An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus

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The V 0 V 1 -ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V 1 part (ABDF) and a proton channel V 0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V 0 V 1 -ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V 0 to acid or 8 M urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A 3 B 3 (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. V 0 V 1 -ATPases (V-ATPases) are the ATPase/ATP synthase superfamily that catalyzes the exchange of the energy between proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1-3). They are widely distributed in different types of eukaryotic cells and some bacteria (2, 4). In eukaryotic cells, V 0 V 1 -ATPases exist in both intracellular compartments and plasma membranes, and are responsible for the acidification of intracellular compartments, renal acidification, born resorption, and tumor metastasis (2). On the other hand, most prokaryotic V 0 V 1 -ATPases produce ATP using the energy of a transmembrane proton electrochemical gradient that is generated by a respiratory chain (4, 5).The overall structure of V 0 V 1 -ATPases is similar to that of F 0 F 1 -ATPases (␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 a 1 b 2 c 10Ϫ14 ), which are responsible for ATP synthesis in mitochondria, chloroplast, and plasma membranes of eubacteria (3, 6). Both are composed of two functional domains, the peripheral catalytic V 1 or F 1 and a membrane embedded ion translocating domain called V 0 or F 0 .The structure and subunit arrangements of F 0 F 1 -ATPases are well characterized. The x-ray structure of F 1 revealed a hexamer of alternating ␣ and ␤ surrounding a central cavity containing a highly ␣-helical ␥ subunit (7). The ␥ and ⑀ subunit constituted a central shaft, which directly contacted with the c subunit ring in F 0 (8). The b subunit has a hydrophobic Nterminal domain anchored in the membrane, and a hydrophilic C-terminal domain forms an elongated peripheral stalk that interacts with the F 1 moiety as a stator (9). The a subunit in F 0 , which consists of a stator part together with b subunits, is situated peripherally to the c subunit ring and plays a crucial role in the proton translocation (3, 10, 11).Like the F 0 F 1 -ATPases, the peripheral V 1 part contains a catalytic core, which is composed of three copies each of A and B subunits. The A subunit contains a catalytic site, and the A and B subunits are arranged alternately, forming a hexameric cylinder. The D subunit, which fills the central cavity of the A 3 B 3 cylinder...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Subunit Arrangement in V-ATPase from Thermus thermophilus

Yokoyama¹,

Nagata

Imamura³

et al. 2003

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“…The sequence corresponding to a 1550-bp region, which is upstream of the atpA gene, containing the termination codon of the atpF gene and the sequence corresponding to a 1750-bp region containing the mutated atpA gene (A-His 8 tags/A-S232A/A-T235S) with its Shine-Dalgarno sequence were cloned in the SphI-SalI and EcoRVEcoRI sites of pUTpyrE, respectively. A pyrE strain, T. thermophilus TTY1, was genetically transformed with the resultant plasmid as described previously (22). Transformants were selected on a minimummedium plate without uracil.…”

Section: Methodsmentioning

confidence: 99%

“…Purification of Mutant V 0 V 1 -ATPase-A mutated V 0 V 1 -ATPase (AHis 8 tags/A-S232A/A-T235S/L-E23C) was constructed by integration vector system (22)(23)(24). pUTpyrE, which carries the pyrE gene cassette, was constructed.…”

