Studies on sodium and hydrogen ion translocation through the F0 part of the sodium-translocating F1F0 ATPase from Propionigenium modestum: discovery of a membrane potential dependent step

Abstract

The purified ATPase of Propionigenium modestum (F1Fo) was incorporated into liposomes, and the F1 part was dissociated. The Fo-liposomes catalyzed proton uptake in response to a potassium diffusion potential (inside negative). Proton translocation was abolished by rebinding F1 to the Fo-liposomes or after incubation with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD). Proton uptake was also sensitive to the presence of external Na+ or Li+ ions and was completely abolished at 2 mM NaCl or 150 mM LiCl, respectively. However, the same concentrations of these salts in the internal volume of the Fo-liposomes were without effect, suggesting that the cation binding site is not accessible from both sides of the membrane simultaneously. An open channel-type of transport through Fo from P. modestum is therefore excluded. The Fo-liposomes also catalyzed Na+ influx or efflux in response to a K+ diffusion potential that was negative on the inside or outside, respectively. These Na+ fluxes could not be created, however, by delta pNa+ of about 60-180 mV. The initial rate of Na+ uptake depended strongly on the size of the membrane potential with no significant conductivity below -40 mV, followed by a proportional increase up to about -115 mV. In the absence of a membrane potential, the Fo-liposomes catalyzed 22Na+ counterflow against a 28-fold concentration gradient. Uptake of 22Na+ into Fo-liposomes against delta pNa+ (counterflow) was completely prevented by imposing an inside-positive potassium diffusion potential of 90 mV. The catalysis of 22Na+ counterflow by Fo from P. modestum is a clear indication of a carrier (transporter)-type mechanism and excludes a channel mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane Topography of the Coupling Ion Binding Site in Na+-translocating F1F0 ATP Synthase

“…The third 80-l portion was added to 650 l of 100 mM Tricine/ KOH buffer, pH 8.9, 21 l of Triton X-100 (20%), 5 mM CaCl 2 , and 14.5 units of phospholipase A 2 and incubated at room temperature overnight. Na ϩ Exchange Experiments-The Na ϩ exchange measurements were performed as described previously (37).…”

Phylogenetic analyses of the homologous transmembrane channel-forming proteins of the F0F1-ATPases of bacteria, chloroplasts and mitochondria

“…F-type ATPases are so efficient that they can turn over an organism's body weight in ATP several times in a single day (Pedersen & Amzel, 1993). Some bacterial ATPases can transport Na+ instead of or in addition to H' , but the process appears to be mechanistically the same (Laubinger & Dimroth, 1988;Gogarten e t al., 1989a, b ;Kluge & Dimroth, 1992;1993 ;Dmitriev e t al., 1993 Phylogenetic analyses of F,F,-ATPases ~ ~~ *The abbreviation used refers to the genus (first upper-case letter) and species (second two lower-case letters) followed by an upper-case letter as follows: B, bacterial protein; C, chloroplast protein; M, mitochondrially encoded mitochondrial protein; N, nuclearly encoded mitochondrial protein ; A, archaeobacterial protein; V, vacuolar-type protein; ?, protein of unknown function; a ' 1 ' or '2' following the source designation is included either when two such proteins are found within a single species (bacterial or mitochondrial proteins) or when the protein has two homologous repeat sequences, each of which is separately analysed (vacuolar-type). Abbreviations are indicated only for those proteins that were included in this study.…”

Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum

“…It was drastically inactivated by DCCD (Fig. 4A), to which several ion transport ATPases are sensitive (Kaestner et al 1988;Kluge and Dimroth 1992;Spruth et al 1995). The purified enzyme is an integral membrane protein and the activity was essentially the same irrespective of the presence of Na + , Li + , K + or Rb + .…”

“…To measure inhibition by sodium vanadate and sodium azide, the ATPase reactions were conducted in 20 mM PIPES buffer (pH 6.5) containing 200 mM KCl, 3 mM MgCl 2 and 2 mM ATP in the presence of each chemical without pretreatment. The activity is expressed as the ratio of each sample compared to the control under each condition The ATPase was drastically inactivated by N,N'-dicyclohexylcarbodiimide (DCCD), 2,4,6-trinitrobenzenesulfonic acid (TNBS) and diethyl pyrocarbonate (DEPC), modifiers of the carboxyl group, amino group and histidine residue, respectively (Haynes et al 1967;Kaestner et al 1988;Wakabayashi et al 1988;Kluge and Dimroth 1992;Spruth et al 1995;Okano et al 1998). Half-maximal inhibition by DCCD occurred at approximately 2 µM (data not shown).…”