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R D Simoni

Publications and source records attributed to R D Simoni.

At least 55 records · Page 3Linked to original sources
PubMed1987

A mutation of the c subunit of the Escherichia coli proton-translocating ATPase that suppresses the effects of a mutant b subunit.

A mutation of the b subunit of the Escherichia coli proton-translocating ATPase and mutations in the gene for the a subunit that suppress its effects have been previously described (Kumamoto, C., and Simoni, R. D. (1986) J. Biol. Chem. 261, 10037-10042). In this paper, we describe the characterization of a new mutation that partially suppresses the effects of the original b mutation. The new suppressor mutation causes the substitution of serine for alanine at position 62 of the c subunit. Biochemical studies of double mutants, carrying both b and c mutations, demonstrate that the c mutation partially restores the function of the enzyme complex.

Adenosine Triphosphate↗
PubMed1986

Genetic evidence for interaction between the a and b subunits of the F0 portion of the Escherichia coli proton translocating ATPase.

A mutation of the b subunit of the Escherichia coli proton translocating ATPase was previously described (Porter, A. C. G., Kumamoto, C., Aldape, K., and Simoni, R. D. (1985) J. Biol. Chem. 260, 8182-8187). This mutation, which causes substitution of aspartic acid for glycine at position 9 (basp9), results in loss of function of the ATPase complex. In this paper we describe the isolation and characterization of two mutations that partially suppress the effects of the basp9 alteration. The suppressor mutations cause amino acid substitutions at position 240 of the a subunit. Membranes derived from strains carrying a suppressor mutation and the basp9 mutation exhibited ATP-dependent proton translocating activity.

Amino Acid Sequence↗
PubMed1986

Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli.

Mutations in the uncB gene which encodes the a subunit of F1F0-ATPase in Escherichia coli were isolated and characterized. Eight mutations caused premature polypeptide chain termination. Two mutations were single amino acid substitutions resulting in the replacements of serine 206 with leucine (ser-206----leu) and histidine 245 with tyrosine (his-245----tyr). The ser-206----leu mutation does not alter F1 binding and allows ATP driven membrane energization at a low level. Stripping of F1 from membranes containing the ser-206----leu mutation does not render the membranes permeable to protons indicating impaired proton conductivity. The his-245----tyr mutation also blocks Fo-mediated proton conduction but has normal F1 binding properties. F1 bound to membranes with both ser-206----leu and his-245----tyr mutant a subunits is sensitive to dicyclohexylcarbodiimide. Apparently, both missense mutations impair proton conduction without altering assembly of the F1F0-ATPase complex. The direct involvement of the a subunit in proton translocation is discussed.

Adenosine Triphosphate↗
PubMed1986

Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli.

Translation of the gene for the b subunit of the Escherichia coli proton-translocating ATPase has been examined. Oligonucleotide-directed site-specific mutagenesis was used to mutate certain nucleotides in the intergenic region between uncE (c) and uncF (b). One of the changes was predicted to lower the stability of a proposed stem structure which blocked the ribosome binding site of the uncF mRNA segment. The result of the mutation is a nearly 3-fold increase in the rate of synthesis of the b polypeptide. Another mutation was introduced which changed the initiation codon for uncF from GUG to AUG. This change resulted in an approximately 2-fold increase in the synthesis rate of the b polypeptide. These results suggest that secondary structure in the mRNA and the use of a less efficient initiation codon play a role in restricting translation initiation of the uncF mRNA segment. These mechanisms may, in part, explain how the polypeptides of the ATPase complex are synthesized in approximately the same relative amounts as they appear in the assembled complex.

Base Sequence↗
PubMed1985

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli.

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.

DNA Restriction Enzymes↗
PubMed1985

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.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1985

Role of the b subunit of the Escherichia coli proton-translocating ATPase. A mutagenic analysis.

A plasmid that carries the uncF gene of Escherichia coli, which codes for the b subunit of the proton-translocating ATPase (F1F0), was mutagenized with hydroxylamine and ethyl methanesulfonate. Mutated plasmids were characterized by complementation analysis and by in vitro transcription/translation. Eleven of the plasmids mutated specifically in uncF were studied in detail: the nucleotide sequences of their uncF genes were determined and their effects on the F1F0 were measured. The results suggest that a C-terminal portion of the b subunit that involves the glycine residue at position 131, is required for the formation of a functional proton pore as well as for the binding of F1 to F0. A mutation in the N-terminal portion of the b subunit, in the glycine residue at position 9, also prevented the formation of a functional proton pore, but had only a small effect on the binding of F1 to F0.

Amino Acid Sequence↗
PubMed1985

The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property.

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.

Amino Acid Sequence↗
PubMed1985

Mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase overaccumulation in three compactin-resistant cell lines.

We have isolated three mammalian cell lines which are resistant to compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The drug resistance in all three cell lines is due to an increase of HMG-CoA reductase activity. Two of the three cell lines overaccumulate HMG-CoA reductase messenger RNA when grown in the presence of compactin. DNA hybridization experiments indicate that both a baby hamster kidney-derived compactin-resistant cell line, C100, and a cell line derived from mouse 3T6 cells, 3T6-40, exhibit amplifications of the HMG-CoA reductase gene. A third compactin-resistant cell line derived from Chinese hamster ovary cells, ML100, does not exhibit an amplification of the HMG-CoA reductase gene, nor does it show an elevated level of HMG-CoA reductase mRNA, comparable to that seen in the other cell lines.

