scholarly journals Direct observation of proton pumping by a eukaryotic P-type ATPase

Science ◽  
2016 ◽  
Vol 351 (6280) ◽  
pp. 1469-1473 ◽  
Author(s):  
S. Veshaguri ◽  
S. M. Christensen ◽  
G. C. Kemmer ◽  
G. Ghale ◽  
M. P. Moller ◽  
...  
Keyword(s):  
Direct Observation ◽  
Proton Pumping ◽  
P Type

Direct observation of inversion capacitance in p-type diamond MOS capacitors with an electron injection layer

2018 ◽  
Vol 57 (4S) ◽  
pp. 04FR01 ◽  
Author(s):  
Tsubasa Matsumoto ◽  
Hiromitsu Kato ◽  
Toshiharu Makino ◽  
Masahiko Ogura ◽  
Daisuke Takeuchi ◽  
...  
Keyword(s):  
Direct Observation ◽  
Electron Injection ◽  
Mos Capacitors ◽  
Electron Injection Layer ◽  
P Type

scholarly journals Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi

2005 ◽  
Vol 392 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Mauricio Vieira ◽  
Peter Rohloff ◽  
Shuhong Luo ◽  
Narcisa L. Cunha-E-Silva ◽  
Wanderley De Souza ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Subcellular Fractionation ◽  
Etiologic Agent ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Intermediate Form ◽  
Confocal Immunofluorescence Microscopy ◽  
P Type

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope–tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

Direct Observation of the Valence Band Edge by in Situ ECSTM-ECTS in p-Type Cu2O Layers Prepared by Copper Anodization

2008 ◽  
Vol 113 (3) ◽  
pp. 1028-1036 ◽  
Author(s):  
Felipe Caballero-Briones ◽  
Juan M. Artés ◽  
Ismael Díez-Pérez ◽  
Pau Gorostiza ◽  
Fausto Sanz
Keyword(s):  
Valence Band ◽  
Direct Observation ◽  
Band Edge ◽  
Valence Band Edge ◽  
P Type

scholarly journals In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase

2018 ◽  
Vol 293 (44) ◽  
pp. 17095-17106 ◽  
Author(s):  
Thao T. Nguyen ◽  
Grzegorz Sabat ◽  
Michael R. Sussman
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Unnatural Amino Acid ◽  
Proton Pumping ◽  
Intramolecular Interactions ◽  
Cross Linking ◽  
Regulatory Domain ◽  
P Type ◽  
Plant Plasma Membrane

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a “head-to-tail” organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.

scholarly journals Targeting the fungal plasma membrane proton pump.

1995 ◽  
Vol 42 (4) ◽  
pp. 481-496 ◽  
Author(s):  
B C Monk ◽  
A B Mason ◽  
T B Kardos ◽  
D S Perlin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Proton Pump ◽  
Fungal Pathogens ◽  
Mutational Analysis ◽  
Proton Pumping ◽  
Proton Pumps ◽  
Essential Enzyme ◽  
Altered Cell ◽  
P Type

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

Sucrose transport-related genes are expressed in both maternal and filial tissues of developing wheat grains

2000 ◽  
Vol 27 (11) ◽  
pp. 1009 ◽  
Author(s):  
Neil Bagnall ◽  
Xin-Ding Wang ◽  
Graham N. Scofield ◽  
Robert T. Furbank ◽  
Christina E. Offler ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Plasma Membranes ◽  
Triticum Turgidum ◽  
Proton Pumping ◽  
Transfer Cells ◽  
Sucrose Transport ◽  
Wheat Grains ◽  
Nucellar Projection ◽  
Vascular Parenchyma ◽  
P Type

In developing wheat grains (Triticum turgidum var. durum cv. Fransawi), post-sieve element transport of phloem-imported photoassimilates (sucrose) includes membrane transport, to and from the grain apoplasm, between symplasmically-isolated maternal and filial tissues. The cellular location and mechanism of these membrane transport steps were explored during rapid grain fill. Genomic Southern analysis indicated the presence of a multigene family of sucrose/H + symporters (SUTs). One or more SUTs were highly expressed in developing grains, as were P-type H + /ATPase(s) and a sucrose binding protein (SBP). Transcripts of these genes were detected in vascular parenchyma, nucellar projection and aleurone cells. Antibodies, raised against a SUT, an H + /ATPase and a SBP, were selectively bound to plasma membranes of vascular parenchyma cells, nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells. The nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells exhibited strong proton pumping activity. In contrast, SUT transport function was restricted to filial tissues containing modified aleurone/sub-aleurone transfer cells. Based on these findings, we conclude that SUTs expressed in maternal tissues do not function as sucrose/H + symporters. Membrane exchange from nucellar projection transfer cells to the endosperm cavity occurs by an as yet unresolved mechanism. Sucrose uptake from the endosperm cavity into filial tissues is mediated by a SUT localised to plasma membranes of the modified aleurone/sub-aleurone transfer cells.

Biogenesis and function of the yeast plasma-membrane H(+)-ATPase

2000 ◽  
Vol 203 (1) ◽  
pp. 155-160 ◽  
Author(s):  
A. Ambesi ◽  
M. Miranda ◽  
V.V. Petrov ◽  
C.W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Animal Cells ◽  
Alpha Helices ◽  
Yeast Plasma Membrane ◽  
High Affinity ◽  
And Function ◽  
P Type

One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

Direct Observation of Heat Exchanger Deposits by Cross Sectioning

1967 ◽  
Vol 25 ◽  
pp. 364-365
Author(s):  
R. W. Anderson ◽  
D. L. Senecal
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Heat Exchanger ◽  
Direct Observation ◽  
Cross Sections ◽  
Preparation Method ◽  
Specimen Preparation ◽  
Flowing Water ◽  
Transmission Electron ◽  
Deposit Layer

A problem was presented to observe the packing densities of deposits of sub-micron corrosion product particles. The deposits were 5-100 mils thick and had formed on the inside surfaces of 3/8 inch diameter Zircaloy-2 heat exchanger tubes. The particles were iron oxides deposited from flowing water and consequently were only weakly bonded. Particular care was required during handling to preserve the original formations of the deposits. The specimen preparation method described below allowed direct observation of cross sections of the deposit layers by transmission electron microscopy.The specimens were short sections of the tubes (about 3 inches long) that were carefully cut from the systems. The insides of the tube sections were first coated with a thin layer of a fluid epoxy resin by dipping. This coating served to impregnate the deposit layer as well as to protect the layer if subsequent handling were required.

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