Section: Methodsmentioning

confidence: 99%

Rotation of the Proteolipid Ring in the V-ATPase

Yokoyama

Nakano

Imamura

et al. 2003

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V 0 V 1 -ATPase catalyzes the interconversion of the energy of proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1-3). Eukaryotic cells adopt V 0 V 1 -ATPase as an ATP hydrolysis-driven proton pump that carries out acidification of cellular compartments such as lysosomes and extracellular fluid in the case of renal acidification, bone resorption, and tumor metastasis (1). On the contrary, in archaea and some eubacteria, a major role of V 0 V 1 -ATPase is to produce ATP that is driven by downhill proton flow across membranes (4, 5). V 0 V 1 -ATPase is a complicated protein complex with a molecular mass of ϳ800 kDa composed of many different subunits arranged into two sectors, V 0 and V 1 (1). We have studied V 0 V 1 -ATPase from a thermophilic eubacterium, Thermus thermophilus, a stable enzyme that allows experimental procedures difficult for enzymes from other sources, and developed the expression systems of subcomplexes and subunits in Escherichia coli (5). The V 1 sector of T. thermophilus, which has ATP hydrolyzing activity by itself, is made up of four different subunits, A (63 kDa), B (53 kDa), D (25 kDa), and F (12 kDa) with a stoichiometry of A 3 B 3 D 1 F 1 (6). The A subunit contains a catalytic site, and the A and B subunits are arranged alternately to form a hexameric cylinder (7). The D subunit fills the central cavity of the cylinder and, together with the F subunit, forms a central stalk (7-9). The V 0 sector is responsible for proton translocation across membranes, and the principal components involved in proton translocation are a highly conserved family of hydrophobic subunits, often termed as proteolipid because of their solubility in organic solvents. The eukaryotic V 0 sector contains three similar but different proteolipid species, which are predicted to contain at least four transmembrane helices, arranged in a ring-like structure (2, 10, 11). On the contrary, single proteolipid proteins with two transmembrane helices, termed L subunit, make a ring-like structure in V 0 sector of T. thermophilus V 0 V 1 -ATPase (4, 7). The V 0 sector of T. thermophilus contains another membrane protein, I subunit (71 kDa), a homolog of yeast Vph1p, that interacts with the proteolipid ring and plays also a critical role in proton translocation (12).V 0 V 1 -ATPase is known to be structurally and evolutionary related to F 0 F 1 -ATP synthase, which is responsible for ATP production in mitochondria, chloroplasts, and many eubacteria (1, 4, 13). F 0 F 1 -ATP synthase is also composed of two sectors, F 0 and F 1 . The c subunit in F 0 is a proteolipid protein composed of two transmembrane helices and forms a ring structure (14). ATP hydrolysis in catalytic sites of F 1 drives rotation of the central stalk subunits, ␥ and ⑀ (15, 16), and this rotation in turn drives the rotation of the F 0 c-ring (16 -18) that is thought to be directly responsible for proton translocation (19). Because of the functional and structural similarity between V 0 V 1 -ATPase and F 0 F 1 -ATP synthase, it...

“…Preparation of V-ATPase-A His 3 tag was introduced to the C terminus of the V 0 -c subunits using an integration vector system (26,27). The recombinant T. thermophilus strain was grown at 70°C for ϳ24 h under strong aeration in a medium containing 8 g of polypeptone, 4 g of yeast extract, and 2 g of NaCl/l.…”

Section: Preparation Of Avi Tag or His Tagmentioning

confidence: 99%

ATP Hydrolysis and Synthesis of a Rotary Motor V-ATPase from Thermus thermophilus

Nakano

Imamura

Toei

et al. 2008

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Vacuolar-type HThe slower k on ATP than k on ADP and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses. Vacuolar-type Hϩ -ATPases (V-ATPases) 2 are found in a wide range of organisms. V-ATPase in eukaryotes functions as an ATP hydrolysis-driven proton pump that carries out acidification of cellular compartments, such as lysosomes, and extracellular fluid in the case of renal acidification, bone resorption, and tumor metastasis (1). A family of V-ATPases is also found in archaea and some eubacteria (the prokaryotic V-ATPase family) (2-7).3 A major role of the V-ATPase in prokaryotes is to produce ATP, a function performed by F 0 F 1 in eukaryotes and most eubacteria. V-ATPase and F 0 F 1 function by a rotary ATP synthase/ATPase mechanism (1). The hydrophilic domain of both V-ATPase and F 0 F 1 (called V 1 and F 1 , respectively) is responsible for ATP synthesis/hydrolysis and is connected via the central rotor and peripheral stator stalks to the transmembrane domain (V 0 and F 0 , respectively), which functions as an ion channel (1, 8). Although composition and arrangement of subunits differ considerably between V-ATPase and F 0 F 1 , they seem to share a common rotary catalysis mechanism, catalyzing the interconversion of the energy from proton translocation across membranes and the energy of ATP synthesis/hydrolysis through rotation of the central rotor subunits (8). It is thought that rotary catalysis is basically reversible (8, 9). When the transmembrane electrochemical gradient of protons (proton motive force (pmf)) is of sufficient strength, pmf drives rotation of a central rotor shaft to synthesize ATP. In contrast, when pmf is weak, the enzymes become an ATPdriven proton pump that rotates in the opposite direction driven by the energy released by ATP hydrolysis. Indeed, it has been shown that yeast V-ATPase, which functions as a proton pump in vivo, is able to catalyze ATP synthesis when exposed to an electrochemical gradient in vitro (10). In addition the F 1 portion of F 0 F 1 can synthesize ATP when the rotor shaft is forced to rotate in a direction opposite that of ATP hydrolysis (11,12). It is well known that the ATP hydrolysis reaction catalyzed by both V-ATPase and F 0 F 1 is highly regulated by a number of different mechanisms to prevent wasteful ATP consumption (1, 13). One such mechanism is Mg-ADP inhibition, whereby Mg-ADP binds into the catalytic site of the V 1 and F 1 domains and thus inhibits ATP hydrolysis (14 -17). Some enzymes appear to be inhibited irreversibly by these mechanisms (18,19).V-ATPase from the thermophilic bacterium, Thermus thermophilus, has been extensively investigated by biochemical and biophysical methods. Subunit rotation coupled to ATP hydrolysis of the V 1 portion has been visualized using a single mole-