Animals↗
PubMed1985

Intracellular transport of phosphatidylcholine to the plasma membrane.

We have used pulse-chase labeling of Chinese hamster ovary cells with choline followed by plasma membrane isolation on cationic beads to study the transport of phosphatidylcholine from the endoplasmic reticulum to the plasma membrane. We have found that the process is rapid (t1/2 [25 degrees C] = 2 min) and not affected by energy poisons or by cytochalasin B, colchicine, monensin, or carbonyl cyanide p-chlorophenylhydrazone. Cooling cells to 0 degree C effectively stops the transport process. The intracellular transport of phosphatidylcholine is distinct in several ways from the intracellular transport of cholesterol (Kaplan, M. R., and R. D. Simoni, 1985, J. Cell. Biol., 101:446-453).

Animals↗
PubMed1985

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane.

We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system.

Animals↗
PubMed1985

The nucleotide sequence of Syrian hamster HMG-CoA reductase cDNA.

We have determined the nucleotide sequence of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase cDNA. Comparison of the deduced amino acid sequence with the homologous sequence from Chinese hamster reveals highly conserved domains which appear to have functional significance. The amino-terminal membrane domain of HMG-CoA reductase exhibits 100% homology. This region may span the endoplasmic reticulum seven times and is thought to be involved in the sterol-regulated degradation of HMG-CoA reductase (Gil et al., 1985; Liscum et al., 1985). The carboxyl terminus contains the active site of the enzyme and exhibits greater than 99% homology. A central region linking these two conserved domains exhibits greater divergence. In this region there is only 85% homology between the two hamster lines, suggesting that this linkage domain has a less stringent structural requirement.

Amino Acid Sequence↗
PubMed1984

Effects of compactin on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in compactin-resistant C100 and wild-type cells.

A cell line, C100, resistant to 225 microM compactin, has been isolated which overproduces 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase approximately 100-fold compared to the parental cell line [E. Hardeman, H. Jenke and R. Simoni (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1516-1520]. It is demonstrated that the overproduction of HMG-CoA reductase in these cells is the result of increased enzyme synthesis due to elevated levels of translatable mRNA. Furthermore, the apparent molecular weight of the in vitro translation product is 94,000, which agrees with the molecular weight of the in vivo synthesized HMG-CoA reductase protomer in C100 cells. However, a comparison of the Staphylococcus aureus V8 proteolysis patterns between the in vitro and in vivo translation products reveals structural differences which suggests in vivo post-translation modification(s). It is also demonstrated unequivocally, by comparing proteolytic cleavage patterns and pulse-chase experiments, that the previously reported 63,000-, 52,000-, and 38,000-Da polypeptides recognized by HMG-CoA reductase antiserum derive from the 94,000-Da protomer as a result of nonphysiological proteolysis. Finally, the types of regulatory mechanisms involved in both the induction and repression of the enzyme in the presence or absence of compactin were determined. Four biochemical parameters of HMG-CoA reductase were examined in variant and parental cells grown in the presence and absence of compactin: enzymatic activity, degradation rate, synthesis rate, and concentration of translatable mRNA. These studies revealed that changes in cellular HMG-CoA reductase content are a function of concurrent changes in the rates of enzyme degradation and synthesis. Changes in enzyme synthesis are due to alterations in the level of translatable mRNA.

Animals↗
PubMed1984

Biogenesis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an integral glycoprotein of the endoplasmic reticulum.

Using a cell line, C100, that overproduces 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34) 100-fold, we have studied the synthesis and insertion of this protein into the endoplasmic reticulum. The enzyme is synthesized on membrane-bound polysomes. It is cotranslationally but not post-translationally inserted into dog pancreatic microsomes. This cotranslational insertion is dependent upon signal recognition particle. HMG-CoA reductase is glycosylated with an oligosaccharide(s) of the "high-mannose" type sensitive to endo-beta-D-N-acetylglucosaminidase H. Partial determination of the NH2-terminal amino acid sequence of the in vitro translation product and the mature polypeptide indicate they are the same and demonstrate there is no cleavage of an NH2-terminal signal sequence.

Amino Acid Sequence↗
PubMed1984

In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli.

The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit. In addition, the epsilon-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0.

Antigen-Antibody Complex↗
PubMed1983

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit.

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.

ATP Synthetase Complexes↗
PubMed1983

Assembly of a functional F0 of the proton-translocating ATPase of Escherichia coli.

We have investigated both structural and functional assembly of the F0 portion of the Escherichia coli proton-translocating ATPase in vivo. Fractionation of E. coli minicells containing plasmids which code for parts of the unc operon shows that each of the F0 peptides a, b, and c insert into the cytoplasmic membrane independent of each other and without the polypeptides which form the F1 portion of the complex alpha, beta, gamma, delta, and epsilon. Assays of membrane energization indicate that, while formation of a functional proton channel requires the presence of all three F0 polypeptides a, b and c, they are not sufficient. Synthesis of both the alpha and beta subunits of the F1 are required for formation of a functional proton channel.

Adenosine Triphosphatases↗