Yasuo Mukohata

Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation

The light-induced changes in pH and ATP level were compared for cell suspensions between strains of Halobacterium halobium differing in pigmentation after growth under the same conditions. Upon illumination, red cells which contained no detectable amount of bacteriorhodopsin showed only a pH increase, which, in the case of purple cells containing bacteriorhodopsin, was followed by a spontaneous pH decrease during illumination. Pre-incubation of cells at 75° for 5 min depressed the pH increase in both cells. Pre-illumination of cells with hydroxylamine depressed the pH decrease in purple cells. Whenever the pH increase was observed, the cellular ATP level increased. The presence of a bacteriorhodopsin different from that in the purple membrane is postulated.

The γ-subunit of ATP synthase from spinach chloroplasts Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the γ‐subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast γ‐subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl‐terminal region of the choloroplast γ‐subunit is highly homologous with those of the γ‐subunits from six other sources (bacteria and mitochondria) sequenced thus far.

ATP synthesis linked to light-dependent proton uptake in a red mutant strain of Halobacterium lacking bacteriorhodopsin

Intact cells of Halobacterium halobium R1mR, a red strain deficient in bacteriorhodopsin, pumped protons only in an inward direction when illuminated, in contrast to R1 cells which showed proton transfer in both directions. The cellular ATP level of R1mR, as well as R1 cells, under nitrogen, was increased upon illumination to the aerobic level. The proton uptake and ATP synthesis observed with both R1 and R1mR occurred even after the majority of bacteriorhodopsin (in R1) had been bleached with NH2OH, but they were abolished by brief heat treatment of the cells. These results suggest a mechanism common to both strains which is responsible for the observed proton uptake and ATP synthesis. When R1mR cells were grown in the presence of nicotine, an inhibitor of carotenoid biosynthesis, both the proton uptake and ATP synthesis were completely depressed, but recovered after all-trans retinal was added externally. The action spectrum by R1mR, NH2OH-treated R1, or nicotinegrown R1mR reconstituted with retinal consistently exhibited a maximum between 580 and 600 nm. Hence the mechanism is independent of bacteriorhodopsin of the purple membrane. Instead, our results indicate the possible presence of a new chromoprotein which involves retinal as the chromophore and participates in the mechanism of cellular ATP synthesis.

Light-induced membrane-potential increase, ATP synthesis, and proton uptake in Halobacterium halobium R1mR catalyzed by halorhodopsin: Effects of N,N′-dicyclohexylcarbodiimide, triphenyltin chloride, and 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847)

Abstract The effects of N,N′ -dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di- tert -butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R 1 mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH + ). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH + and partly Δψ. After R 1 mR was treated with DCCD, TPT abolished ΔH + almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R 1 mR, ATP is synthesized by a H + -ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H + -ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.

AUSTRALIAN Halobacteria and THEIR RETINAL‐PROTEIN ION PUMPS*

Abstract– 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.

Archaerhodopsin-2, from Halobacterium sp. aus-2 further reveals essential amino acid residues for light-driven proton pumps☆

We have isolated a retinal protein which differs from bacteriorhodopsin and archaerhodopsin and pumps out as many protons in the light as those proton pumps. We tentatively named it archaerhodopsin-2. We have cloned and sequenced the gene that encodes archaerhodopsin-2. The gene consists of 780-bp nucleotides for 259 amino acids with a molecular mass of 27,937 Da. The amino acid sequence of archaerhodopsin-2 is 56% identical to bacteriorhodopsin and 88% to archaerhodopsin, with a few gaps of a few amino acids in both cases. Although the amino acid sequence of archaerhodopsin has revealed 157 conserved residues common to bacteriorhodopsin, the sequence of archaerhodopsin-2 reduces that number to 133. Of these, 38 amino acids are also common to chloride pumps and 24 to all bacterial retinal proteins known to date.

The halobacterial H+-translocating ATP synthase relates to the eukaryotic anion-sensitive H+-ATPase.

The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102, 797-802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102, 591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vulgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca2+-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.

Modification of one lysine by pyridoxal phosphate completely inactivates chloroplast coupling factor 1 ATPase.

Chemical modification of chloroplast coupling factor 1 (CF,) in situ caused energy-transfer inhibition [l-3] of photophosphorylation and revealed essential arginyl [ 1,4] and lysyl [2,3] residues. When CF 1 in situ was modified by pyridoxal phosphate (PLP) [3] , the a, fl and y subunits were equally labeled and phosphorylation was inhibited. For 50% inhibition, 1 mol PLP was found in each of these 3 subunit fractions per mol CF 1 _ Since CFr has an allotopic nature [5] , it was of interest to examine PLP modification on isolated CFr , which was thus isolated from thylakoids, modified by PLP, then assayed for the heat-activated Ca”-ATPa.se [6] . It was found that modification of only 1 lysyl residue/CFr inactivated the Ca*‘-ATPase completely. For 50% inhibition, 0.5 mol PLP was found in each of the (Y and /3 subunit fractions per mol CFr , but none in the y subunit fraction.

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.

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.