Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine.

The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin.

Carboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and in mixed DMPC/CHAPS/SDS micelles at neutral pH, thereby affecting the opsin shift and the photocycle of the regenerated chromophore. On the contrary, after solubilization with SDS, aO-1 and bO were modified by DCCD and NCD-4, which decreased the chromophore regeneration. In particular, the reaction of aO-1 in SDS with NCD-4 proceeded in a 1:1 ratio at neutral pH. The fluorescence and CD spectra indicated that the modified site was located in the hydrophobic, asymmetrical region. Lysyl-endopeptidase digestion of NCD-4 modified aO-1 produced a fluorescent fragment and amino acid sequence analysis showed that Asp85 or Asp96 in helix C is a probable candidate for the modified residue at present. Kinetic CD measurements revealed that the introduction of N-acylurea at an Asp residue in helix C did not affect the formation of the transient intermediate but inhibited the side chain packing during refolding.

Halobacterial rhodopsins.

Following the discovery of the bacteriorhodopsin proton pump in Halobacterium halobium (salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteriorhodopsin).

Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation.

The amino acid sequences of 25 archaeal retinal proteins from 13 different strains of extreme halophiles were analyzed to establish their molecular phylogenetic relationship. On the basis of amino acid sequence similarity, these proteins apparently formed a distinct family designated as the archaeal rhodopsin family (ARF), which was not related to other known proteins, including G protein-coupled receptors. The archaeal rhodopsin family was further divided into four clusters with different functions; H+ pump (bacteriorhodopsin), Cl- pump (halorhodopsin), and two kinds of sensor (sensory rhodopsin and phoborhodopsin). These four rhodopsin clusters seemed to have occurred by gene duplication(s) before the generic speciation of halophilic archaea, based on phylogenetic analysis. Therefore, the degrees of differences in amino acid sequences within each cluster simply reflected the divergent evolution of halophilic archaea. By comparing the branch lengths after speciation points of the reconstituted tree, we calculated the relative evolution rates of the four archaeal rhodopsins bacteriorhodopsin:halorhodopsin:sensory rhodopsin: phoborhodopsin to be 5:4:3:10. From these values, the degrees of functional and structural restriction of each protein can be inferred. The branching topology of four clusters grouped bacteriorhodopsin and halorhodopsin versus sensory rhodopsin and phoborhodopsin by likelihood mapping. Using bacteriorhodopsin (and halorhodopsin) as an outgroup, the gene duplication point of sensory rhodopsin/phoborhodopsin was determined. By calculating the branch lengths between the gene duplication point and each halophilic archaea speciation point, we could speculate upon the relative evolution rate of pre-sensory rhodopsin and pre-phoborhodopsin. The evolution rate of pre-sensory rhodopsin was fivefold faster than that of pre-phoborhodopsin, which suggests that the original function of the ancestral sensor was similar to that of phoborhodopsin, and that sensory rhodopsin evolved from pre-sensory rhodopsin by the accumulation of mutations. The changes in evolution rate by gene duplication and functional differentiation were demonstrated in the archaeal rhodopsin family using the gene duplication date and halobacterial speciation date as common time stamps.

An insertion or deletion in the extramembrane loop connecting helices E and F of archaerhodopsin-1 affects in vitro refolding and slows the photocycle.

Upon addition of retinal, archaeopsin-1 expressed in Escherichia coli (ecaO-1002) regenerated the chromophore in dimyristoyl phosphatidylcholine (DMPC), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) mixed micelles as efficiently as the same opsin prepared from halobacteria. Introduction of an insertion or a deletion of five amino acids into the surface loop connecting helices E and F changed the secondary and tertiary structures of ecaO-1002 in SDS, and diminished regeneration of the chromophore. The effect of the insertion and deletion on the in vitro refolding was specific to archaeopsin because the same insertion introduced at the corresponding position of bacterioopsin (bO) did not affect chromophore regeneration. The photocycle of the regenerated ecaR-1002 decreased in DMPC/CHAPS/SDS mixed micelles compared with that of aR-1 in the claret membrane, which was consistent with the reported behavior of bO. Unexpectedly, the insertion and deletion in loop EF perturbed the photocycle of the regenerated ecaR-1002. The accumulation of long-lived N- and O-like intermediates suggested that the insertion and deletion slowed down the proton uptake steps at the cytoplasmic surface.

Identification of proteolipid from an extremely halophilic archaeon Halobacterium salinarum as an N,N'-dicyclohexyl-carbodiimide binding subunit of ATP synthase.

