scholarly journals Photosensitive phosphoproteins in Halobacteria: regulatory coupling of transmembrane proton flux and protein dephosphorylation.

1981 ◽  
Vol 91 (3) ◽  
pp. 895-900 ◽  
Author(s):  
E N Spudich ◽  
J L Spudich
Keyword(s):  
Protein Phosphorylation ◽  
Action Spectrum ◽  
Atp Synthesis ◽  
Energy Coupling ◽  
Proton Flux ◽  
Protonmotive Force ◽  
Proton Efflux ◽  
Ph Gradients ◽  
Protein Dephosphorylation ◽  
Reversible Protein Phosphorylation

A photoregulated reversible protein phosphorylation system controlled by the halobacterial rhodopsins was recently reported. The results presented in this paper identify the initial steps in the pathway from the absorption of light to the photoregulated protein phosphorylation and dephosphorylation reactions. Action spectrum, biochemical, and genetic analyses show that the proton pump bacteriorhodopsin mediates light-induced dephosphorylation of three photoregulated phosphoproteins. Light absorbed by bacteriorhodopsin is used to establish a proton efflux from the cells. The increase in the inwardly directed protonmotive force (pmf) from this efflux induces dephosphorylation of the three phosphoproteins, as demonstrated by the effects of the protonophore CCCP and of artificially imposed transmembrane pH gradients. Upon darkening the cells, cessation of the proton efflux through bacteriorhodopsin causes a decrease in pmf, which induces rephosphorylation of the proteins. Pmf appears to function as a regulator rather than a driving force in this system. Measurements of pmf-driven ATP synthesis in our conditions indicate the regulation of protein phosphorylation by pmf is probably not a consequence of proton flux through the H+ ATPase, a known energy coupling structure in these cells. The properties of this system may indicate the existence of a pmf detector which regulates kinase or phosphatase activity; i.e., a regulatory coupling device.

scholarly journals The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1978 ◽  
Vol 174 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Douglas B. Kell ◽  
Philip John ◽  
Stuart J. Ferguson
Keyword(s):  
Membrane Potential ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Energy Coupling ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Electron Mediator ◽  
Succinate Oxidation ◽  
Phosphorylation Potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S14CN−, and the transmembrane pH gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN′N′-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (ΔGp=ΔG0′+RTln[ATP]/ [ADP][Pi]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO3 the protonmotive force was not detectable (<60mV) but ΔGp was not altered. This result may indicate either that there is no relationship between the protonmotive force and ΔGp, or that for an unidentified reason the equilibration of SCN− or methylamine with the membrane potential and the pH gradient is prevented by NO3− in this system.

scholarly journals Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

2009 ◽  
Vol 8 (7) ◽  
pp. 922-932 ◽  
Author(s):  
Jens Boesger ◽  
Volker Wagner ◽  
Wolfram Weisheit ◽  
Maria Mittag
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Protein Phosphorylation ◽  
Proteome Analysis ◽  
Physiological Status ◽  
Phosphorylation Sites ◽  
Cell Organelles ◽  
Flagellar Proteins ◽  
Reversible Protein Phosphorylation ◽  
Cilia And Flagella

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

Molecular Features of Energy Coupling in the F0F1 ATP Synthase

Physiology ◽  
1999 ◽  
Vol 14 (1) ◽  
pp. 40-46
Author(s):  
Robert K. Nakamoto
Keyword(s):  
Atp Synthase ◽  
Catalytic Domain ◽  
Atp Synthesis ◽  
Energy Coupling ◽  
F0f1 Atp Synthase ◽  
Molecular Features ◽  
Rotary Mechanism ◽  
Transport Domain

H+ translocation is coupled to ATP synthesis in the F0F1 ATP synthase via a rotary mechanism. Catalytic turnover, site-site cooperativity, and H+ transport obligatorily involve rotation of a set of subunits. The transport domain in the membranous F0 and the catalytic domain in the F1 are mechanisms designed for generating torque.

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