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Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

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Structural model of human V-ATPase.Peripheral stalk subunits (E, G, C, aNT, and H) are emphasized using colors in the schematic model.
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pone-0055704-g001: Structural model of human V-ATPase.Peripheral stalk subunits (E, G, C, aNT, and H) are emphasized using colors in the schematic model.

Mentions: In mammals, vacuolar ATPases (V-ATPases) are found within endomembrane systems where their ATP hydrolysis-driven proton pump function has important roles in crucial cellular processes. Their malfunction can, therefore, cause diverse pathophysiological states in humans [1]–[6]. Eukaryotic V-ATPases are multiprotein complexes consisting of at least 14 different polypeptide chains. The sophisticated complex has a structure consisting of membrane-integrated Vo and cytoplasmic extrinsic V1 domains. Vo and V1 domains are linked by a connecting region consisting of a central shaft and multiple peripheral stator elements. The V1 domain contains three copies each of A, B, E, and G subunits and one copy each of C, D, F, and H subunits (Figure 1). It has a head group consisting of three nucleotide-binding A-B dimers responsible for ATP hydrolysis and two peripheral and central stalks consisting of the remaining V1 subunits, with distinct functions in the rotary mechanism by which the V-ATPase couples ATP hydrolysis to proton transport. The central stalk serves as a shaft that couples the energy of ATP hydrolysis to the rotation of a ring of proteolipid subunits in Vo. Conversely, the peripheral stalks serves to prevent rotation of the A3B3 head during ATP hydrolysis and acts as a structural link between V1 and Vo, thus providing physical support for the whole complex.


Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Structural model of human V-ATPase.Peripheral stalk subunits (E, G, C, aNT, and H) are emphasized using colors in the schematic model.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g001: Structural model of human V-ATPase.Peripheral stalk subunits (E, G, C, aNT, and H) are emphasized using colors in the schematic model.
Mentions: In mammals, vacuolar ATPases (V-ATPases) are found within endomembrane systems where their ATP hydrolysis-driven proton pump function has important roles in crucial cellular processes. Their malfunction can, therefore, cause diverse pathophysiological states in humans [1]–[6]. Eukaryotic V-ATPases are multiprotein complexes consisting of at least 14 different polypeptide chains. The sophisticated complex has a structure consisting of membrane-integrated Vo and cytoplasmic extrinsic V1 domains. Vo and V1 domains are linked by a connecting region consisting of a central shaft and multiple peripheral stator elements. The V1 domain contains three copies each of A, B, E, and G subunits and one copy each of C, D, F, and H subunits (Figure 1). It has a head group consisting of three nucleotide-binding A-B dimers responsible for ATP hydrolysis and two peripheral and central stalks consisting of the remaining V1 subunits, with distinct functions in the rotary mechanism by which the V-ATPase couples ATP hydrolysis to proton transport. The central stalk serves as a shaft that couples the energy of ATP hydrolysis to the rotation of a ring of proteolipid subunits in Vo. Conversely, the peripheral stalks serves to prevent rotation of the A3B3 head during ATP hydrolysis and acts as a structural link between V1 and Vo, thus providing physical support for the whole complex.

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

Show MeSH
Related in: MedlinePlus