Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure-function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His(211) were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure-function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein.
- MeSH
- cirkulární dichroismus MeSH
- Escherichia coli genetika metabolismus MeSH
- iontové kanály chemie genetika metabolismus MeSH
- konformace proteinů MeSH
- liposomy MeSH
- mangan metabolismus MeSH
- metoda terčíkového zámku MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie genetika metabolismus MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- proteiny přenášející kationty chemie genetika metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- substituce aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH(in)) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH(in) for three prototrophic wild-types. The new construct was also used to determine the pH(in) in strains differing in the activity of the plasma membrane Pma1 H(+)-ATPase and the influence of glucose on pH(in). We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.
- MeSH
- buněčná membrána genetika metabolismus MeSH
- cytosol metabolismus MeSH
- draslík metabolismus MeSH
- druhová specificita MeSH
- homeostáza MeSH
- koncentrace vodíkových iontů MeSH
- plazmidy MeSH
- pufry MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika fyziologie MeSH
- zelené fluorescenční proteiny chemie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C3(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character. Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.
- MeSH
- ABC transportéry fyziologie MeSH
- financování organizované MeSH
- fruktosa farmakologie MeSH
- glukosa farmakologie MeSH
- karbocyaniny farmakologie MeSH
- kultivační média MeSH
- membránové potenciály účinky léků MeSH
- mnohočetná fungální léková rezistence fyziologie MeSH
- Saccharomyces cerevisiae - proteiny fyziologie MeSH
- Saccharomycetales fyziologie růst a vývoj účinky léků MeSH
- MeSH
- adenosintrifosfát metabolismus MeSH
- butyráty metabolismus MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- lipidové dvojvrstvy metabolismus MeSH
- membránové proteiny fyziologie chemie metabolismus MeSH
- proteolipidy metabolismus MeSH
- protony MeSH
- transportní proteiny fyziologie chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- amelanotický melanom farmakoterapie patologie MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky terapeutické užití MeSH
- melanom experimentální farmakoterapie patologie MeSH
- nádory kůže farmakoterapie patologie MeSH
- porfyriny terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- krysa rodu rattus MeSH
- mastné kyseliny metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- mitochondrie metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- MeSH
- erytrocyty chemie MeSH
- gravitace MeSH
- lidé MeSH
- separace buněk metody MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
