The ATP/Substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/ substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate ( glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mM glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ ADP ratio on the ATP/ substrate stoichiometry determinations could be eliminated

Acetylation of core histones in response to HDAC inhibitors is diminished in mitotic HeLa cells

Histone acetylation is a key modification that regulates chromatin accessibility. Here we show that treatment with butyrate or other histone deacetylase (HDAC) inhibitors does not induce histone hyperacetylation in metaphase-arrested HeLa cells. When compared to similarly treated interphase cells, acetylation levels are significantly decreased in all four core histones and at all individual sites examined. However, the extent of the decrease varies, ranging from only slight reduction at H3K23 and H4K12 to no acetylation at H3K27 and barely detectable acetylation at H4K16. Our results show that the bulk effect is not due to increased or butyrate-insensitive HDAC activity, though these factors may play a role with some individual sites. We conclude that the lack of histone acetylation during mitosis is primarily due to changes in histone acetyltransferases (HATs) or changes in chromatin. The effects of protein phosphatase inhibitors on histone acetylation in cell lysates suggest that the reduced ability of histones to become acetylated in mitotic cells depends on protein phosphorylation

The ATP/Substrate Stoichiometry of the ATP-binding Cassette (ABC) Transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mM glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.

Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine β-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress. C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuAΔ12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was ~0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg2+ and Ba2+) were more effective in activating OpuA than NH4+, K+, Na+, or Li+, consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb+ and Cs+ were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb+ and Cs+ were no longer inhibitory in OpuAΔ12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuAΔ12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Isotope-based discrimination between the infrared modes of plastosemiquinone anion radicals and neutral tyrosyl radicals in photosystem II

Photosystem II (PSII) conducts the light-driven oxidation of water and reduction of plastoquinone. Difference Fourier transform infrared (FT-IR) spectroscopy can be used to obtain information about structural changes which occur in protein and cofactors when charge separation occurs. The focus of this work was the assignment of vibrational lines to two different species in PSII: the tyrosyl radical, Z, and the plastosemiquinone anion radical, QA-. Difference FT-IR experiments were conducted with cyanobacterial PSII samples, in which the tyrosine ring was uniformly 13C-labeled, in which tyrosine was 13C-labeled at carbon 4, and in which plastoquinone was methyl-deuterated. At 80 K, difference FT-IR spectra reflect the oxidation of chlorophyll/ carotenoid and the one-electron reduction of QA; no significant D or Z contribution to the spectrum is observed under these conditions. At 264 K, difference FT-IR spectra reflect the oxidation of redox-active tyrosines Z and D; no significant QA- contribution is observed under these conditions. At 80 K, isotope-induced shifts were observed in spectral features at 1482 and 1469 cm-1 upon deuteration of plastoquinone. At 264 K, isotope-induced shifts were observed in a 1478 cm-1 line upon 13C- labeling of tyrosine, but little change was observed upon plastoquinone deuteration. These data support the assignment of a positive 1478 cm-1 line to a tyrosyl radical vibrational mode and positive 1482 and 1469 cm-1 lines to plastosemiquinone anion vibrational modes. Hybrid Hartree-Fock/density functional calculations of p-cresyl radical\u27s vibrational frequencies and isotopic frequency shifts support this assignment. © 2000 American Chemical Society