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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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Interactions between E1G1 and H.(A) Possible mode of E1G1-H binding interaction in vitro. Using a Biacore system, the KD values for affinity of H-E1G1 was estimated to be 48 nM. (B) Gel filtration profile of E1G1/H complex formation (red) in comparison to E1G1 (green) and H (purple) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel Border colors indicate samples corresponding to the color scheme used in 3B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and H interaction. For complex formation, equimolar amounts of E1G1 and H proteins were mixed and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and H are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1H complex band eluted from the native gel in panel X (lane 3). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fractions eluted using buffer B; lane 2, E1G1 complex bound with His-tagged H subunit eluted using buffer C. (F) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (H) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to H ligand on a CM5 sensor chip.
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pone-0055704-g003: Interactions between E1G1 and H.(A) Possible mode of E1G1-H binding interaction in vitro. Using a Biacore system, the KD values for affinity of H-E1G1 was estimated to be 48 nM. (B) Gel filtration profile of E1G1/H complex formation (red) in comparison to E1G1 (green) and H (purple) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel Border colors indicate samples corresponding to the color scheme used in 3B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and H interaction. For complex formation, equimolar amounts of E1G1 and H proteins were mixed and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and H are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1H complex band eluted from the native gel in panel X (lane 3). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fractions eluted using buffer B; lane 2, E1G1 complex bound with His-tagged H subunit eluted using buffer C. (F) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (H) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to H ligand on a CM5 sensor chip.

Mentions: We observed similarly stable complex formation of E1G1 and H subunits (Figure 3A). Again, the equimolar complex of E1G1 and H eluted earlier with an apparent molecular weight of approximately 220 kDa, which is much higher than that calculated (Figure 3B). Complex formation was confirmed by analyzing the fractions by SDS-PAGE. Densitometric analysis of the gel filtration fractions of E1G1-H indicated that the binding stoichiometry of H vs. E1G1 was 1∶1.2 (Figure 3C and Figure S1). The anomalous early elution of these stator subunits alone (especially E1G1) or as a complex (E1G1-C1 or E1G1-H) can be interpreted as the effect of the elongated structure of these subunits, and is consistent with previous finding in studies on yeast [10], [25], [26].


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)

Interactions between E1G1 and H.(A) Possible mode of E1G1-H binding interaction in vitro. Using a Biacore system, the KD values for affinity of H-E1G1 was estimated to be 48 nM. (B) Gel filtration profile of E1G1/H complex formation (red) in comparison to E1G1 (green) and H (purple) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel Border colors indicate samples corresponding to the color scheme used in 3B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and H interaction. For complex formation, equimolar amounts of E1G1 and H proteins were mixed and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and H are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1H complex band eluted from the native gel in panel X (lane 3). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fractions eluted using buffer B; lane 2, E1G1 complex bound with His-tagged H subunit eluted using buffer C. (F) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (H) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to H ligand on a CM5 sensor chip.
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getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g003: Interactions between E1G1 and H.(A) Possible mode of E1G1-H binding interaction in vitro. Using a Biacore system, the KD values for affinity of H-E1G1 was estimated to be 48 nM. (B) Gel filtration profile of E1G1/H complex formation (red) in comparison to E1G1 (green) and H (purple) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Gel Border colors indicate samples corresponding to the color scheme used in 3B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of E1G1 and H interaction. For complex formation, equimolar amounts of E1G1 and H proteins were mixed and incubated on ice for 1 h (lane 3). Bands corresponding to one molar amount of E1G1 and H are visible in lanes 1 and 2, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1H complex band eluted from the native gel in panel X (lane 3). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fractions eluted using buffer B; lane 2, E1G1 complex bound with His-tagged H subunit eluted using buffer C. (F) Real-time binding evaluation was performed using a Biacore system. Sensorgrams for the binding of various concentrations of the analyte (E1G1) to the ligand (H) are shown. The inset curve shows the steady-state binding isotherm for binding of E1G1 at various concentrations to H ligand on a CM5 sensor chip.
Mentions: We observed similarly stable complex formation of E1G1 and H subunits (Figure 3A). Again, the equimolar complex of E1G1 and H eluted earlier with an apparent molecular weight of approximately 220 kDa, which is much higher than that calculated (Figure 3B). Complex formation was confirmed by analyzing the fractions by SDS-PAGE. Densitometric analysis of the gel filtration fractions of E1G1-H indicated that the binding stoichiometry of H vs. E1G1 was 1∶1.2 (Figure 3C and Figure S1). The anomalous early elution of these stator subunits alone (especially E1G1) or as a complex (E1G1-C1 or E1G1-H) can be interpreted as the effect of the elongated structure of these subunits, and is consistent with previous finding in studies on yeast [10], [25], [26].

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