Atsuko Iwamoto-Kihara

Subunit Rotation of Vacuolar-type Proton Pumping ATPase RELATIVE ROTATION OF THE G AND c SUBUNITS

Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy

The structure of Escherichia coli F0F1‐ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping‐mode operation. The majority of F0F1‐ATPases were visualized as spheres with a calculated diameter of , and a height of from the membrane surface. F0 sectors were visualized as two different ring‐like structures (one with a central mass and the other with a central hollow of depth) with a calculated outer diameter of . The two different images possibly represent the opposite orientations of the complex in the membranes. The ring‐like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

ATP synthase FoF1 (α3β3γδɛab2c10–14) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F1 β or membrane sector Fo a subunit when the Fo c subunit ring was immobilized; a biotin-tag was introduced onto the β or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the β or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the α or β subunit. These results established that the γɛc10–14 and α3β3δab2 complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis.

Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector STOCHASTIC FLUCTUATION AND A KEY DOMAIN OF THE β SUBUNIT

A complex of γ, ϵ, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the γ subunit of the Escherichia coli F1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the β subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (βS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120° positions during the stepped revolution. The pause positions were probably not at the “ATP waiting” dwell but at the “ATP hydrolysis/product release” dwell, since the ATP concentration used for the assay was ∼30-fold higher than the Km value for ATP. A βGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of βS174F. The revertant (βG149A/βS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant βG149A/βS174L. These results indicate that the domain between β-sheet 4 (βSer-174) and P-loop (βGly-149) is important to drive rotation.

Halotolerant Cyanobacterium Aphanothece halophytica Contains an Na+-dependent F1F0-ATP Synthase with a Potential Role in Salt-stress Tolerance

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H+-ATP synthase, Aphanothece halophytica contains a putative F1F0-type Na+-ATP synthase (ApNa+-ATPase) operon (ApNa+-atp). The operon consists of nine genes organized in the order of putative subunits β, ϵ, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa+-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa+-ATPase exhibited Na+-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na+ ion protected the inhibition of ApNa+-ATPase by N,N′-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na+-loaded inverted membrane vesicles. Expression of the ApNa+-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na+-dependent F1F0-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.

A unique mechanism of curcumin inhibition on F1 ATPase

ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F1 sector. Previously, we found that F1 ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F1 ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F1 and a mutant lacking part of the piceatannol binding site (γΔ277-286) indicates that curcumin binds to F1 differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F1 mutants with defective β-γ subunit interactions (γMet23 to Lys) or β conformational changes (βSer174 to Phe). These results confirm that smooth interaction between each β subunit and entire γ subunit in F1 is pertinent for rotational catalysis.

ATP-dependent rotation of mutant ATP synthases defective in proton transport

During ATP hydrolysis, the γϵc10 complex (γ and ϵ subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the α3β3δab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.

A unique F-type H⁺-ATPase from Streptococcus mutans: an active H⁺ pump at acidic pH.

We have shown previously that the Streptococcus mutans F-type H(+)-ATPase (F(O)F(1)) c subunit gene could complement Escherichia coli defective in the corresponding gene, particularly at acidic pH (Araki et al., (2013) [14]). In this study, the entire S. mutans F(O)F(1) was functionally assembled in the E. coli plasma membrane (SF(O)F(1)). Membrane SF(O)F(1) ATPase showed optimum activity at pH 7, essentially the same as that of the S. mutans, although the activity of E. coli F(O)F(1) (EF(O)F(1)) was optimum at pH≥9. The membranes showed detectable ATP-dependent H(+)-translocation at pH 5.5-6.5, but not at neutral conditions (pH≥7), consistent with the role of S. mutans F(O)F(1) to pump H(+) out of the acidic cytoplasm. A hybrid F(O)F(1), consisting of membrane-integrated F(O) and -peripheral F(1) sectors from S. mutans and E. coli (SF(O)EF(1)), respectively, essentially showed the same pH profile as that of EF(O)F(1) ATPase. However, ATP-driven H(+)-transport was similar to that by SF(O)F(1), with activity at acidic pH. Replacement of the conserved c subunit Glu53 in SF(O)F(1) abolished H(+)-transport at pH 6 or 7, suggesting its role in H(+) transport. Mutations in the SF(O)F(1) c subunit, Ser17Ala or Glu20Ile, changed the pH dependency of H(+)-transport, and the F(O) could transport H(+) at pH 7, as the membranes with EF(O)F(1). Ser17, Glu20, and their vicinity were suggested to be involved in H(+)-transport in S. mutans at acidic pH.

Elastic rotation of Escherichia coli F(O)F(1) having ε subunit fused with cytochrome b(562) or flavodoxin reductase.

Intra-molecular rotation of FOF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FOF1 with the ε subunit connected to a globular protein [cytochrome b562 (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.