ATP synthesis in an extremely halophilic archaeon, Halobacterium salinarum, was inhibited by N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), a fluorescent analog of N,N'-dicyclohexylcarbodiimide (DCCD). By tracing the fluorescent signal, a hydrophobic 8-kDa protein (proteolipid) was purified from the halobacterial membrane as one of the most DCCD-reactive proteins and its N-terminal amino acid sequence was determined. The gene encoding the proteolipid was found in the region upstream of the genes encoding the two major subunits of halobacterial A-type ATPase [K. Ihara and Y.Mukohata (1991) Arch. Biochem. Biophys. 286, 111-116]. Halobacterial proteolipid was more similar in size to the proteolipid of F-type ATPase than that of V-type ATPase. However, multiple amino acid sequence alignment of proteolipids showed a higher degree of relatedness between V-type and A-type ATPase proteolipids. Together with the recent finding of a triplicate proteolipid encoding gene from the methanogenic archaeon Methanococcus jannaschii [C. J. Bult et al. (1996) Science 273, 1058-1073], proteolipids from archaea seem to have diverse characteristics in comparison with those from eubacteria or from eukaryotes.

Dual roles of DMPC and CHAPS in the refolding of bacterial opsins in vitro.

The bacterial opsins can be refolded to regenerate the chromophore by transfer from SDS to DMPC/CHAPS/SDS mixed micelles in the presence of retinal. A sequential refolding model has been proposed for bacterioopsin [Booth et al. (1995) Nature Struct. Biol. 2, 139-143]. However, the roles of DMPC and CHAPS in the refolding process are not clear. In this study we measured the effects of DMPC and CHAPS on the refolding of bacterial opsins in vitro by CD and fluorescence spectroscopy. In contrast to in experiments in the presence of large amounts of DMPC, the process of retinal binding pocket formation was a rate-determining step in overall chromophore regeneration with relatively low concentrations of DMPC. CHAPS triggered alpha-helix formation and long-range interactions between the helices within 1 s by providing a suitable hydrophobic environment for bacterial opsins. This CHAPS-induced transient molten globule-like structure would be identical to I1 postulated by Booth et al., to which DMPC bound and induced the proper packing of the side chains to form a retinal binding pocket. If DMPC was not present, CHAPS induced another conformation change in bacterial opsins, which led to denaturation. DMPC dependence of chromophore regeneration and the maintenance of the retinal binding pocket suggested that retinal binding pocket formation was part of the large structure changes during stable apoprotein formation.

Novel bacterial rhodopsins from Haloarcula vallismortis.

New bacterial rhodopsins of the cruxrhodopsin (cR) tribe were identified in a type strain Haloarcula vallismortis. The genes encoding a bacteriorhodopsin-like ion pump (named cR-3), a halorhodopsin-like ion pump (chR-3) and a sensor rhodopsin (csR-3) were cloned and sequenced. Together with the data for vsRII (Seidel et al., Proc. Natl. Acad. Sci. USA 92, 3036-3040 (1995); cpR-3 in our notation), the primary structures of a set of four rhodopsins are now all known only in this species. They are separated by almost the same distances in homology, suggesting that they have derived from a single ancestral rhodopsin. The degree of conservation in the amino acid sequence of each helix showed that helices C and G are relatively well conserved in all rhodopsins, whereas helices DEF are conserved especially in sensor rhodopsin-I, possibly because these helices are needed for interaction with the transducer protein (Htr).

The light-driven proton pump, cruxrhodopsin-2 in Haloarcula sp. arg-2 (bR+, hR-), and its coupled ATP formation.

Haloarcula sp. arg-2, a natural bacterial isolate from Andes heights, has a light-driven proton pump but not a light-driven anion pump. We have cloned and sequenced the gene encoding for the proton pump which has been named cruxrhodopsin-2. The gene consists of 768 bp encoding 255 amino acids with a molecular mass of 27,544 Da. The deduced amino acid sequence of cruxrhodopsin-2 is 77%, 50%, 48% and 48% identical to those of cruxrhodopsin-1, bacteriorhodopsin, archaerhodopsin-1 and archaerhodopsin-2, respectively. The charged amino acids important for the proton pump function were conserved among all these molecules. Cruxrhodopsin-2 accounted for 0.05 nmol/mg protein in arg-2, which was 20-30-fold less than the proportion of bacteriorhodopsin in Halobacterium salinarium R1M1. In contrast to R1M1, under anaerobic conditions, arg-2 showed light-induced proton extrusion concomitant with an increase in ATP level without transient proton uptake. Dicyclohexylcarbodiimide enhanced the rate and extent of proton extrusion and inhibited ATP formation in the light. The apparent stoichiometry of H+/ATP was estimated to be more than three in this natural bR+hR- strain.

