Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membranespanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membraneanchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membranespanning proton transporters.diffusion ͉ fluorescence ͉ membrane protein ͉ pH ͉ proton transfer E nergy metabolism in living organisms involves translocation of protons across biological membranes through specific membrane-spanning proton transporters, thereby generating a proton-motive force (⌬p)where ⌬ is the electrical potential, R is the gas constant (J⅐K Ϫ1 ⅐mol Ϫ1 ), T is the absolute temperature (K), F is the Faraday constant (C mol Ϫ1 ), and ⌬pH is the pH difference between the two bulk solutions on either side of the membrane. The proton-motive force is used, e.g., by ATP synthase to produce ATP. It originally was proposed that ⌬p only depends on the solution properties and that the membrane only acts as a passive barrier isolating the two compartments (1). Over the years, evidence has accumulated indicating that the scenario is more complicated and also that the membrane surface and the water layer near the surface play major roles in the process (for a recent review, see ref.2). One important finding in this respect is the alkaliphilic bacteria, which live in environments having 2-3 units higher pH than that of the bacterial cytosol. Assuming a passive role of the membrane, these bacteria would in principle not be able to maintain a ⌬p large enough to account for their ATP production (3). To resolve this apparent paradox, the idea of a localized proton circuit along the membrane surface, originally proposed by Williams (4), has been exploited (2, 5-7) (see also ref. 8 for a theoretical analysis as well as refs. 9 and 10). According to this idea, protons ejected by a membrane-spanning proton transporter are transferred along the membrane to the proton acceptor (e.g., ATP synthase) faster than they are released to the bulk solution. Most likely, this process is facilitated by surface-b...

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of sitedirected mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the ''peroxy'' state, P r) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH Ϸ 7.5. The movement of the Lys is proposed to regulate proton transfer by ''shutting off'' the protonic connectivity through the K-pathway after initiation of the O 2 reduction chemistry. This ''shutoff'' prevents a short-circuit of the protonpumping machinery of the enzyme during the subsequent reaction steps.flow-flash ͉ proton pumping ͉ cytochrome aa3 ͉ flash photolysis ͉ gating ͉ R. sphaeroides

Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.