Hybrid motor with H(+)- and Na(+)-driven components can rotate Vibrio polar flagella by using sodium ions. (17/762)

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.  (+info)

Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge. (18/762)

Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V). While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na(+)-coupled transport. Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier. Eight revertant substitutions were isolated from the R52S strain: Asp-19-->Gly, Asp-55-->Asn, Pro-60-->Gln, Trp-116-->Arg, Asn-244-->Ser, Ser-247-->Arg, Asn-248-->Lys, and Ile-352-->Val. Two revertants were also isolated from the R52V strain: Trp-116-->Arg and Thr-338-->Arg revertants. The R52Q strain yielded an Asp-55-->Asn substitution and a first-site revertant, Lys-52 (R52K). The R52K strain had transport properties similar to those of the wild type. Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116-->Arg, Ser-247-->Arg, and Asn-248-->Lys revertants regained significant Na(+)-coupled accumulation. In general, downhill melibiose transport in the presence of Na(+) was better in the revertant strains than in the parental mutants. Three revertant strains, Asp-19-->Gly, Asp-55-->Asn, and Thr-338-->Arg strains, required a high Na(+) concentration (100 mM) for maximal activity. Kinetic measurements showed that the N248K and W116R revertants lowered the K(m) for melibiose, while other revertants restored transport velocity. We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII. We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I).  (+info)

Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance. (19/762)

Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations. The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.  (+info)

Porins in the cell wall of Mycobacterium tuberculosis. (20/762)

Lipid bilayer experiments indicated that the cell wall of Mycobacterium tuberculosis contains at least two different porins: (i) a cation-selective, heat-sensitive 0.7-nS channel which has a short-lived open state and is probably composed of 15-kDa subunits and (ii) a 3-nS, >60-kDa channel with a long-lived open state, resembling porins from fast-growing mycobacteria.  (+info)

Trafficking of Glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels. (21/762)

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement ('translocation') of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K(+)-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.  (+info)

Comparative study of effects of impact tone and steady state tone exposure: EP and concentration of K+ ion and Na+ ion. (22/762)

To test the adequacy of equal energy principle (EEP), guinea pigs were exposed to impact tone. The changes in electrophysiological data, namely endocochlear potential (EP) and the change in K+ ion and Na+ ion concentrations in the endolymph were investigated. The frequency of impact tone was 1 pulse/second or 1 pulse/3 seconds. The steady state tone had Leq24h = 100, 95, 90 or 85 dB, and impact tone had Leq24h = 95, 90 or 85 dB. The results are the following. Both steady state and impact tone exposure cause changes of electrophysiological data. The effects on the absolute value of negative EP induced by impact tone exposures were smaller than that of steady state tone of the same Leq. The rate of pulses was also an important factor for impact tone exposure. Impact tone exposure of 1 pulse/second caused smaller absolute value of negative EP than that of 1 pulse/3 seconds. The K+ ion concentration time course in the endolymph remained similar to the control (Exp. 1) only in Exp. 8 (85 dB; the lowest steady state noise exposure in our experiments), but no decrease in the K+ ion concentration was detected in the other experiments, suggesting an alteration in the K+ ion flow. The Na+ ion concentration time course was also influenced showing no increase in Na+ ion concentration compared to the control (Exp. 1c) and the lowest steady-state exposure experiment (Exp. 8c). Our experimental results suggest that both the K+ ion and Na+ ion movement are altered by tone exposure. We found also that the different types of noise exposure with the same Leq value does not exhibit the same changes. Leq24h is not an accurate damage risk criteria.  (+info)

Ca2+-activated non-selective cation current in rabbit ventricular myocytes. (23/762)

Oscillatory currents (OCs) were studied in isolated rabbit ventricular myocytes with whole cell mode voltage clamp using Na+-free intracellular and extracellular solutions under conditions where K+ currents were anticipated to be eliminated or minimized. All OCs were dependent on release of Ca2+ from the sarcoplasmic reticulum (SR) because they were associated with intracellular Ca2+ ([Ca2+]i) transients, and were suppressed by high concentrations of BAPTA (20 mmol l-1) or pretreatment with the SR antagonist agents ryanodine (10 micromol l-1) or thapsigargin (1 micromol l-1). The reversal potential (Vrev) for OCs shifted with changes in the calculated Vrev for Cl- (ECl) but was between ECl and the calculated Vrev for elemental monovalent cations (ECat), indicating that more than one Ca2+-activated current contributed to OCs. Addition of the Ca2+-activated Cl- current (ICl(Ca)) antagonist, niflumic acid, shifted the OC Vrev to ECat, suggesting that ICl(Ca) and a Ca2+-activated non-selective cation current (ICAN) contributed to the observed OCs. A reduced niflumic acid-insensitive Ca2+-activated OC persisted following marked symmetrical reduction of Cl- in the intracellular and extracellular solutions. Subsequent removal of all extracellular monovalent cations, by N-methyl-D-glucamine (NMDG) substitution, eliminated OCs and the inward holding current suggesting that ICAN and ICl(Ca) accounted for all or most of the Ca2+-activated OC in the absence of Na+. The OC Vrev was equal to ECl in the absence of monovalent elemental cations. Under these conditions niflumic acid eliminated all OCs. Macroscopic OC is partially due to ICAN in rabbit ventricular myocytes.  (+info)

De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. (24/762)

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.  (+info)