The novel ion pump rhodopsins from Haloarcula form a family independent from both the bacteriorhodopsin and archaerhodopsin families/tribes.

Extreme halophiles newly collected from Argentine salt flats were characterized, in one of which, Haloarcula (sp. arg-1), light-driven retinal protein ion pumps were found. The proton pump, cruxrhodopsin-1, shows amino acid sequence homologies of 52% to bacteriorhodopsin and 48% to archaerhodopsin-1. The anion pump, cruxhalorhodopsin-1, identified partially as a 394bp polymerase chain reaction product, shows homologies of 70% to halorhodopsin, and 72% to pharaonis halorhodopsin. The ion pumps (and possibly sensors still to be found) in Haloarcula sp. arg-1, which constitute the cruxrhodopsin-1 family, are distinct from the bacteriorhodopsin and the archaerhodopsin families/tribes.

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.

pH dependence of the absorption spectra and photochemical transformations of the archaerhodopsins.

Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium. These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCl, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.

Comparative studies on ion pumps of the bacterial rhodopsin family.

Bacteriorhodopsin (proton pump), halorhodopsin (anion pump), sensory rhodopsin and phoborhodopsin (photosensors) are found in Halobacterium salinarium (halobium). In some other strains, other sets of rhodopsin pumps and sensors have been found. Here, these bacterial rhodopsins are classified according to their amino acid sequence homologies, and their host genera are assigned on the basis of 16S rRNA sequence comparison. Haloarcula is the host for cruxrhodopsins and a new genus (temporarily "Halorubra") is the host for archaerhodopsins. Difference in the all-trans:13-cis ratios of retinal in two proton pumps (bacteriorhodopsin and archaerhodopsin-2) at equilibrium states in the dark was ascribed to only one amino acid residue in the retinal pocket. This predicted methionine-145 in bacteriorhodopsin was point-mutated to phenylalanine as in archaerhodopsin-2. The mutated bacteriorhodopsin (M145F) became to show the same dark-adapted isomer ratio that archaerhodopsin-2 shows. Chimeric proton pumps were made by exchanging genes of one or more helix regions of two similar pumps (archaerhodopsin-1 and -2) in order to know structural delicacy of the inter-helix space. Preliminary results show that some photochemical properties depend on one helix or one distinct amino acid residue on the helix. Such new lines initiated by our archaerhodopsins are discussed for studying structure and function of these unique bacterial rhodopsins.

Archae-opsin expressed in Escherichia coli and its conversion to purple pigment in vitro.

Archae-opsin-1 (aO-1) has been expressed efficiently as a fusion protein with 13 heterologous amino acids at the amino terminus of the mature aO-1 in Escherichia coli under the control of T7 promoter. The E. coli-expressed aO-1, designated as aO-1002, which was located in the membrane fraction, was extracted with 8 M urea and partially purified by gel filtration chromatography in the presence of SDS. When all-trans retinal was added, aO-1002 in dimyristoylphosphatidylcholine and detergent-mixed micelles was converted to a purple pigment with lambda max at 558 nm at 20 degrees C via a 435/460 nm intermediate. Conversion of the intermediate to purple pigment was the rate-limiting step and proceeded as a two-state transition, because an isosbestic point was seen at 485 nm. Similar spectral changes were also observed in the regeneration process of hydroxylamine-bleached claret membranes and aO-1 isolated from claret membranes. Thus, the polypeptide of aO-1002 is considered to fold and form a retinal binding pocket in phospholipid and detergent micelles similarly to aO-1 isolated from the halobacterial membranes. Purple pigment showed a light-driven proton-pumping activity when reconstituted into phosphatidylcholine liposomes.

Photophosphorylation elements in halobacteria: an A-type ATP synthase and bacterial rhodopsins.

Photophosphorylation in halobacteria is carried out by two rather simple elements: an A-type ATP synthase and light-driven ion-pumping bacterial rhodopsins. The unique features of halobacterial ATP synthase, mostly common to archaebacteria (A-type), and of new members of the bacteriorhodopsin family are introduced along with studies performed in the authors' laboratory. This is the story of how we found that the A-type ATP synthase is close to V-type ATPase but far from F-type ATPase, although all three ATPases are believed to have the same ancestor. Archaerhodopsins, the new members of the proton-pumping retinal proteins, were found in Australian halobacteria and have been used in a comparative study of bacterial rhodopsins.

