which lambda cI857 S7 was inserted into bglB. The lambda phage was induced, and asnA+ transducing phage that carried unc were selected. Transducing phage carrying mutations in the uncA, B, D, E, and F genes were used for complementation analysis with a collection of unc mutants, including mutants which had been reported previously but not genetically characterized. Some mutations gave a simple complementation pattern, indicating a single defective gene, whereas other mutations gave more complex patterns. Two mutants (uncE105 and uncE107) altered in the proteolipid (omega) subunit of F0 were not complemented by any of the lambda unc phage, even though both mutants had a fully functional F1 ATPase and therefore normal A and D genes. Hence, only limited conclusions can be drawn from genetic complementation alone, since it cannot distinguish normal from abnormal genes in certain classes of unc mutants. The lambda unc phage proved to be essential in characterizing several mutants defective in F0-mediated H+ translocation. The unc gene products were overproduced by heat induction of the lysogenized lambda unc phage to determine whether all the F0 subunits were in the membrane. Two mutants that gave a simple complementation pattern, indicative of one defective gene, did not assemble a three-subunit F0. The uncB108 mutant was shown to lack the chi subunit of F0 but to retain psi and omega. Trace amounts of an altered omega subunit and normal amounts of chi and psi were found in the uncE106 mutant. A substitution of aspartate for glycine at residue 58 of the protein was determined by DNA sequence analysis of the uncE gene cloned from the lambda uncE106 phage DNA. One of the omega-defective, noncomplementing mutants (uncE107) was shown to retain all three F0 subunits. The uncE gene from this mutant was also sequenced to confirm an asparagine-for-aspartate substitution at position 61 (the dicyclohexylcarbodiimide-binding site) of the omega subunit.

Bacteriophage lambda↗
PubMed1982

Native bare zone assemblage nucleates myosin filament assembly.

Native myosin filaments from rabbit psoas muscle are always 1.5 micrometer long. The regulated assembly of these filaments is generally considered to occur by an initial antiparallel and subsequent parallel aggregation of identical myosin subunits. In this schema myosin filament length is controlled by either a self-assembly or a Vernier process. We present evidence which refines these ideas. Namely, that the intact myosin bare zone assemblage nucleates myosin filament assembly. This suggestion is based on the following experimental evidence. (1) A native bare zone assemblage about 0.3 micrometer long can be formed by dialysis of native myosin filaments to either a pH 8 or a 0.2 M-KCl solution. (2) Upon dialysis back to 0.1 M-KCl, bare zone assemblages and distal myosin molecules recombine to form 1.5 micrometer long bipolar filaments. (3) The bare zone assemblage can be separated from the distal myosin molecules by column chromatography in 0.2 M-KCl. Upon dialysis of the fractionated subsets back to 0.1 M-KCl, the bare zone assemblage retains its length of about 0.3 micrometer. However, the distal molecules reassemble to form filaments about 5 micrometers long. (4) Filaments are formed from mixes of the isolated subsets. The lengths of these filaments vary with the amount of distal myosin present. (5) When native filaments, isolated bare zone assemblages or distal myosin molecules are moved sequentially to 0.6 M-KCl and then to 0.1 M-KCl, the final filament lengths are all about 5 micrometers. The capacity of the bare zone assemblage to nucleate filament assembly may be due to the bare zone myosin molecules, the associated M band components or both.

Animals↗