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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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Ternary interactions of E1G1, C1, and a1NT.(A) Possible model of ternary binding interaction of E1G1, C1 and a1NT. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of E1G1/C1/a1NT mixture (red) in comparison to E1G1 (green), C1 (blue) and a1NT (yellow) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 5B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the E1G1/C1/a1NT mixture. A 2∶1∶2 molar ratio of E1G1:C1:a1NT proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1 and a1NT proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the strong band eluted from the native gel in panel X (lane 4), suggesting the presence of C1, E1, and G1 in the complex. An unbound a1NT band was observed at the expected position. (E) Model showing the binding mode interpreted from the Biacore data (inset) where ligand was C1: X, the binding model of E1G1 as analyte (10 µM protein). Y, the binding model of a1NT as analyte (10 µM protein). Z, the binding model of E1G1 and a1NT as analytes (10 µM of each protein). The inset shows the sensorgrams of X, Y, and Z binding interactions (See details in the text).
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pone-0055704-g005: Ternary interactions of E1G1, C1, and a1NT.(A) Possible model of ternary binding interaction of E1G1, C1 and a1NT. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of E1G1/C1/a1NT mixture (red) in comparison to E1G1 (green), C1 (blue) and a1NT (yellow) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 5B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the E1G1/C1/a1NT mixture. A 2∶1∶2 molar ratio of E1G1:C1:a1NT proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1 and a1NT proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the strong band eluted from the native gel in panel X (lane 4), suggesting the presence of C1, E1, and G1 in the complex. An unbound a1NT band was observed at the expected position. (E) Model showing the binding mode interpreted from the Biacore data (inset) where ligand was C1: X, the binding model of E1G1 as analyte (10 µM protein). Y, the binding model of a1NT as analyte (10 µM protein). Z, the binding model of E1G1 and a1NT as analytes (10 µM of each protein). The inset shows the sensorgrams of X, Y, and Z binding interactions (See details in the text).

Mentions: It is thought that a key feature of the reversible disassembly of eukaryotic V-ATPases is located on the V1–Vo interface, involving Cfoot, the distal lobe of aNT, and one EG heterodimer (Figure 5A). Gel filtration analysis of an E1G1-a1NT-C1 ternary complex revealed a sharp peak with a distinct shoulder (Figure 5B). From the SDS-PAGE profile of the fractions (Figure 5C), we determined that the major peak corresponded to C1-E1G1, and the shoulder corresponded to the unbound a1NT subunit. Both E1G1 and C1 subunits showed low affinity for the a1NT subunit (Table 1), which explains the failure to form a stable ternary complex. By basic native gel electrophoresis, we observed two bands corresponding to E1G1-C1 and unbound a1NT (Figure 5D).


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)

Ternary interactions of E1G1, C1, and a1NT.(A) Possible model of ternary binding interaction of E1G1, C1 and a1NT. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of E1G1/C1/a1NT mixture (red) in comparison to E1G1 (green), C1 (blue) and a1NT (yellow) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 5B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the E1G1/C1/a1NT mixture. A 2∶1∶2 molar ratio of E1G1:C1:a1NT proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1 and a1NT proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the strong band eluted from the native gel in panel X (lane 4), suggesting the presence of C1, E1, and G1 in the complex. An unbound a1NT band was observed at the expected position. (E) Model showing the binding mode interpreted from the Biacore data (inset) where ligand was C1: X, the binding model of E1G1 as analyte (10 µM protein). Y, the binding model of a1NT as analyte (10 µM protein). Z, the binding model of E1G1 and a1NT as analytes (10 µM of each protein). The inset shows the sensorgrams of X, Y, and Z binding interactions (See details in the text).
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getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g005: Ternary interactions of E1G1, C1, and a1NT.(A) Possible model of ternary binding interaction of E1G1, C1 and a1NT. Dotted arrows indicate weak and solid arrows (black) strong binding. (B) Gel filtration profile of E1G1/C1/a1NT mixture (red) in comparison to E1G1 (green), C1 (blue) and a1NT (yellow) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 5B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the E1G1/C1/a1NT mixture. A 2∶1∶2 molar ratio of E1G1:C1:a1NT proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1 and a1NT proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the strong band eluted from the native gel in panel X (lane 4), suggesting the presence of C1, E1, and G1 in the complex. An unbound a1NT band was observed at the expected position. (E) Model showing the binding mode interpreted from the Biacore data (inset) where ligand was C1: X, the binding model of E1G1 as analyte (10 µM protein). Y, the binding model of a1NT as analyte (10 µM protein). Z, the binding model of E1G1 and a1NT as analytes (10 µM of each protein). The inset shows the sensorgrams of X, Y, and Z binding interactions (See details in the text).
Mentions: It is thought that a key feature of the reversible disassembly of eukaryotic V-ATPases is located on the V1–Vo interface, involving Cfoot, the distal lobe of aNT, and one EG heterodimer (Figure 5A). Gel filtration analysis of an E1G1-a1NT-C1 ternary complex revealed a sharp peak with a distinct shoulder (Figure 5B). From the SDS-PAGE profile of the fractions (Figure 5C), we determined that the major peak corresponded to C1-E1G1, and the shoulder corresponded to the unbound a1NT subunit. Both E1G1 and C1 subunits showed low affinity for the a1NT subunit (Table 1), which explains the failure to form a stable ternary complex. By basic native gel electrophoresis, we observed two bands corresponding to E1G1-C1 and unbound a1NT (Figure 5D).

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