HALOBACTERIAL A-ATP SYNTHASE IN RELATION TO V-ATPase.

The head piece separated from the A-ATP synthase of Halobacterium halobium hydrolyses ATP. This A1-ATPase is inhibited by nitrate but not by other chaotropic anions. The nitrate inhibition is noncompetitive with respect to ATP, reversible, and partially protected by chloride. In contrast, ATP synthase in situ (A1Ao-ATPase) is not inhibited by nitrate but apparently is inhibited by stronger chaotropic reagents, such as thiocyanate and trichloroacetate, which make the vesicle membrane permeable to protons. The mode of action of nitrate and chaotropic anions seems to differentiate A-ATPases from V-ATPases. Other strains of Halobacterium, Haloferax, Haloarcula, Halococcus and Natronobacterium, contain at least two polypeptides immunochemically similar to the two major subunits, (&agr;) (86x10(3 )Mr on SDS-PAGE) and &bgr; (64x10(3 )Mr), of the A-ATPase of Halobacterium halobium. When solubilized, membrane vesicles of these halobacteria hydrolyse ATP. Their ATPases are commonly sensitive to nitrate. They require high concentrations of the supporting salt but depend differently on chloride or sulfate/sulfite. The A-ATPases of Halobacteriaceae appear to diverge with respect to salt preference.

Australian Halobacteria and their retinal-protein ion pumps.

Halophiles collected in Western Australia have been found to be examples of extremely halophilic rod-shaped archaebacteria, members of the genus Halobacterium. Most of them contain retinal proteins, and these proteins differ from one another and also from both bacteriorhodopsin (bR) and halorhodopsin [and sensory rhodopsins (sR)] isolated from Halobacterium salinarium (halobium), as revealed by their peptide maps and amino acid sequences. However, these retinal proteins still have the ability to pump protons or chloride ions in the light. These new ion pumps, designated archaerhodopsins (aR) [Mukohata et al. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345], are almost identical in terms of their molecular sizes and transient photochemical properties to the ion pumps identified previously. Differences are found in the: (1) apparent extinction coefficient of dark/light-adapted aR-2; (2) titration profiles at acidic pH of the absorption spectra of all aRs; and (3) circular dichroism spectra, which are influenced by the coexistent isoprenoid bacterioruberin. The amino acid sequences of two proton pumps from the Australian halobacteria, namely aR and aR-2, are approximately 90% homologous and both sequences are about 60% homologous with that of bR. Hydropathy plots suggest that these pumps also have a seven-helical structure similar to that of bR. The amino acid residues are highly conserved in the helical regions, in particular in the case of helices C and G (91 and 84%, respectively), among the three proton pumps.

Expression of Maize Ferredoxin cDNA in Escherichia coli: Comparison of Photosynthetic and Nonphotosynthetic Ferredoxin Isoproteins and their Chimeric Molecule.

Maize (Zea mays L.) has two types of ferredoxin (Fd) differentially expressed in photosynthetic and nonphotosynthetic organs. A cDNA fragment encoding the mature polypeptide of Fd III, an Fd isoprotein of the nonphotosynthetic type, was expressed in Escherichia coli, and the Fd was synthesized as a holo-form assembled with the [2Fe-2S] cluster, which was completely identical with authentic Fd III prepared from maize roots. This expression system made it possible to prepare Fd present at fairly low levels in plants in amounts sufficient for functional and structural studies. Comparison of electron transfer activity of Fd III with that of Fd I, an Fd isoprotein of the photosynthetic type, showed that Fd III was superior as an electron acceptor from NADPH, and Fd I was superior as an electron donor for NADP(+), in reactions catalyzed by Fd-NADP(+) reductase from maize leaf. The circular dichronism spectra of the two Fds also indicated a subtle difference in the geometry of their iron-sulfur clusters. These results are consistent with the view that photosynthetic and nonphotosynthetic Fds may be functionally differentiated. An artificial chimeric Fd, Fd III/Fd I, whose amino-terminal and carboxylterminal halves are derived from the corresponding regions of Fd III and Fd I, respectively, showed an activity and CD spectrum significantly similar to those of Fd I. This suggests that 18 amino acid substitutions between Fd III and Fd III/Fd I alter the properties of Fd III so that they resemble those of Fd I.

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.