scholarly journals Conserved Asp684in Transmembrane Segment M6 of the Plant Plasma Membrane P-type Proton Pump AHA2 Is a Molecular Determinant of Proton Translocation

2003 ◽  
Vol 278 (20) ◽  
pp. 17845-17851 ◽  
Author(s):  
Morten J. Buch-Pedersen ◽  
Michael G. Palmgren
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
Proton Translocation ◽  
Transmembrane Segment ◽  
Molecular Determinant ◽  
P Type ◽  
Plant Plasma Membrane

scholarly journals A Systematic Mutagenesis Study of Ile-282 in Transmembrane Segment M4 of the Plasma Membrane H+-ATPase

2005 ◽  
Vol 280 (23) ◽  
pp. 21785-21790 ◽  
Author(s):  
Å. Staffan Fraysse ◽  
Anders L. B. Møller ◽  
Lisbeth R. Poulsen ◽  
Bernd Wollenweber ◽  
Morten J. Buch-Pedersen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proton Translocation ◽  
Carbonyl Oxygen ◽  
Saturation Mutagenesis ◽  
Proton Pumps ◽  
Side Chains ◽  
Transmembrane Segment ◽  
Alanine Scanning Mutagenesis ◽  
P Type

Homology models of plasma membrane H+-ATPase (Bukrinsky, J. T., Buch-Pedersen, M. J., Larsen, S., and Palmgren, M. G. (2001) FEBS Lett. 494, 6–10) has pointed to residues in transmembrane segment M4 as being important for proton translocation by P-type proton pumps. To test this model, alanine-scanning mutagenesis was carried out through 12 residues in the M4 of the plant plasma membrane H+-ATPase AHA2. An I282A mutation showed apparent reduced H+ affinity, and this residue was subsequently substituted with all other naturally occurring amino acids by saturation mutagenesis. The ability of mutant enzymes to substitute for the yeast proton pump PMA1 was found to correlate with the size of the side chain rather than its chemical nature. Thus, smaller side chains (Gly, Ala, and Ser) at this position resulted in lower H+ affinity and lowered levels of H+ transport in vivo, whereas substitution with side chains of similar and larger size resulted in only minor effects. Substitutions of Ile-282 had only minor effects on ATP affinity and sensitivity toward vanadate, ruling out an indirect effect through changes in the enzyme conformational equilibrium. These results are consistent with a model in which the backbone carbonyl oxygen of Ile-282 contributes directly to proton translocation.

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.

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